List of Abbreviations
AZC
azetidine-2-carboxylic acid
BiP
binding protein
BSA
bovine serum albumin
CHLPEP
chloroplast transit peptide
cpn60
chaperonin 60
cpn10
co-chaperonin 10
DIECA
diethyldithiocarbamic acid
DTT
dithiothreitol
EDTA
ethylenediaminetetraacetic acid
EPPS
N-(2-hydroxymethyl) piperazine-N’-3-propanesulfonic acid
ER
endoplasmic reticulum
GRP
glucose-regulated protein
HA
haemmagglutinin
Hsc
heat shock cognate
HSE
heat shock element
HSF
heat shock factor
Hsp
heat shock protein
IEF
isoelectric focusing
IPG
immobilized pH gradient
kD
kilodaltons
LSU
rubisco large subunit
MES
2-(N-morpholino) ethanesulfonic acid
1
PAGE
polyacrylamide gel electrophoresis
pI
isoelectric point
pre-SSU
precursor rubisco small subunit
PMSF
phenylmethlsulfonyl fluoride
PVPP
polyvinylpolypyrrolidone
rbcL
gene for the rubisco large subunit
rbcS
gene for the rubisco small subunit
rcm
reductive carboxymethylation
rubisco
ribulose-1, 5-bisphosphate carboxylase/oxygenase
RuBP
ribulose-1, 5-bisphosphate
SDS
sodium dodecyl sulfate
SDS-PAGE
sodium dodecyl sulfate polyacrylamide gel electrophoresis
sHsp70
stroma-localized Hsp70
smHsp
small heat shock protein
SSU
rubisco small subunit
tris
tris(hydroxymethyl)methylamine
2
Abstract
After exposure to stressful conditions, cells increase expression of heat shock proteins, a
set of proteins that have been implicated in the protection of protein structure and
function. A common consequence of the stresses that induce the heat shock response may
be the unfolding of proteins. Therefore, the expression of heat shock proteins may be
induced by the presence of misfolded proteins. The present experiment examined the role
of misfolded proteins in the induction of heat shock response in Nicotiana tabacum L. var.
Petit Havana transformed with the rubisco large and small subunit genes from the diatom
Phaeodactylum tricornutum. The foreign algal rubisco subunits are highly expressed but
fail to fold and assemble properly into functional holoenzyme in the tobacco chloroplast.
Content of heat shock proteins was assessed by immunoblotting of the mature leaf tissue
extracts, from both the supernatant and pellet fractions. The content of Hsp60 was much
greater in the transgenic plants (and heat-shocked plants) than in the non-transformed
control plants; immunoreactivity was confined to the supernatant fraction. An increase in
soluble Hsp93 was also observed in the transgenic plants relative to the controls.
Although heat shock substantially increased the levels of small Hsps and Hsp101, these
proteins were not detected in the transgenic plants. There were no noticeable differences
in the soluble content of cytoplasmic Hsp70 and stromal Hsp70 between the transgenic
and control plants. Subsequent 2-D Western analysis detected the presence of Hsp70
isoforms in the transgenic plants but did not resolve its induction level relative to the
controls. Taken together, these results provide support for the hypothesis that the
presence of misfolded proteins induces the production of heat shock proteins; however
the response is restricted to certain classes of heat shock proteins.
3
Introduction
In response to stressful conditions, cells increase expression of a set of proteins
called the heat shock proteins (Hsps). Hsps serve to protect cellular proteins from
denaturation, aggregation and also irreversible damage (Becker and Craig, 1994; Boston
et al., 1996; Wang et al., 2004). In addition, many constitutively-expressed molecular
chaperones (Hscs), besides guiding the folding events of nascent polypeptides, are
involved in the stabilization and refolding of non-native structures of proteins (Boston et
al., 1996; Wang et al., 2004). Therefore, Hscs are also often characterized as Hsps. The
realization that this defense mechanism, termed the “heat shock response”, may be
signaled by protein denaturation, is appealing. In the present experiment, we provide an
opportunity to examine this in vivo cellular stress response in higher plants, which to our
knowledge has not yet been elucidated.
For this paper, I will begin with an overview of the general function and
regulation of Hsps, followed by a brief discussion on plant-specific Hsps from five
families: Hsp60, Hsp70, Hsp90, Hsp100 and small Hsps. Next, I will focus on evidence
that Hsps are induced by the presence of misfolded proteins. Then, I will introduce
ribulose-1, 5-bisphosphate carboxylase/oxygenase (rubisco), the key enzyme in the
Calvin cycle. Specifically, I will provide the current understanding and research on the
molecular chaperones involved in the folding and assembly pathway of rubisco. I will
also present examples of tobacco transformation experiments that aim to construct
rubisco with enhanced kinetic properties in the higher plant, but failed due to unknown
folding requirements of the foreign enzyme. In conclusion, I will state the hypothesis of
the present experiment and describe the procedures taken to validate my theory.
General Functions of Hsps
Hsps play an essential role in normal cellular homeostasis and the stress response
(Pirkkala et al., 2001; Vierling, 1991; Wang et al., 2004). In the former, Hsps are
produced constitutively as molecular chaperones, termed the heat shock cognate (Hscs).
Under non-stressed conditions, Hscs guide the proper folding and assembly of nascent
polypeptides during translation and/or after they emerge from the ribosomes (Boston et
al., 1996; Miernyk, 1999). During these co- and post-translational processes, Hscs bind to
4
the exposed hydrophobic regions of the partially-folded polypeptides to prevent
unfavorable interactions that may lead to incorrect folding and/or aggregation. Hscs then
direct the polypeptides into the correct formation of tertiary structures (Boston et al.,
1996; Miernyk, 1999). Hscs also function in protein transport. Hscs maintain precursors
in a partially-unfolded, transport-competent state for entry into organelles. This import
process involves ATP hydrolysis, which acts to drive the unidirectional movement of
precursor proteins into subcellular compartments such as mitochondria, chloroplast and
etc. (Becker and Craig, 1994; Boston et al., 1996).
During cellular stress, proteins lose some or all of their native structure. In this
situation, Hsps bind to the partially-denatured proteins to block aggregation and
irreparable damage (Becker and Craig, 1994; Miernyk, 1999; Wang et al., 2004). Again,
the hydrophobic stretches of the unfolded proteins are targeted by Hsps to destabilize
improper interactions. Hsps are also involved in the solubilization of thermallyaggregated proteins (Glover and Tkach, 2001; Wang et al., 2004). Hsps disassemble the
protein aggregates to produce intermediates that are capable of refolding or are targeted
for degradation.
Negative Regulation of Hsps
First observed in Drosophila, the heat shock response is now described in a wide
range of species, including Homo sapiens, Saccharomyces cerevisiae, Arabidopsis
thaliana and Escherichia coli (Ananthan et al., 1986; Vierling, 1991). This stress-coping
mechanism is characterized by elevated expression of Hsps, along with the concomitant
termination of normal protein synthesis (Schöffl et al., 1998). For eukaryotes, the current
model of synthesis of Hsps is based on the negative regulation of heat shock factors
(HSFs) activity by Hsp70 and feedback control. Prior to heat shock, Hsp70s are found in
a binary complex with HSFs. However, during cellular stress, these Hsps are recruited to
stabilize the partially-denatured proteins to prevent further damage (Howarth and
Ougham, 1993; Schöffl et al., 1998). At this stage, the free, unbound HSFs undergo
oligomerization, move into the nucleus and bind to the promoter regions of Hsp genes
termed the heat shock elements (HSEs) (Howarth and Ougham, 1993; Scharf et al., 1998).
The active HSFs then induce the downstream expression of Hsps. The HSFs continue to
5
upregulate the production of Hsps until the synthesis of excess levels of Hsp70 shuts off
the activity of HSFs, and consequently, the heat shock response (Howarth and Ougham,
1993; Schöffl et al., 1998).
Hsps and Hscs in Plants
Hsps comprise a large family of highly-conserved proteins that are broadly
designated according to their molecular weight. In plants, five major families of Hsps are
recognized: the heat shock protein 60 (Hsp60), the heat shock protein 70 (Hsp70), the
heat shock protein 90 (Hsp90), the heat shock protein 100 (Hsp100) and small heat shock
protein (smHsp) (Boston et al., 1996; Wang et al., 2004). As in other eukaryotes, many
of these heat shock proteins are also constitutively expressed as molecular chaperones
(Hscs). All plant Hscs and Hsps are encoded by the nuclear genome (Boston et al., 1996;
Wang et al., 2004).
Hsp70
Hsp70s are characterized by their high degree of conservation. A comparison of
amino acid sequence for cytoplasmic Hsc70 and Hsp70 homologues of Saccharomyces
cerevisiae, Arabidopsis, maize, petunia, soybean, pea and human origins revealed them
as 71.0% identical and 91.0% similar (Vierling, 1991). In prokaryotes, eg. Escherichia
coli, Hsp70 is known as the DnaK homologue. Hsp70 members also participate in diverse
cellular functions. As Hscs, they are involved in assisting the folding of de novo
synthesized polypeptides, higher-order assemblies, translocation of precursor proteins,
and degradation of damaged proteins (Boston et al., 1996; Sung et al., 2001; Wang et al.,
2004; Zhang and Glaser, 2002). Stress-induced Hsp70s function to promote refolding and
prevent aggregation of partially-denatured proteins, as well as tag irreversibly-damaged
proteins for proteolysis. Hsc70/Hsp70 operates though iterative cycles of substratebinding and release, which are coupled with its intrinsic ATPase activity (Boston et al.,
1996; Sung et al., 2001; Wang et al., 2004; Zhang and Glaser, 2002).
The functions performed by individual Hsp70 members are most likely
determined by their subcellular localization and different expression profiles. In plants,
different members of the HSP70 gene family encode proteins that are targeted to different
6
cellular compartments, including the cytosol, mitochondria, chloroplast and endoplasmic
reticulum (ER) (Vierling, 1991). The expression pattern for most of these genes can be
broadly divided into three categories: heat-induced, expressed-constitutively but not heatinduced and expressed constitutively with additional heat induction (DeRocher and
Vierling, 1995). In particular, the accumulation profiles of Hsp70s in the pea (Pisum
sativum) leaves and mung bean (Vigna radiata L. Wilczek var. radiata) seeds were
studied using two-dimensional Western analysis (DeRocher and Vierling, 1995; Wang
and Lin, 1993). DeRocher and Vierling (1995) showed that within the isoelectric points
(pIs) of 5.4-5.7, two 72 kDa polypeptides were induced by heat stress, in addition to the
three 70 kDa polypeptides detected at the basal level in pea leaves. A thermally-induced
Hsp70 from mung bean seeds was also found to migrate at the pI of 5.6 and
distinguishable from its constitutive counterpart (Wang and Lin, 1993).
Hsp60 (chaperonin 60)
Found in mitochondria and chloroplasts, the Hsp60 family is composed of two
distinct members: chaperonin 60 (cpn60) and co-chaperonin 10 (cpn10) (Boston et al.,
1996; Dickson et al., 2000; Vierling, 1991). More specifically, chloroplast cpn60 is
known as the ribulose-bisphosphate carboxylase (rubisco) binding protein, an oligomeric
protein that was first discovered in a bound complex with the nascent rubisco large
subunit (LSU). Cpn60 comprises the α and β subunits and acts in concert with cpn21, a
co-chaperonin that is twice the size of any other known cpn10 found in bacteria and
mitochondria. In prokaryotes, the chaperonins are referred as the GroEL (cpn60) and
GroES (cpn10) homologues (Boston et al., 1996; Dickson et al., 2000; Vierling, 1991).
The chaperonins are expressed constitutively and increase slightly under heat
stress (Boston et al., 1996; Vierling, 1991). In the presence of ATP, chaperonins function
to assist the folding and assembly of plastid proteins. Among the chloroplast-localized
proteins that have been shown to associate with cpn60 are the rubisco small and large
subunits, Reiske Fe-S protein, glutamine synthetase, β-subunit of ATP synthase,
chloramphenicol acetyltransferase, pre-β-lactamase, and the light-harvesting chlorophyll
a/b binding protein (Lubben et al., 1989; Madueño et. al., 1993).
7
Small Hsp
Ubiquitous in nature, the small Hsps (smHsps) are low molecular weight Hsps
that range from 17-30 kDa (Heckathorn et al., 1998; Waters et al., 1996; Vierling, 1991).
In plants, there are at least six recognized families, each of which is found in a distinct
cellular compartment (ie. cytosol, chloroplast, mitochondria and ER) (Wang et al., 2004;
Waters et. al., 1996). SmHsps are not expressed at detectable levels under normal
physiological conditions but are produced in response to heat and other stresses
(Heckathorn et al., 1998; Wang et al., 2004). In particular, the chloroplast smHsp is
known as the most heat-responsive of all smHsps (Heckathorn et al., 1998).
In contrast to other higher molecular weight Hsps, smHsps are not themselves
able to refold non-native proteins (Lee and Vierling, 2000). Rather, they act to stabilize
and prevent the aggregation of partially-denatured proteins. The subsequent refolding of
these proteins is then mediated by ATP-dependent chaperones such as the Hsp70 system.
During acute stress, smHsps also function in the protection of photosystem II, a highly
thermolabile component of the electron transport chain in plants (Heckathorn et al., 1998).
Hsp90
Like the Hsp70 family, Hsp90 is an abundant, highly-conserved group of proteins
(Sangster and Queitsch, 2005; Wang et al., 2004). It comprises 1-2% of total cellular
proteins and shares 63-71% amino acid sequence identities with Hsp90s from yeast and
animal origin (Krishna and Gloor, 2001; Wang et al., 2004). In plants, Hsp90s are
localized to different cellular compartments, including the cytosol, ER, mitochondria, and
chloroplast.
Present at high levels under normal growth conditions, Hsp90 is moderately
induced when cells are exposed to stress (Krishna and Gloor, 2001; Wang et al., 2004).
Hsp90s are essential for plant survival and function as part of a multichaperone complex
in the folding, activation and trafficking of signaling molecules such as protein kinases.
During heat stress, Hsp90s help to prevent protein aggregation and misfolding (Vierling,
1991). In addition, Hsp90s participate in several disease resistance pathways, including
the R (Resistance)-gene mediated defense signaling (Sangster and Queitsch, 2005). Also,
in Arabidopsis, Hsp90s act as a buffer to sustain the functions of mutated proteins that are
8
involved in the signal-transduction network and morphogenesis. The expression of such
genetic variations is suppressed by the “buffering” effect of Hsp90s under normal
physiological conditions. However, when the plant is faced with environmental assaults,
Hsp90s are diverted to function in other important cellular processes for survival, and
consequently, the effects of these hidden mutations/variants are released. As such, the
loss of the Hsp90 “buffer” system may confer a selective advantage to Arabidopsis under
stress conditions, as well as contribute to its evolutionary adaptation (Sangster and
Queitsch, 2005; Wang et al., 2004).
Hsp100 (Clp)
Members of the Hsp100 family belong to a larger class of proteins, Clp (Vierling,
1991; Wang et al., 2004; Young et al., 2001). In particular, two subclasses of the Clp
proteins, the cytosolic ClpB and stroma ClpC, are well-characterized. Also known as the
yeast Hsp104 and plant Hsp101 homologues, ClpB is essential for thermotolerance
(Young et al., 2001). In higher plants, Hsp101 is expressed at low, sometimes
undetectable levels under normal growth conditions but is strongly induced upon heat
stress (Queitsch, 2000; Young et al., 2001). Hsp101 functions to solubilize protein
aggregates generated by thermal stress before releasing them in a refolding-competent
state to the Hsp70 system (Glover and Tkach, 2001; Wang et al., 2004). Also, Hsp101
works in conjunction with the proteolytic ClpP to degrade proteins that are irreversibly
damaged (Wang et al., 2004).
In contrast, ClpC is constitutively-expressed in higher plants, with little additional
induction during heat stress (Agarwal et al., 2001; Clarke, 1996). Also known as Hsp93,
it is present as a soluble protein in the chloroplast stroma and is also a component of the
translocation complex (Constan et al., 2004; Nielsen et al., 1997; Zhang and Glaser,
2002). While in association with the chloroplast inner envelope, Hsp93 is believed to
facilitate the import process of precursor proteins in an ATP-dependent manner. In
addition, it is speculated that Hsp93 participates in the folding and/or degradation of
plastid polypeptides, especially those that are related to oxygenic photosynthesis (Clarke,
1996).
9
Hypothesis: Induction of Hsps by Misfolded Proteins
The expression of Hsps is commonly associated with a variety of physical and
chemical stresses such as temperature upshift, extreme pH levels, cellular treatment with
ethanol, amino acid analogs, etc. (Ananthan et al, 1986; Becker and Craig, 1994). All of
these phenomena are linked to protein denaturation, misfolding and sometimes
aggregation. This discovery leads to the idea that the initiation of a heat shock response
may be a function of protein denaturation. Therefore, the presence of misfolded proteins
may serve as a cellular indicator for the expression of Hsps.
To date, several studies have been performed to examine the putative role of
misfolded proteins in the signaling of heat shock response. Toad (Xenopus laevis)
oocytes carrying a Drosophila Hsp70 reporter construct were co-injected with purified
bovine β-lactoglobulin or bovine serum albumin (BSA) (Ananthan et al, 1986). Coinjection of proteins destabilized by reductive carboxymethylation (rcm) stimulated the
expression of Hsp70 gene. However, this signal was not observed when the proteins were
introduced in their native, unmodified form. This experiment indicated that purified
proteins are only competent to activate the Hsp70 reporter gene when denatured, but not
in their native state. Comparing the results with that of a heat shock positive control,
Ananthan et al. (1986) further observed that the degree of Hsp70 induction is regulated
by the amount of denatured bovine β-lactoglobulin or bovine BSA present in the oocytes.
Thus, Ananthan et al. (1986) established that proteins, when in their non-native state, can
induce the production of Hsp70 in a concentration-dependent fashion.
Next, Lee and Hahn (1988) cultivated Chinese hamster ovary fibroblasts in the
presence of diamide, a sulfhydryl oxidizing agent, at 37°C for 1 hour and then exposed
them to a heat challenge at 45°C for 45 minutes. The cells exhibited a rise in cellular
survival by almost three orders of magnitude compared to those grown without diamide.
These results indicated that diamide-treated cells acquired some form of resistance to heat
shock. Presumably, thermotolerance was developed due to the increased production of
Hsps. This prediction was verified by the detection of at least three Hsps at 110 kD, 89
kD and 70 kD by [35S]methionine pulse-labeling. The findings of Lee and Hahn (1988)
were later supported by Freeman et al. (1995), who showed that diamide-induced protein
thiol oxidation in Chinese hamster ovary cells activated the DNA-binding ability of HSF
10
and subsequently the expressions of Hsp70 and Hsc70. Taken together, the results of Lee
and Hahn (1988) and Freeman et al. (1995) demonstrated that cellular proteins with nonnative disulfide bridges can act as a signal for the induction of the heat shock response.
In another experiment, simian cells were transfected with either the wild-type or
mutant forms of the influenza virus haemmagglutinin (HA) (Kozutsumi et al., 1988). The
latter was misfolded and thus blocked from exiting the endoplasmic reticulum (ER).
Simian cells carrying the mutant construct exhibited an increase in the mRNA and
translation product of a Hsp70-related protein, the 78 kD glucose-regulated protein (GRP)
or binding protein (BiP), while the cells carrying the wild type protein did not. These
results indicated that the expression of GRP78 is correlated to the incorrect folding of the
HA mutant protein (Kozutsumi et al., 1988). Therefore, the presence of misfolded
proteins can induce the production of Hsps-related protein in the ER.
Trotter et al. (2001, 2002) showed that misfolded proteins induce the expression
of Hsps in yeast cells treated with sublethal concentrations of azetidine-2-carboxylic acid
(AZC), a compound that competitively inhibits the incorporation of proline during
protein synthesis, causing improper backbone formation and folding. Specifically, the
expression of small Hsps and other HSF-dependent genes were upregulated. The results
suggested that proteins distorted by AZC evoke the heat shock response through a similar
mechanism as heat stress, which involves the binding of HSFs to the HSEs of
temperature-sensitive proteins. This activation of HSF, however, was found later to be
selective, as treatment with canavanine (an arginine analog that is less potent than AZC
in causing the misfolding of proteins) did not yield a similar stress response. Misfolded
proteins caused by canavanine treatment were not competent to induce the full
complement of HSE-regulated proteins, presumably because HSF was not robustly
activated (Trotter et al., 2001, 2002). Thus, the extent of misfolding may be critical for
incorrectly-folded proteins to act as a signal for the induction of heat shock response.
Using Chinese hamster lung cells, McDuffee et al. (1997) observed that proteins
containing non-native disulfides produced by menadione, a redox-cycling compound, can
also serve as a signal for the induction of heat shock response. Compared to diamide,
menadione is more potent in causing oxidative stress, resulting in the accumulation of
denatured proteins as insoluble, aggregated complexes. Upon treatment, the lung cells
11
activated HSF-1, accumulated Hsp70 mRNA, and increased the synthesis of Hsp70. This
experiment indicated that denatured protein aggregates are capable of eliciting a heat
shock response (McDuffee et al., 1997).
Challenging the findings of all the above researchers except that of McDuffee et
al. (1997), Mifflin and Cohen (1994) showed that denatured protein aggregates are the
true, effective stress inducer, rather than proteins that misfolded but did not form
aggregates. Mifflin and Cohen (1994) demonstrated that of all the potential stress
response inducers (eg. rcm-BSA, rcm-β-lactoglobulin, α-casein, oxidized RNase A, etc.)
investigated, only the chemically-modified, unheated BSA is capable of triggering the βgalactosidase activity of Hsp70 reporter gene present in Xenopus laevis oocytes. The
BSA inducer was later recognized as denatured protein aggregates that tend to
disassemble into smaller particles when heated. Thus, denaturation per se is insufficient
for stress response induction. Only the introduction of aggregated, misfolded proteins is
capable of inducing the synthesis of Hsps.
Implications of the Research Findings
While evidence clearly supports the role of denatured proteins in the induction of
heat shock response, it is still debatable whether the response is initiated by the presence
of any misfolded protein, or only specific proteins that achieved a threshold in their
degree of denaturation and/or become aggregated (Mifflin and Cohen, 1994; Trotter et al.,
2002). It is also unknown whether the amount of denatured protein and/or aggregated
protein affects the initiation of heat shock response. Furthermore, it is unclear how the
complement of Hsps is selectively and/or differentially induced when a specific
denatured protein is present. Lastly, the exact nature of mechanisms that are involved in
evoking the heat shock response remain to be investigated.
Rubisco Synthesis and Assembly
The form of misfolded protein used in the present experiment is ribulose-1, 5bisphosphate carboxylase/oxygenase (rubisco). Rubisco is the key enzyme for carbon
dioxide assimilation in the Calvin cycle. It also catalyzes the oxygenation of ribulose-1,
5-bisphosphate (RuBP), in which O2 competes with CO2 as a substrate at the same active
12
site (Hartman and Harpel, 1994; Lorimer, 1981; Spreitzer, 1999). Two major isoforms of
rusbico, Form I and Form II, are recognized, based on structural divergence (Kellogg and
Juliano, 1997; Whitney et al., 2001). The Form II enzyme occurs in some bacteria and
dinoflagellates as a simple assembly of two LSUs. The Form I rubisco is found in most
bacteria (including cyanobacteria), algae, and higher plants. It is composed of eight 50-55
kD LSUs that are arranged as tetramers of dimers in a barrel fashion; this barrel core is
then capped at each end by four 12-18 kD SSUs. For the hexadecameric Form I isoform,
two subclasses “green” and “red” have been discovered, with the latter being more
kinetically-efficient (Whitney et al., 2001). Additionally, these two subtypes differ in
their manner of inheritance. “Green” rubisco is encoded by rbcL and rbcS in the plastidic
and nuclear genomes, respectively, while “red” rubisco is encoded in the chloroplast
genome as a simple bicistronic operon. Nevertheless, it is believed that both subclasses of
the Form I rubisco are descended from a common cyanobacterial ancestor and
presumably share similar synthesis, folding, and assembly machineries (Whitney et al.,
2001).
Fate of rubisco SSU during and after translocation into chloroplast stroma
In higher plants such as tobacco, “green” rubisco subunits are expressed (Whitney
et al., 2001). The nuclear-encoded SSU is synthesized on cytoplasmic polysomes as a
precursor protein (pre-SSU) carrying a phosphorylated amino-terminal transit peptide
(Gutteridge and Gatenby, 1995; Houtz and Portis, 2003; Soll and Schleiff, 2004). The
pre-SSU is then transported into the chloroplast stroma, where LSU resides. During the
import process, the transit peptide interacts with chloroplast Hsp70(s) (Houtz and Portis,
2003; Ivey et al., 2000; Becker et al., 2005). Specifically, this group of chaperones is
believed to act as a translocase, directing the ATP-dependent, unidirectional movement
of pre-SSU across the chloroplast envelope (Ivey et al., 2000). This molecular motor
model is supported by algorithm findings, which indicated that over 95% of the members
from the chloroplast transit peptide (CHLPEP) database contain at least one potential
Hsp70 recognition domain (Ivey et al., 2000). Also, in a native gel shift assay, the fulllength pea pre-SSU transit peptide is found to exhibit an overall higher affinity for DnaK
than any of the synthetic peptides (20-mers) corresponding to the N-terminal (1-20),
13
middle (21-40) and C-terminal (41-60) thirds of the transit peptide, suggesting the
cooperative presence of several Hsp70 binding domains on its intact form (Ivey et al.,
2000).
Ivey and Bruce (2000) speculated that multiple binding sites of Hsp70 on the preSSU transit peptide permit an incoming precursor to simultaneously engage with more
than one chaperone molecule, allowing the entry of the stroma-targeted transit peptide in
an unraveled, import-competent state. In fact, at least four different Hsp70 homologues:
Com70 (outer envelope), IAP70 (intermembrane space), stroma Hsp70 (stroma) and
lumenal Hsc70 (thylakoid lumen) have been identified as members of this essential
chloroplast protein import machinery (Ivey and Bruce, 2000; Marshall et al., 1990; Rial
et al., 2000; Schnell et al., 1994; Zhang and Glaser, 2002; Figure 1). Also, ClpC (Hsp93),
a stromal Hsp100 homolog, is described as a molecular chaperone within the chloroplast
translocation complex (Akita et al., 1997; Nielsen et al., 1997). Nielsen et al. (1997)
reported that co-immunoprecipitation of Hsp93 and pre-SSU only occurred under
conditions that supported either the binding or translocation of a precursor protein. Such
interaction was found to decrease with time during the import process, indicating that
pre-SSU is associated with Hsp93 as a functional translocation intermediate (Nielsen et
al., 1997). The addition of ATP was also shown to destabilize the association of ClpC
from the precursor-containing complex, further suggesting that ClpC has a role as a
molecular motor in chloroplast protein import (Nielsen et. al., 1997).
After its uptake into the chloroplast, the transit sequence of pre-SSU is cleaved by
a stroma peptidase. The mature protein then associates with chloroplast chaperonins
60/21 before entering the rubisco assembly pathway (Barraclough and Ellis, 1980;
Gatenby et al., 1988; Gutteridge and Gatenby, 1995; Houtz and Portis, 2003).
Rubisco LSU folding in the chloroplast stroma
The maturation pathway of rubisco LSU is best characterized with respect to its
chaperonin-mediated folding. In isolated chloroplasts, newly-synthesized LSUs have
been isolated bound to a large oligomeric protein of over 600 kD comprising 60 kD
subunits known as chaperonin 60 (cpn60) (Gutteridge and Gatenby, 1995; Houtz and
Portis, 2003). The complex between cpn60 and LSUs is shown to dissociate upon ATP
14
addition, a process that releases the folded LSUs for its assembly with the mature, folded
SSUs to form the rubisco holoenzyme (Viitanen et al., 1995).
To date, there are several lines of evidences suggesting the putative role of
Hsp70 chaperones in rubisco LSU folding, which is presumed to occur prior to the
chaperonin-mediated folding. For instance, Brutnell et al. (1999) noted the accumulation
of rbcL transcripts but not LSUs in maize plants lacking Bsd2, a gene that encodes for
Hsp40, a co-chaperone for Hsp70. This gene ablation appeared to induce exclusively the
misregulation of rbcL, since the steady-state levels of rbcS transcripts were found to be
similar for both wild type and maize mutants. In a separate experiment, E. coli dnaK null
mutants transformed to express either the Rhodospirillum rubrum (Form II or dimeric) or
Chromatium vinousum (Form I or hexadecameric) rubisco showed an increase in
aggregated, insoluble rubisco LSUs. However, when DnaK levels were reestablished to
normal levels through plasmid induction, soluble LSUs and rubisco activity were
recovered in both transformants (Checa and Viale, 1997). These findings appear to
underscore the essential role of Hsp70 in rubisco LSU synthesis and folding.
Assembly of Rubisco Subunits
The exact requirements for rubisco assembly are unclear, although researchers
postulated that the process requires the presence of chaperonin 60, co-chaperonin cpn10
and MgATP (Gutteridge and Gatenby, 1995; Schmidt et al., 1994). The highly
aggregation-prone rubisco folding intermediates are thought to be stabilized by the above
factors, which cooperatively mediate the kinetic partitioning of the rubisco folding
intermediates between the misfolded and correctly-folded states (Gatenby and Viitanen,
1994). In a later step, the co-chaperonin cpn10 acts to discharge the bound rubisco
holoenzyme complex from the chaperonin system (Gutteridge and Gatenby, 1995;
Viitanen et al., 1995). Again, ATP hydrolysis is needed to drive the system towards
dissociation.
Transgenic Manipulation in Higher Plants - Tobacco
Current rubisco research is focused on altering the discrimination between its
competing substrates CO2 and O2, or in other words, its specificity factor, Φ (Parry et al.,
15
2003). This parameter is found to vary by at least a factor of 10 among divergent species
(Jordan and Ogren, 1981, 1983; Read and Tabita, 1992). Significant differences in
relative specificities are also documented from enzyme paralogs among closely-related
organisms (Kanevski et al., 1999). Exploiting these natural variations in the catalytic
properties of rubisco, nuclear or chloroplast transformation of foreign rbcL/S has been
carried out to improve the photosynthesis of higher plants (Andrews and Whitney, 2003;
Parry et al., 2003).
Tobacco lines were transformed with the sunflower Helianthus annuus rbcL,
replacing the endogenous, plastome-encoded gene (Kanevski et al., 1999). The hybrid
enzyme, comprising the sunflower LSU and tobacco SSU, is expressed at 30% of the
wild-type level of rubisco and found to be catalytically active. Nevertheless, it exhibits
compromised affinities for both RuBP and CO2 and a four-fold decrease in turnover rate,
even though its specificity factor is comparable to that of wild-type tobacco. Binding
incompatibility at the interface between the large and small subunits is proposed to
account for the unfavorable change in kinetic parameters (Kanevski et al., 1999).
Next, the research team of Whitney et al. (2001) inserted rbcLS operons of
Galdieria sulphururia and Phaeodactylum tricornutum separately into the inverted repeat
region of the tobacco plastid genome, leaving the endogenous rbcL gene unaltered.
Specifically, this transformation is aimed at assessing the feasibility of expressing the
kinetically-efficient Form I “red” enzymes from these two algae in higher plants. In both
cases, the foreign rbcLS transcripts are abundantly translated. However, no algal LSUs
are detected in the soluble, supernatant fractions of leaf extracts, suggesting problems
with folding and/or assembly and also a complete lack of foreign rubisco activity. In
addition, the corresponding algal SSUs are mostly recovered in the insoluble pellet.
Hence, Whitney et al. (2001) concluded that the non-green algal rubisco subunits are not
recognized by the tobacco chloroplast chaperones, although the reasons remain unclear.
The special requirements of “red” subclass rubisco for efficient folding and assembly in
higher plants chloroplasts would thus have to be further defined before one can
successfully exploit the attractive kinetic properties of red enzyme (Whitney et al., 2001).
Whitney and Andrews (2001) next replaced the native rbcL of tobacco with the
homologous gene from Rhodospirillum rubrum. Active rubisco protein was recovered,
16
reflecting the simpler dimeric structure and consequential simplicity in subunit assembly
of the α-proteobacterium holoenzyme. More specifically, the functional assembly of R.
rubrum rubisco demonstrated that the enzyme homolog from a phylogenetically-distant
organism can operate in higher plant chloroplast without severe problems (Whitney and
Andrews, 2001). Therefore, the prospects for the successful replacement of higher plant
rubisco with the Form I “red” enzyme remain promising.
Clearly, the introduction of a high specificity factor rubisco into tobacco
continues to be a major challenge. Attempts to engineer or discover the properties of a
better rubisco may be futile if one cannot transfer the better enzyme to a compatible host.
Therefore, future studies are required to define the special folding and assembly
requirements of such rubisco. The endeavor, if successful, would represent a significant
step towards molecular manipulation of the rubisco in higher plants eg. tobacco,
promising great agronomic benefits.
Present Experiment
Research investigating the induction of heat shock proteins (Hsps) has indicated
that the common link among all physical and chemical stresses that evoke the heat shock
response may be the denaturation of proteins. Thus, expression of Hsps may be triggered
by the presence of misfolded proteins. The present experiment examines the role of
misfolded proteins in the induction of the heat shock response in Nicotiana tabacum L.
Petit Havana transformed with rubisco genes from the diatom Phaeodactylum
tricornutum. The foreign algal rubisco subunits are highly expressed but fail to fold and
assemble properly into functional holoenzyme in the tobacco chloroplast (Whitney et al.,
2001). This introduces a form of misfolded protein in the transgenic N. tabacum plants,
which I used as a model for testing the induction of the expression of Hsps in vivo. If the
presence of misfolded proteins induces the expression of Hsps, then the transformed
tobacco plants should contain higher levels of some or all Hsps than the non-transformed
control plants.
To support the above hypothesis, I tested the transgenic tobacco plants for
evidence of expression of Hsps using antibodies directed against small Hsp, Hsp60,
Hsp70, Hsp93 (ClpC) and Hsp101. By exploring the expression profiles of different
17
families of Hsps, I showed whether the transformed plants exhibit a selective or similar
response in upregulating their levels of Hsps. As a comparison, I also performed similar
immunoblot assays on the leaf extracts of a heat-shocked plant (positive control), and a
non-heat-shocked, non-transformed plant (negative control).
Materials and Methods
General Procedures
Plant Growth
Non-transformed Nicotiana tabacum L. var. Petit Havana and transgenic plants (Whitney
et al., 2001) used in the study were raised to maturity under ambient light and
temperature conditions in the greenhouse of Franklin and Marshall College. Whitney et
al. (2001) generated the transgenic tobacco plants by inserting rubisco genes from the
diatom Phaeodactylum tricornutum into the endogenous chloroplast genome.
Approximately 2 months after cotyledon emergence, sets of three leaf discs (1.5 cm
diameter each) were harvested, quick-frozen in liquid nitrogen, and stored at -80°C until
use. Tissue samples were taken from young, fully-expanded leaves 5, 6 or 7 of the
tobacco plants, numbered beginning from the first leaf over 2 cm wide at the apical
meristem. For heat shock studies, a tobacco plant was placed for four hours in a growth
chamber to achieve a leaf temperature of 44-45°C. Positive control samples were
collected immediately following the heat shock treatment and again after four hours of
recovery at ambient temperature in the greenhouse.
Antibodies
Primary antibodies raised against specific Hsps in rabbit or mouse were used to detect the
expression of heat shock proteins in N. tabacum plants. Anti-(plant/algal)Hsp60 antibody
(EnVirtue Biotechologies, Inc., Harrisonburg, VA; AB-H100-P, 1:5000 dil.) raised in
rabbit crossreacts with plant Hsp60 and the a and b isoforms of the chloroplast Hsp60. Dr.
Scott Heckathorn provided the polyclonal rabbit antisera (Heckathorn et al., 1998)
specific to the methionine-rich domain of chloroplast small Hsp (smHsp-met, 1:1000 dil.)
and to the highly-conserved -helical region of general small Hsps (smHsp-α, 1:2000
dil.). A monoclonal mouse anti-Hsp70 (Stressgen Biotechnologies Corp., San Diego, CA;
18
SPA-820; 1:1000 dil.), raised against the human antigen, detects both the constitutive and
inducible forms
of
Hsp70.
Another monoclonal
mouse antibody (Stressgen
Biotechnologies Corp., San Diego, CA; SPA-810; 1:1000 dil.), also raised against the
human antigen, is known to react exclusively with the inducible Hsp70 in vertebrate
species. Antibodies raised against pea stromal Hsp70 (sHsp70) (1:2000 dil.) and pea
Hsp93 (ClpC) (1:2000 dil.) in rabbit are gifts from Dr. John Froehlich (Nielsen et al.,
1997) and found to crossreact with the Arabidopsis thaliana homologs. Anti-Hsp101
(1:2000 dil.) rabbit antiserum is raised against wheat Hsp101 but detects homologs from
a wide range of plant species (gift from Dr. Daniel Gallie; Young et al., 2001). Secondary
alkaline phosphatase-conjugated goat anti-rabbit (Southern Biotechnology Assoc., Inc.,
Birmingham, AL; 4010-04, 1:1000 dil.) and goat anti-mouse antibodies (Sigma, St. Louis,
MO; A3562, 1:30000 dil.) were used to develop immunoblots. All antibodies were
diluted in 5% non-fat dry milk in TBS (100 mM Tris-HCl pH 7.5 and 0.9% (w/v) NaCl).
One-dimensional Gels
Protein Extraction
To prepare soluble (supernatant) and insoluble (pellet) protein fractions, the excised
tobacco leaf disks (a set of 3 with total area 5.3 cm2) were ground in 300 μL of extraction
buffer [50 mM EPPS-NaOH (HEPPS) pH 8.0, 0.5 mM Na2EDTA•2H2O, 5 mM
MgCl2•6H2O, 5 mM dithiothreitol (DTT), 1% (w/v) polyvinylpolypyrrolidone (PVPP), 1
mM Na-DIECA, 2 mM benzamidine, 2 mM ε-aminocaproic acid, and 0.5 mM
phenylmethylsulfonic acid (PMSF)] (Whitney et al., 2001) in a glass homogenizer on ice
until the tissue was completely disintegrated. The leaf extract was then centrifuged at
15,800 x g for 5 min at 4°C and the supernatant was collected. The supernatant fraction
was diluted 3:1 with 4X SDS-PAGE sample buffer [8% (w/v) SDS, 40% (v/v) glycerol,
140 mM Tris-HCl, 0.4 mM EDTA free acid and 0.002% (w/v) bromophenol blue] then
kept at -20°C (Anonymous, 1994). The pellet was washed by resuspension in 1 mL
extraction buffer (minus PVPP). One third of the suspension was spun at 15,800 x g for 5
min at 4°C, while the remaining portion was stored at -20°C for future use. The
supernatant was then removed from the centrifuged sample and discarded. The resulting
19
pellet was washed twice with 0.5 mL extraction buffer (minus PVPP) and resuspended in
180 μL 1X SDS-PAGE sample buffer and then stored at -20°C.
SDS-PAGE and Western Blotting
To test for evidence of expression of heat shock proteins, protein samples were first
treated with 0.5 M DTT stock at 1:10 dilution then incubated for 10 min at 70°C.
Samples were centrifuged and supernatants were loaded onto a pre-cast 4-12%
polyacrylamide gradient SDS mini-gel (Invitrogen, Carlsbad, CA). The amount of protein
loaded from the supernatant sample was derived from leaf areas between 10.56 mm2 and
13.92 mm2. The protein loaded for the pellet samples was also derived from the same
area. Electrophoresis was performed with either MES running buffer (50 mM MES, 50
mM Tris base pH 7.3, 3.5 mM SDS and 1.0 mM EDTA free acid) or MOPS running
buffer (50 mM MOPS, 50 mM Tris Base pH 7.3, 3.5 mM SDS and 1.0 mM EDTA free
acid) (Invitrogen, Carlsbad, CA) at 170 V for approximately 1 hr at 4°C. When the gel
run was completed, proteins were transferred onto a nitrocellulose membrane (0.45 μm
pore size) with cold (4°C) transfer buffer [25 mM bicine, 25 mM bis-tris (free base), 1
mM EDTA free acid, 0.05 mM chlorobutanol pH 7.2, 10% (v/v) methanol and 0.01%
(v/v) NuPAGE antioxidant] (Invitrogen, Carlsbad, CA). The membrane was then blocked
with 5% non-fat dry milk in TBS (100 mM Tris-HCl pH 7.5 and 0.9% (w/v) NaCl) on a
rotary shaker at room temperature for 1 hr before incubation with an anti-Hsp primary
antibody as specified in each experiment. The following steps were also performed at
room temperature. After four rinses for 10 min each with TTBS [100 mM Tris-HCl pH
7.5, 0.9% (w/v) NaCl and 0.1% (v/v) Tween-20], the immunoblot was developed with an
appropriate alkaline phosphatase-conjugated secondary antibody as specified for 1 hr.
The immunoblot was then washed four more times for 10 min each with TTBS and then
rinsed once for 15 min with distilled water before incubation in alkaline phosphate
substrate solution (Roche, Indianapolis, IN). The colorimetric reaction was terminated by
rinsing in distilled water.
Analysis of Hsp70 Isoforms
20
Since at least two Hsp70 isoforms (constitutive and inducible) were known to be
expressed in higher plants, attempts were made to resolve the single, broad polypeptide
band observed for soluble supernatant of the heat-shocked positive control, transformed
and non-transformed control leaf extracts in one-dimensional (mini-gels) Western
analysis. Two approaches were used: (1) A monoclonal mouse antibody raised against a
Hsp70 isolated from human HeLa cells (Stressgen Biotechnologies Corp., San Diego, CA;
SPA-810, 1:1000 dil.) and known to detect exclusively with the inducible isoform in a
variety of vertebrates was employed to examine the induction level of Hsp70 in
transgenic tobacco plant. (2) SDS-PAGE was performed using a full-sized (14 cm x 14
cm), single-concentration polyacrylamide gel [resolving: 8% (v/v) 40% acrylamide/bisacrylamide mix, 0.1% (w/v) SDS, 0.1% (v/v) ammonium persulfate and 0.05% (v/v)
TEMED; stacking: 5% (v/v) 40% acylamide/bis-acrylamide mix, 0.1% (w/v) SDS, 0.1%
(v/v) ammonium persulfate and 0.01% (v/v) TEMED] run in tris-glycine electrophoresis
buffer [25 mM Tris, 250 mM glycine pH 8.3 and 0.1% (w/v) SDS] to distinguish the
constitutive and inducible Hsp70 isoforms (Sambrook et al., 1989). Prior to the gel run,
protein extracts were treated with 4X SDS-PAGE sample buffer (8% (w/v) SDS, 40%
(v/v) glycerol, 250 mM Tris-HCl, 0.0125% (w/v) bromophenol blue and 20% (v/v) βmercaptoethanol) before incubation at 100°C for 3 min (Anonymous, 1994). The amount
of protein loaded from the supernatant sample was derived from leaf areas between 21.65
mm2 and 28.87 mm2. The protein loaded for the pellet sample was also derived from the
same area. Proteins were subsequently transferred onto a nitrocellulose membrane (0.45
μm pore size) with cold (4°C) transfer buffer [12 mM tris base, 96 mM glycine pH 8.3
and 20% (v/v) methanol] (Invitrogen, Carlsbad, CA). Steps for membrane blocking and
immunoblotting were performed as described above.
Two-dimensional Gels
The following sample preparation, first-dimension or isoelectric focusing (IEF),
equilibration, second-dimension or SDS-PAGE and staining steps were performed using
the Bio-Rad (Hercules, CA) system, unless otherwise noted.
Protein Extraction
21
The soluble supernatant of the leaf extract for transformed plant was subjected to twodimensional PAGE to further resolve the constitutive and inducible forms of Hsp70. Six
tobacco leaf discs, weighing approximately 160 mg, were first ground to a fine powder in
liquid nitrogen using a mortar and pestle. The tissue powder and liquid nitrogen
suspension was then transferred into a N2-cooled microcentrifuge tube. After all the N2
had evaporated, 400 μL of lysis buffer was added into the sample on ice. Protein extracts
were then prepared using the ReadyPrep Protein Extraction kit (Soluble/Insoluble),
according to manufacturer’s directions. The soluble fraction was further isolated and
quantitatively precipitated using the ReadyPrep 2-D Cleanup kit, before resuspension in
250 μL of 2D rehydration/sample buffer [2 M urea, 50 mM DTT, 2% (w/v) 3-[(3cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 0.2% (w/v) 100X
Bio-Lyte® 3/10 ampholyte and 0.002% (w/v) bromophenol blue]. The concentration of
the soluble sample was then determined using the RC DC Protein Assay kit.
Isoelectric Focusing (IEF) – First Dimension
The supernatant fraction corresponding to 195 μg of soluble proteins was diluted in 2D
rehydration/sample buffer to a final volume of 200 μL prior to loading onto a 11 cm pH
4-7 immobilized pH gradient (IPG) strip. A sample for 2D SDS-PAGE standards (BioRad, Hercules, CA) with a pI range of 4.5 to 8.5 was also prepared and loaded onto a
strip. IPG strips loaded with protein samples were then overlayed with 2-3 mL of mineral
oil for rehydration overnight. IEF was performed on the next day using the PROTEAN
IEF cell, according to the manufacturer’s instructions. After the first dimension
separation was complete, IPG strips were incubated sequentially in equilibration buffer I
[6 M urea, 2% SDS, 375 mM Tris-HCl pH 8.8, 20% glycerol and 2% (w/v) DTT] and
equilibration buffer II [6 M urea, 2% SDS, 375 mM Tris-HCl pH 8.8, 20% glycerol, 2%
(w/v) DTT and 0.5 g iodoacetamide] before placing onto a 8-16% SDS-PAGE gel for
second dimension analysis.
SDS-PAGE – Second Dimension
Second dimension analysis was performed in tris-glycine running buffer (25 mM tris base,
192 mM glycine, 0.1% SDS pH 8.3). After electrophoresis was complete, the gel with 2D
22
SDS-PAGE standards was stained in Coomassie Brilliant Blue R-250 stain (0.25 g/100
mL 40% methanol: 10% acetic acid solution), followed by multiple destainings using the
5% methanol: 7% acetic acid solution. For the remaining gels, proteins were transferred
onto a nitrocellulose membrane (0.45 μm pore size) with cold (4°C) transfer buffer [25
mM Tris Base, 192 mM glycine pH 8.3 and 20% (v/v) methanol]. Steps for membrane
blocking and immunoblotting were then performed as described above.
Results
Transgenic N. tabacum plants are impaired in their growth and leaf appearance.
The transformed and non-transformed control tobacco plants were grown in the
greenhouse at various times as needed during the research project and their appearances
were recorded approximately 2 months after seed germination. All tobacco transformants
were shorter in height compared to the control plants (Figure 2). Also, the transformed
plants had smaller leaves than the non-transformed control counterpart (Figure 3). In
addition, the transgenic plant had a preponderance of yellowish-green leaves (instead of
the robust, green leaves present on the control plant), suggesting an overall reduction in
the chlorophyll content.
The induction of Hsps in transformed tobacco plants is selective across different families.
To investigate the expression of Hsps in tobacco plants due to the presence of misfolded
protein, leaf samples were harvested, extracted and separated by centrifugation into
supernatant and pellet fractions (Whitney et al., 2001). Protein samples were then
subjected to one-dimensional gel electrophoresis using either the MES or MOPS running
buffer (unless indicated otherwise) before immunodetection with primary anti-Hsps
antibodies. Selected classes of Hsps are induced in the transformed tobacco plants,
relative to the non-transformed controls. Figures 4-6 and 8-12 showed the immunoblots
of which each has been performed at least 3 times and are a summary of the results
accumulated from Summer 2003 through Spring 2004.
Multiple Hsp60 isoforms are induced by the presence of misfolded algal rubisco in the
transformed tobacco plants.
23
The transgenic tobacco plants produced higher levels of Hsp60 compared to the nontransformed control plants [Figure 4; GM (s), C (s)]. Immunoreactivity was not detected
in the insoluble pellet fractions [GM (p), C (p)]. The heat-shocked positive control plants
also expressed a roughly similar amount of Hsp60 compared to that of the transformed
plants; again, the reaction was observed primarily within the supernatant fraction [HS (s)].
Compared to the single band (65 kD) observed for the control plant supernatant sample,
both heat-shocked and transgenic plants exhibited multiple bands (60-65 kD), suggesting
the presence of more than one Hsp60 isoform [Figure 4; HS (s), GM (s), C (s)].
Hsp70 is abundantly expressed in transgenic plants, but the constitutive and inducible
forms remained elusive.
Hsp70 was abundantly expressed in the transgenic plant; nevertheless, its content in the
supernatant fraction was comparable to that in the supernatant fraction of the nontransformed control plants [Figure 5; GM (s), C (s)]. For both samples, the molecular
weights were determined to span from 70-72 kD. The single, broad bands shown in the
two lanes preclude the recognition and analysis of both constitutive and stress-inducible
Hsp70 isoforms detectable by the anti-Hsp70 antibody (Stressgen Biotechnologies Corp.,
San Diego, CA; SPA-820). Additionally, the antibody reacts with Hsp70 members from
different cellular compartments, ie. the cytosol, chloroplast, mitochondria and ER. Large
amounts of Hsp70 (70-72 kD) were also recovered in the supernatant fraction of the heatshocked plant [HS (s)], which appeared slightly more than that detected for the
transformed plant [GM (s)]. No immunoreactivity was observed in the pellet fractions of
any plant samples [Figure 5; HS (p), GM (p), C (p)].
Stroma-localized Hsp70 is present at high levels in both the transformed and control
tobacco plants.
As a further step in analyzing the expression of Hsp70, plant samples were probed with
an antibody that reacts specifically with a stroma-localized Hsp70 (sHsp70) (gift from Dr.
John Froehlich). As shown in Figure 6 [GM (s)], a stroma-localized 78 kD Hsp70
homologue was present at high levels in the supernatant fraction of the transformed
plants. The soluble sHsp70 content of the transformed plant appeared similar as the basal
24
level expression exhibited by the control supernatant sample [GM (s), C (s)]. This
expression of sHsp70 did not seem to change even after the tobacco plant was exposed to
heat stress, as the band intensities for the supernatant samples of all three plants were
strong and comparable [HS (s), GM (s), C (s)]. In contrast, the reaction was faint for the
pellet fraction of heat-shocked and control plants, but appeared more intense for the
transgenic plant (Figure 6; HS (p), GM (p), C (p)]).
N. tabacum proteins do not crossreact with an antibody that detects exclusively the
inducible Hsp70.
The previous attempt at investigating the induction levels of Hsp70 in response to
misfolded rubisco failed (Figure 5). The constitutive and stress-inducible isoforms of
plant Hsp70 that are known to differ by 2 kD could not be resolved on a 4-12%
polyacrylamide SDS-gradient mini-gel. Moreover, Hsc70 has a strong basal level
expression. This prompted a search for a plant antibody that can detect exclusively the
inducible Hsp70 isoform in order to determine the differences (if any) in the expression
levels of Hsp70 between the transgenic and non-transformed control plant. An extensive
literature search failed to uncover any antibodies designed to react solely against the plant
inducible Hsp70. An antibody that was developed against a human antigen and known to
react exclusively with the inducible Hsp70 among vertebrate species was identified
(Stressgen Biotechnologies Corp., San Diego, CA; SPA-810). Sequence alignment was
subsequently performed between the heat-inducible Hsp70 members from human
(SWISSPROT: HS71_HUMAN) and tobacco (GBPLN: 30025965_30025966) to predict
the conservation of amino acid residues corresponding to the epitope region (436-503 aa.)
of the antibody of interest (Figure 7). The epitope region was found to be highly
conserved, suggesting that the antibody is likely to crossreact with the plant antigen.
However, contrary to prediction, immunoblots probed with the antibody produced no
reaction in either the supernatant and pellet fractions of any plants (data not shown).
The Hsp70 isoforms remain inseparable in a high-resolution, single-concentration
polyacrylamide gel.
The previous steps taken to examine the expression levels of inducible Hsp70 in the
transgenic tobacco plant fell short of achieving the objective. The next course of action
25
was to separate the proteins using a large-format, single concentration (8%)
polyacrylamide gel run in the tris-glycine electrophoresis buffer. The use of this
discontinuous buffer system that consists of two different gel layers (stacking and
resolving) is practical because it concentrates the samples into a very small volume and
greatly improves the resolution of protein bands that are close together (Sambrook et al.,
1989). Figure 8 presents the results of protein separation using this discontinuous buffer
system. As shown in Figure 5, the transgenic plant supernatant sample had a band (70-72
kD) that is similar in intensity to that for the control plant sample, confirming that soluble
Hsp70 is expressed in great amounts in both plants [GM (s), C (s)]. The single band in
both samples also appeared broad and unresolved. Compared to the transgenic and nontransformed plant, the heat-shocked positive control plant expressed slightly more
Hsp70s, as indicated by the strong, unresolved band in the supernatant fraction [HS (s)].
The pellet fraction of the heat-shocked plant showed very little reaction with the antiHsp70 antibody, while no bands were observed in the transformed and non-transformed
control pellet samples. In sum, the findings of the single-concentration (8%) gel appeared
to confirm the immunoblot in Figure 5, but did not resolve the induction levels of Hsp70
(Figure 8).
The expression of ClpC (Hsp93) is induced in the transgenic plants but not upregulated
in the heat-shocked plants.
Transgenic tobacco plants expressed greater amounts of soluble ClpC (Hsp93) compared
to the non-transformed control and heat-shocked plants [Figure 9; HS (s), GM (s), C (s)].
This immunoprecipitation reaction appeared as a broad band for the transformed plant
supernatant sample, while the reaction for similar fractions of the non-transformed
control and heat-shocked plants was limited to a single, narrow band. ClpC (Hsp93) was
also present in roughly similar amounts across the pellet samples of all three plants [HS
(p), GM (p), C (p)]. However, the overall band intensities corresponding to the pellet
fractions were weaker than those for the supernatant samples. All bands in the
supernatant and pellet fractions were found to migrate to the same position on gel, which
was determined to be approximately 93-94 kD (Figure 9).
26
Hsp101 is not induced in the transformed plants.
Hsp101 was not present at a noticeable level in either the transformed or control tobacco
plants [Figure 10; GM (s), C (s), GM (p), C (p)]. However, strong bands were observed
to migrate at a position slightly above 100 kD in both of the heat-shocked positive control
samples [HS (s), HS (p)], indicating that Hsp101 is heat-responsive.
Small Hsps are not detected in the transgenic plants.
The transgenic plants did not produce a reaction in the supernatant and pellet fractions
when probed with antibodies targeted against the small Hsps [Figures 11 and 12; GM (s),
GM (p) for both figures]. This absence of signal was also observed for the nontransformed control plant [Figures 11 and 12; C (s), C (p) for both figures]. In contrast,
the supernatant and pellet samples of heat-shocked plant showed strong bands when
probed with smHsp-α, an antibody that is designed to react with the highly conserved αhelical region of most small Hsps, regardless of cellular localization [Figure 11; HS (s),
HS (p)]. The molecular weights determined for the supernatant and pellet fraction of the
heat-shocked plant were 18-23 kD and 20-22 kD, respectively. Interestingly, the strong
reaction exhibited by the heat-shocked plant samples were not observed when the
immunoblot was probed with smHsp-met, an antibody that detects specifically the
chloroplast-localized small Hsps [Figure 12; HS (s), HS (p)].
The presence of Hsp70 isoforms in the transgenic tobacco plants is detectable through
two-dimensional gel analysis.
Previous attempts to investigate the induction levels of Hsp70 using one-dimensional
gradient and non-gradient gel analysis, as well as an antibody that reacts exclusively with
the inducible human Hsp70, did not yield definitive results. The inconclusive findings
prompted the study of proteins using a two-dimensional gel analysis, where constitutive
and stress-inducible Hsp70s could potentially be resolved sufficiently by their differences
in protein pIs and molecular weight. The soluble fraction of protein samples was isolated
from the extraction procedure (Bio-Rad, Hercules, CA) for two-dimensional analysis
because Hsp70 was previously recovered almost exclusively in the supernatant fraction.
Immunodetection with the anti-Hsp70 antibody that reacts with both the constitutive and
27
induced isoforms was employed. Figures 13-16 summarize the work performed from Fall
2004 through Spring 2005.
Hsp70 isoforms in the soluble heat-shocked and transgenic plant samples migrate at a pI
range of 5.0-5.3.
Figure 13 shows the Coomassie stain of a two-dimensional electrophoretic protein pattern
of 2-D SDS-PAGE standards on a 8-16% SDS-polyacrylamide gel. The protein spots
detected enabled the determination of pI and molecular weight of a protein of interest.
Figures 14-16 presents the immunoblots of soluble transformed, heat-shocked, and nontransformed control samples subjected to two-dimensional analysis. The transformed
tobacco plants expressed detectable mixtures of 70 and 72 kD of Hsp70 isoforms, evident
by the faint, “smudge”-like bands that extended from a pI of 5.0 to 5.3 (Figure 14). No
other bands were observed at pIs outside of this range. The soluble heat-shocked plant
sample exhibited a similar reaction within the same pI range, but with a stronger signal
(Figure 15). Compared to Figure 14, the signal of the heat-shocked sample appeared
clearer as horizontal, double “streak”-like bands, which migrated at the position between
70 kD and 100 kD. For both samples (Figures 14 and 15), bands appeared darker toward
the left end of the pI range 5.0-5.3.
The soluble non-transformed control plant samples exhibit an absence of reaction.
Contrary to the prediction from one-dimensional analysis (Figures 5 and 8), the soluble
control plant sample did not produce a reaction in its two-dimensional immunoblot. This
absence of reaction precluded analysis and comparison of the basal level expression of
Hsp70 with its putative induction in the transformed plant.
Discussion
Overexpression of misfolded P. tricornutum rubisco stresses the transgenic tobacco
plants.
Both the transformed and non-transformed N. tabacum plants were raised under the same
light and temperature conditions, but the former exhibited a reduction in height, leaf size
28
and chlorophyll content. This observation gave rise to the speculation that the transgenic
plants were experiencing an unusual stress. Whitney et al. (2001) concluded that the
transformed plants were stressed by the overexpression of misfolded P. tricornutum
rubisco subunits, which caused a decrease in soluble leaf protein and endogenous rubisco
content per unit leaf area. Denatured foreign rubisco is not active and hence, the overall
growth of the transgenic plant was affected. The lower chlorophyll content of the
transformed plants might be due to a reduction in photosynthesis, which limits the rate of
plastid protein synthesis and thus the formation of chlorophyll complexes (Whitney et al.,
2001).
The induction of certain classes of Hsps in transgenic tobacco plants provides support for
the hypothesis of the experiment
The presence of denatured P. tricornutum rubisco in the transgenic tobacco plants was
conclusive. All foreign LSU and a vast majority of foreign SSU were found in the pellet
fractions of leaf extracts, indicating that they are not properly folded and thus present as
insoluble aggregates (Whitney et al., 2001; corroborated by Claire Marie Filone). Protein
samples were prepared from the transformed and control leaf tissue to test for the
evidence of the expression of Hsps in response to the in vivo presence of misfolded
rubisco subunits in the chloroplast stroma. If the misfolded foreign rubisco acts as a
signal to elicit the heat shock response, then my hypothesis states that the transformed
tobacco plants should exhibit an increase of some or all Hsps compared to the nontransformed control plants. Collectively, the results provided support for my hypothesis.
The presence of misfolded proteins in transgenic tobacco plants induces the production of
Hsps, but this response appears specific and is restricted to only certain classes of Hsps.
Hsp60 is upregulated but does not appear to be associated with the misfolded algal
rubisco.
As shown in Figure 4, the transformed tobacco plants produced greater amounts of
soluble Hsp60 than the non-transformed control plants. Hsp60 is expressed constitutively
as a molecular chaperone but is induced by misfolded algal rubisco subunits, which are
only present in the transformed tobacco plants. Therefore, the results provided support to
29
my hypothesis. In addition, the multiple bands observed to migrate at 60-65 kD in the
transgenic plant supernatant sample suggested the presence of more than one Hsp60
isoform, which is logical. Plant cells contain Hsp60 isoforms in the chloroplasts and
mitochondria (Boston et al., 1996; Vierling, 1991). The anti-Hsp60 antibody employed
crossreacts with different plant cpn60 isoforms. Thus, the detection of multiple bands
indicated that the upregulation of Hsp60 in the transgenic tobacco plant may involve an
increase in cpn60 from more than one subcellular compartment. This is consistent with
the speculation of Whitney et al. (2001) that the presence of denatured foreign rubisco in
the chloroplast stroma might have caused stress to the surrounding cellular environment.
The lack of reaction in the pellet fraction of the transgenic plant sample
suggested that the Hsp60, though induced, was not associated with the foreign rubisco
subunits, which were almost exclusively found in the insoluble pellet fraction (Whitney
et al., 2001). Logically, if Hsp60 is engaged with the misfolded algal rubisco, then both
proteins should appear in the same fraction during SDS-PAGE analysis. It is possible to
speculate on other explanations for this observation. One possible scenario is epitope
masking, where the Hsp60-foreign rubisco subunits complex was present in the pellet
fraction but remained resistant to SDS treatment. While in a bound complex, Hsp60 did
not produce a signal with the antibody because its epitope was hidden. Therefore, the
only reaction that occurred was with the soluble, unattached Hsp60. However, the
proposed interaction seems extremely unlikely, since SDS treatment did dissociate the
aggregates of algal rubisco LSUs and SSUs to produce the predicted bands on a Western
analysis (Whitney et al., 2001). Moreover, I have no reason to expect that the Hsp60foreign rubisco subunits complex, if it exists, would be less-sensitive to SDS-treatment
than the aggregates of foreign rubisco subunits. Thus, the current findings argued in favor
of the complete absence of Hsp60 in the insoluble, pellet fraction, suggesting that
misfolded foreign rubisco was totally rejected from the tobacco chaperonin system.
The presence of multiple bands in the heat-shocked supernatant sample was
intriguing. Previous experiments conducted by Prasad and Halleberg (1990) and Hartman
et al. (1992) showed that maize mitochondrial cpn60 and barley chloroplast cpn60 were
responsive to heat-stress. However, multiple bands were not observed in their
mitochondrial or chloroplastic fractions analyzed on the SDS-PAGE. The results of the
30
present experiment therefore suggested that the heat-shocked tobacco plant induced a
mixture of cpn60 isoforms. Proteolysis appeared unlikely in this case since a single band
was observed in the control plant sample.
Significance of the study of Hsp70 induction.
The transgenic tobacco plants expressed a great amount of Hsp70 (Figures 5 and 8).
However, I could not conclude if induction took place in the transformed line because the
control plants also produced a strong, broad band on the immunoblot. For both
immunoblots, the antibody employed was developed to react with both the mammalian
Hsc70 and Hsp70 (Stressgen Biotechnologies Corp., San Diego, CA; SPA-820). In
higher plants, at least two Hsp70 isoforms (constitutive and inducible) are known to be
expressed; they differ by 2 kD in molecular weight (DeRocher and Vierling, 1995; Wang
and Lin, 1993). Also, members of the plant Hsp70 family reside in different cellular
compartments, including the cytosol, mitochondria, chloroplast and ER (Vierling, 1991).
Based on the expression pattern of Hsp70, it was predicted that if the anti-Hsp70
antibody crossreacted with the tobacco proteins, the one-dimensional immunoblot would
yield two close-by but distinguishable bands using adequate gel resolution. In a twodimensional analysis, cytoplasmic Hsp70s are predicted to migrate at a pI range of 5.45.7 (DeRocher and Vierling, 1995).
The experiment is significant because the induction levels of Hsp70 homologues
(if any) would be indicative of the degree of “stress” experienced by the transgenic
tobacco plant cell, since Hsp70 is the negative regulator of the activity of HSF (Schöffl et
al., 1998). Moreover, Whitney et al. (2001) suggested that the stress imposed by the
misfolded foreign rubisco subunits in the chloroplast may have been “delocalized”
(Whitney et al., 2001). This “delocalized” stress may potentially lead to the upregulation
of Hsp70 isoforms in other cellular compartment, which in turns affects the activity of
HSF and the production of other Hsps, thereby providing additional support to my
hypothesis.
One-dimensional Western analysis fails to provide conclusive findings on the putative
induction of Hsp70.
31
Figure 5 shows the results of protein electrophoresis using the MOPS running buffer.
Gradient mini-gels are usually of limited resolution, which explained the appearance of a
single, broad polypeptide band in the transgenic supernatant sample. The band also failed
to resolve in the non-transformed control and heat-shocked supernatant samples,
precluding further Hsp70 analysis. The strong signal exhibited by the mammalian Hsp70
specific antibody with tobacco leaf tissues, however, raised hope that an antibody that
was designed to react solely with the inducible Hsp70 in vertebrate species could produce
a similar crossreaction (Stressgen Biotechnologies Corp., San Diego, CA; SPA-810). The
latter antibody would then lead to insights on the putative induction of Hsp70 by
misfolded rubisco in the transgenic tobacco plants since the constitutive forms would
remain undetected and thus would not interfere with the inducible signals. Furthermore,
the development of an antibody directed against a plant heat-inducible Hsp70 has yet to
be successful (Dr. Charles Guy, personal communication). Nevertheless, despite the high
degree of conservation shown at the epitope region (Figure 7), immunoblots of tobacco
leaf tissue did not yield a reaction with the antibody of interest. Increasing protein load
during SDS-PAGE did not appear to rectify the problem. Protein separation was next
performed on a large format gel that favors greater resolution. However, when the
immunoblot
was
probed
with
the
original
anti-Hsp70
antibody
(Stressgen
Biotechnologies Corp., San Diego, CA; SPA-820), the bands still remained inseparable
(Figure 8). Thus, the use of an alternative antibody or a high-resolution gel failed to result
in a separation of Hsp70 isoforms in any samples.
The one-dimensional immunoblots probed with anti-Hsp70 were comparable to
the Hsp60 results with respect to a possible interaction with the misfolded rubisco
subunits, which are present as insoluble pellet (Whitney et al., 2001; Figures 5 and 8).
Similar to Hsp60, Hsp70 was only detectable in the transgenic supernatant and not the
pellet fraction (Figures 5 and 8). Again, Hsp70 may or may not have associated with the
algal rubisco subunits and epitope-masking remained a possibility, though unlikely, in
explaining the presence of Hsp70 and algal rubisco subunits in separate fractions.
Two-dimensional Western analysis detects the presence of Hsp70 isoforms in the
transgenic plants but does not resolve its induction level relative to the controls.
32
A two-dimensional analysis of protein was suggested as a remedy to resolve the
induction level of Hsp70 in the transgenic tobacco plants. Soluble samples of tobacco
plants were subjected to two-dimensional separation based on protein pI and molecular
weight (Bio-Rad, Hercules, CA). The transformed tobacco plants appeared to express a
mixture of Hsp70 isoforms that migrated at a pI range of 5.0-5.3 (Figure 14),
approximately 0.3 pH units more acidic than that observed by DeRocher and Vierling
(1995) with pea leaves. DeRocher and Vierling (1995) employed the pea HSP71.2
antiserum, an antibody that was predicted to react with many Hsp70 homologues, but
most strongly with cytoplasmic Hsp70s. As such, our findings appeared to suggest that
the anti-Hsp70 antibody had reacted with the chloroplast Hsp70s, which were known to
migrate at a more acidic position than the cytoplasmic counterparts. Additional
experiments would have to be performed to verify this discrepancy, which could be due
to species differences or a technical matter, since the anti-Hsp70 antibody employed was
designed to react with both Hsc70 and Hsp70, regardless of cellular compartment.
In addition, the separation of inducible and constitutive Hsp70 homologues in the
soluble transgenic sample was unclear as the bands were faint and appeared as a
“smudge”, rather than “streak-like”. Nevertheless, the signal appeared suggestive of the
presence of inducible Hsp70 isoforms, which were migrating at 72 kD (DeRocher and
Vierling, 1995). The analysis of results was further complicated by the lack of reaction
shown by the immunoblot of the soluble control plant sample (Figure 16). This absence
of signal was unexpected, since Hsp70 is expressed abundantly in unstressed plants
(Boston et al., 1996; Vierling, 1991; Figure 5 and 8).
However, we could derive insights from the immunoblot of the heat-shocked
soluble sample, which might or might not resemble that of the transformed plant samples.
The stronger reaction observed in Figure 15 indicated that distinct Hsp70 homologues
were present at a greater abundance in the soluble heat-shocked plant sample compared
to that observed in the soluble transgenic sample. Also, the double “streak-like” signal
pattern was also seen in the two dimensional immunoblot results of DeRocher and
Vierling (1995). The “top” streak represents the heat-induced 72 kD polypeptides, while
the “bottom” streak corresponds to the constitutively-expressed 70 kD polypeptides
(DeRocher and Vierling, 1995). However, in the soluble heat-shocked plant sample, the
33
signal on two-dimensional immunoblot was observed to span from 70-100 kD, in contrast
to the 70-72 kD detected in one-dimensional Western analysis (Figures 5, 8, 15).
Ostensibly, the bands migrated higher than expected. Hence, additional experiments
would have to be performed to investigate this observation.
To improve the detection of Hsp70 by 2-D gels, future steps should involve the
the addition of ampholytes into protein samples prior to loading, as well as the loading of
increased amounts of proteins for IEF analysis (Bio-Rad, Hercules, CA). During the IEF
step, ampholytes assist the “focusing” of a protein to its pI value on the immobilized pH
gradient strip, enabling it to remain fixed in position and not drift towards either electrode.
Thus, sharper bands may result when the immunoblot is probed with the antibody. Also,
increased loading of soluble proteins may enhance detection with the antibody.
The preliminary findings showed that Hsp70 isoforms in the transgenic tobacco
plant are detectable through 2-D gel analysis. However, we failed to provide conclusive
evidence on the induction levels of Hsp70 in the transgenic tobacco plants.
Stroma-localized Hsp70 and Hsp93 are expressed in the transgenic tobacco plants.
The hypothesis of the experiment was further examined by investigating the expression
of sHsp70 (S78 in peas) and Hsp93, two major stroma molecular chaperones (Constan et
al., 2004; Nielsen et al., 1997; Zhang and Glaser, 2002). While Hsp93 was suggested by
the previous research findings as a bona fide component of the chloroplast protein
translocation complex, the role of sHsp70 in the import process remained ambiguous
(Nielsen et al., 1997). Figure 6 shows that the transgenic tobacco plant produced high
levels of soluble stroma-localized Hsp70 (sHsp70), which appeared similar in amount to
that detected in the control plant supernatant sample. However, sHsp70 was also present
as a weak band in the pellet sample of the transgenic plants. This immunoprecipitation
reaction was not observed in the control pellet sample. It is possible that the detectable
amount of sHsp70 that was present in the transgenic pellet sample could simply be
shifted from its soluble supernatant portion. On the other hand, the results also suggested
that the sHsp70 may be associated with denatured, insoluble rubisco subunits prior to
SDS treatment. However, the interaction was minimal, since sHsp70 was present at a
much higher level in the soluble supernatant sample of the transgenic plant. The levels of
34
soluble sHsp70 did not appear to change after heat-stress, consistent with the findings by
Marshall et al. (1990), who performed an SDS-PAGE analysis using proteins from
isolated pea chloroplasts. Ostensibly, the results suggested that sHsp70 is not heatinducible.
Figure 9 shows the induction of Hsp93 in the transgenic plant supernatant sample
relative to its control. This induction phenomenon was not observed in the heat-shocked
plants compared to the control. Clarke (1996), on the other hand, reported that Hsp93 is
only slightly induced during heat stress among higher plants and cyanobacteria. The
upregulation of Hsp93 in the transgenic plant, however, appeared to suggest that this
stroma-localized Hsp100 homolog was induced by the presence of misfolded algal
rubisco, thus verifying my hypothesis. The single, broad band observed for the transgenic
supernatant sample further suggested the possible presence of multiple isoforms, as
observed for Hsp60. This prediction could be verified by improving the gel resolution.
Hsp101 is not induced by misfolded algal rubisco.
Figure 10 shows the lack of expression of Hsp101 in the transgenic tobacco plant.
Hsp101 is also not expressed constitutively. However, the intense band observed for the
heat-shocked positive control samples suggested that Hsp101 is heat-responsive. This
finding was consistent with the research of Queitsch (2000) and Young et al. (2001), who
postulated that Hsp101 confers an important thermotolerance effect and is highly induced
at elevated temperatures for both maize and Arabidopsis. Hsp101 functions to dissociate
thermally-denatured protein aggregates and hence is dispensable at normal physiological
conditions (Glover and Tkach, 2001; Wang et al., 2004). In the transgenic tobacco plants,
expression of Hsp101 was not detected because the denatured foreign rubisco may be
misfolded but did not form large-sized insoluble aggregates, as demonstrated by the
analysis of immunogold labeling (Whitney et al., 2001). However, if Whitney et al.
(2001) were mistaken in concluding about the non-native nature of misfolded rubisco
subunits and that aggregation did occur, then the epitope-masking explanation would
apply to describe the absence of reaction in the transgenic tobacco plant sample.
Small Hsps are not detected in the transgenic tobacco plants.
35
Transgenic tobacco plants also showed a lack of expression of small Hsps (Figures 11
and 12). Like Hsp101, small Hsps were not present in the control samples. The small
Hsps were strongly induced by elevated temperature, evident from the intense bands
observed in the supernatant and pellet samples of the heat-shocked positive control plants
probed with smHsp-α (Figure 11). However, this reaction was not reproduced in the
immunoblot probed with smHsp-met. No immunodetection was observed even when the
experiment was performed again using a different sample of antibody, suggesting that the
lack of reaction was not due to technical problems. Heckathorn et al. (1998), on the other
hand, detected the presence of chloroplast small Hsps when using smHsp-met antibody
on a heat-stressed tomato plant. One possible reason that could account for the absence of
immunoreaction was the heat-shock treatment employed, in which the tomato plant was
exposed to gradual temperature increase (25 °C for two hour, 42 °C for six hours,
followed by 25 °C for two hours again), while the tobacco plants used in the present
experiment were incubated for 4 hours at a leaf temperature of 44-45 °C. Perhaps the
gradual temperature change induces the production of chloroplast-localized small Hsps.
Another possible explanation was that heat-shocked tobacco plants did not show
an increase in the chloroplast-localized small Hsps. This scenario appeared unlikely,
because chloroplast smHsps are the most heat-responsive of all Hsps (Heckathorn et al.,
1998). Therefore, I attributed the lack of reaction to the failure of smHsp-met, which was
raised against a synthetic oligopeptide antigen, to crossreact with the tobacco plant
proteins.
Nevertheless, since the smHsp-α antibody, which is designed to react with any
small Hsps, recognizes tobacco proteins, the lack of signal in the transformed plant
samples indicated that no small Hsps are induced by the presence of misfolded rubisco.
Subsequent findings of the transgenic plants with the smHsp-met antibody did not
provide additional information, although it is still puzzling that the antibody did not
produce a reaction with any of the tobacco plant samples.
Conclusion
The presence of misfolded rubisco in the transgenic Nicotiana tabacum plant induces the
expression of Hsps. However, the response is limited selected classes of Hsps, including
36
Hsp60 and Hsp93. These results provide support for my hypothesis that misfolded
proteins could act as a cellular signal for the induction of heat shock response.
37
polyribosome
mRNA
Translated polypeptide (pre-SSU)
Hsc70
Nucleus
Hsc70
Translocating pre-SSU
P
14-3-3
Com70
Chloroplast
34
Outer envelope
Intermembrane space
64
Toc
160
75
IAP70
110
22
Inner envelope
Stroma
pre-SSU transit peptide
is cleaved
SPP
20
?
55
sHsp70
(S78) CGE
CDJ
40
Tic
ClpC
(Hsp93)
Mature SSU
Figure 1. Current model of the chloroplast protein translocation complex (adapted from
Keegstra and Froehlich, 1999; Zhang and Glaser, 2002). Higher plant rubisco SSU is
encoded by the nuclear genome and translated as pre-SSU on the cytoplasmic
polyribosomes before import into the chloroplast stroma. Newly-synthesized pre-SSU is
phosphorylated in the cytosol and interacts with Hsc70 and the 14-3-3 protein complex,
which guide the precursor protein toward the outer envelope of chloroplast. The
precursor protein begins its entry through the “channel” formed by Toc (translocon of the
outer chloroplast envelope) complex, IAP70 and Tic (translocon of the inner chloroplast
envelope) before reaching the stroma compartment. The Hsp70 members involved in the
translocation process are Hsc70 (cytosol), Com70 (outer envelope), IAP70
(intermembrane space) and sHsp70 (S78, as it is known in peas) (stroma). Hsc70 guide
the folding of nascent pre-SSU upon release from the polyribosomes. Com70 is believed
to be involved in the unfolding of pre-SSU before the precursor protein begins its entry
into the chloroplast. IAP70 and sHsp70 (S78) are predicted to provide the driving force
for the precursor protein to move across the outer and inner chloroplast envelopes. The
final step in the import process of precursor protein into the chloroplast stroma is also
believed to be facilitated by ClpC (Hsp93), which acts in an ATP-dependent manner.
SPP, stroma processing peptidase, is responsible for cleaving the transit peptide from preSSU to yield the mature polypeptide.
38
Figure 2. Transformed (right) and
non-transformed control (left)
Nicotiana tabacum L. Petit
Havana plants, at age 2 months,
raised under similar light and
temperature conditions in the
greenhouse at the Fackenthal
Building, Franklin and Marshall
College. The differences in plant
height, leaf color and width were
noted.
Figure 3. Leaf samples belonging to
the non-transformed control N.
tabacum (left) and its transgenic
counterpart (right). The differences
in leaf color and width were noted.
39
HS (s) GM (s) C (s) HS (p) GM (p) C (p)
Figure 4. Immunoblot of heat-shocked, transgenic and non-transformed control leaf
samples probed with antibody to Hsp60. Supernatant samples are loaded as follows: heatshocked positive control, HS (s), transformed, GM (s) and non-transformed control, C (s)
respectively. Pellet samples are loaded as follows: heat-shocked positive control, HS (p),
transformed, GM (p) and non-transformed control, C (p) respectively. A total of 3
replicates were performed for the immunodetection with anti-Hsp60 antibody. Leaf
proteins were extracted into supernatant and pellet fractions as described by Whitney et
al. (2001). The samples were run on a pre-cast 4-12% polycrylamide gradient SDS gel
using MES running buffer (Invitrogen, Carlsbad, CA). Each lane was loaded with
supernatant or pellet derived from 10.56 mm2 of leaf.
HS (s) GM (s)
C (s)
HS (p) GM (p) C (p)
Figure 5. Immunoblot of heat-shocked, transgenic and non-transformed control leaf
samples probed with antibody to Hsp70. Supernatant samples are loaded as follows: heatshocked positive control, HS (s), transformed, GM (s) and non-transformed control, C (s)
respectively. Pellet samples are loaded as follows: heat-shocked positive control, HS (p),
transformed, GM (p) and non-transformed control, C (p) respectively. A total of 3
replicates were performed for the immunodetection with anti-Hsp70 antibody. Leaf
samples were prepared, loaded and electrophoresed as specified in Figure 4.
40
HS (s) GM (s) C (s)
HS (p) GM (p) C (p)
Figure 6. Immunoblot of a heat-shocked, transgenic and non-transformed control leaf
samples probed with antibody to stroma-localized Hsp70. Supernatant samples are loaded
as follows: heat-shocked positive control, HS (s), transformed, GM (s) and nontransformed control, C (s) respectively. Pellet samples are loaded as follows: heatshocked positive control, HS (p), transformed, GM (p) and non-transformed control, C (p)
respectively. A total of 6 replicates were performed for the immunodetection with antisHsp70 antibody. Leaf samples were prepared, loaded and electrophoresed as specified in
Figure 4.
Figure 7. A comparison of amino acid sequences between the heat-inducible Hsp70
proteins of human (SWISSPROT: HS71_HUMAN) and tobacco (GBPLN:
30025965_30025966). The epitope region for the anti-Hsp70 antibody of interest,
spanning 436-503 aa., is underlined. For the dual sequence alignment, the symbols and
meanings are as followed: * (fully-conserved residues) and : (highly-conserved residues).
Blank indicates there is no conservation between residues.
41
HS (s)
GM (s)
C (s)
HS (p)
GM (p)
C (p)
Figure 8. Immunoblot of a heat-shocked, transgenic and non-transformed control leaf
samples assayed on the large-format, 8% non-gradient gel and probed with antibody to
Hsp70. Supernatant samples are loaded as follows: heat-shocked positive control, HS (s),
transformed, GM (s) and non-transformed control, C (s) respectively. Pellet samples are
loaded as follows: heat-shocked positive control, HS (p), transformed, GM (p) and nontransformed control, C (p) respectively. A total of 3 replicates were performed for the
immunodetection with anti-Hsp70 antibody. Leaf proteins were extracted into
supernatant and pellet fractions as described by Whitney et al. (2001). The samples were
run on a single concentration (8%) polyacrylamide SDS gel using tris-glycine running
buffer (Sambrook et al., 1989). Each lane was loaded with supernatant or pellet derived
from 21.65 mm2 of leaf.
HS (s)
GM (s)
C (s)
HS (p)
GM (p) C (p)
Figure 9. Immunoblot of heat-shocked, transgenic and non-transformed control leaf
samples probed with antibody to Hsp93 (ClpC). Supernatant samples are loaded as
follows: heat-shocked positive control, HS (s), transformed, GM (s) and non-transformed
control, C (s) respectively. Pellet samples are loaded as follows: heat-shocked positive
control, HS (p), transformed, GM (p) and non-transformed control, C (p) respectively. A
total of 4 replicates were performed for the immunodetection with anti-Hsp93 antibody.
Leaf samples were prepared, loaded and electrophoresed as specified in Figure 4.
42
HS (s)
GM (s) C (s)
HS (p) GM (p)
C (p)
Figure 10. Immunoblot of heat-shocked, transgenic and non-transformed control leaf
samples probed with antibody to Hsp101. Supernatant samples are loaded as followed:
heat-shocked positive control, HS (s), transformed, GM (s) and non-transformed control,
C (s) respectively. Pellet samples are loaded as followed: heat-shocked positive control,
HS (p), transformed, GM (p) and non-transformed control, C (p) respectively. A total of 5
replicates were performed for the immunodetection with anti-Hsp101 antibody. Leaf
samples were prepared, loaded and electrophoresed as specified in Figure 4.
HS (s)
GM (s)
C (s)
HS (p) GM (p)
C (p)
Figure 11. Immunoblot of heat-shocked, transgenic and non-transformed control leaf
samples probed with antibody to smHsp-α. Supernatant samples are loaded as follows:
heat-shocked positive control, HS (s), transformed, GM (s) and non-transformed control,
C (s) respectively. Pellet samples are loaded as follows: heat-shocked positive control,
HS (p), transformed, GM (p) and non-transformed control, C (p) respectively. A total of 5
replicates were performed for the immunodetection with anti-smHsp-α antibody. Leaf
samples were prepared, loaded and electrophoresed as specified in Figure 4.
43
HS (s)
GM (s)
C (s)
HS (p)
GM (p)
C (p)
Figure 12. Immunoblot of heat-shocked, transgenic and non-transformed control leaf
samples probed with antibody to smHsp-met. Supernatant samples are loaded as follows:
heat-shocked positive control, HS (s), transformed, GM (s) and non-transformed control,
C (s) respectively. Pellet samples are loaded as follows: heat-shocked positive control,
HS (p), transformed, GM (p) and non-transformed control, C (p) respectively. A total of 5
replicates were performed for the immunodetection with anti-smHsp-met antibody. Leaf
samples were prepared, electrophoresed and loaded as specified in Figure 4.
mwt.
(kD) 4.0
Protein pI
7.0
98
64
6.4
5.4
50
5.0
36
22
5.9
4.5
Figure 13. Coomassie stain of a 8-16% SDS-polyacrylamide gel showing the twodimensional electrophoretic protein pattern of 2-D SDS-PAGE standards separated in the
PROTEAN IEF cell. A total of 4.0 μL standards was applied and protein separation was
performed according to the manufacturer’s instructions (Bio-Rad, Hercules, CA). The
protein spots detected from left to right are as followed: soybean trypsin inhibitor, pI 4.5,
21.5 kD; bovine muscle actin, pI 5.0, 43.0 kD; bovine serum albumen (BSA), pI 5.4, 66.2
kD; bovine carbonic anhydrase, pI 5.9, 31.0 kD and hen egg white conalbumin type 1, pI
6.4, 76.0 kD.
44
mwt.
(kD)
5.0
Protein pI
5.4
98
64
50
Figure 14. Immunoblot of a transformed leaf sample assayed using the two-dimensional
gel and probed with antibody to Hsp70. Leaf proteins were extracted into the soluble and
insoluble fractions according to manufacturer’s instructions (Bio-Rad, Hercules, CA).
Soluble sample that corresponded to 195 μg proteins was loaded onto a pH 4-7 IPG strip,
rehydrated overnight and subjected to IEF in a first-dimension analysis. The IPG strip
was then placed onto a 8-16% SDS-PAGE gel for second-dimension separation of
proteins in tris-glycine running buffer.
mwt.
(kD)
5.0
Protein pI
5.4
98
64
50
Figure 15. Immunoblot of a heat-shocked leaf sample assayed using the two-dimensional
gel and probed with antibody to Hsp70. Leaf proteins were extracted into the soluble and
insoluble fractions according to manufacturer’s instructions (Bio-Rad, Hercules, CA).
The soluble sample was prepared and separated in a two-dimensional analysis as
described in Figure 10.
mwt.
(kD)
5.0
Protein pI
5.4
98
64
50
Figure 16. Immunoblot of a non-transformed control leaf sample assayed using the twodimensional gel and probed with antibody to Hsp70. Leaf proteins were extracted into the
soluble and insoluble fractions according to manufacturer’s instructions (Bio-Rad,
Hercules, CA). The soluble sample was prepared and separated in a two-dimensional
analysis as described in Figure 10.
45
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51
Acknowledgments
I would like to thank Dr. Carl Pike for his guidance, patience and support. I would also
like to express my appreciation to Dr. Scott Heckathorn, Dr. John Froehlich and Dr.
Daniel Gallie for kindly donating their antibodies. My gratitude extends to Dr. Robert
Jinks, Abby Rudolph and Vessela Petrova, who had helped me in one way or the other
throughout the project. I also thank the Howard Hughes Medical Institute and Franklin
and Marshall College for their financial support.
52
Appendix A
RECIPE FOR TOBACCO LEAF EXTRACTION BUFFER
Based on Whitney et al. (2001) Plant Journal 26: 535-547 and Whitney et al. (1999)
Plant Physiology 121, 579-588, as well as notes from CSP ANU notebook on buffer used
by Hiromi Nakano.
50 mM EPPS (HEPPS) pH 8.0 with NaOH (1.2615 g/100mL)
0.5 mM Na2EDTA.2H2O (0.01861 g/100mL)
5 mM MgCl2.6H20 (1.10166 g/100mL)
Above 3 components are combined to yield 100 mL working stock solution.
The following are added just before use.
5 mM DTT (dithiothreitol)
Dissolve 7.7 mg in 50 µl water (1 M); then add 25 µL per 5 mL working solution.
1% (w/v) polyvinylpolypyrrolidone
See extraction protocol.
1 mM Na-DIECA
Stock solution is 0.1 M (22 mg/mL); add 50 µL per 5 mL working solution.
2 mM benzamidine
Stock solution is 0.1 M (15.7 mg/mL); add 100 µL per 5 mL working solution.
mM -aminocaproic acid
Stock solution is 0.1 M (13.1 mg/mL); add 100 µL per 5 mL working solution.
1.0 mM phenylmethylsulfonic acid (PMSF)
Stock solution is 0.1 M (17.4 mg/mL 2-propanol); dilute 1:100 (10 µL per 1 mL
working solution) only at the moment of grinding.
In the Nakano formulation, 2 mM Na2EDTA.2H2O and 10 mM MgCl2.6H20 were used.
PVPP was added at 1%. The buffer was HEPES pH 7.4. Na-DIECA was added at the
suggestion of Dean Price.
The last four stock solutions are prepared in small aliquots and stored frozen. They are
thawed before use and kept on ice. PMSF stock may have to be gently warmed to
dissolve.
53
Appendix B
EXTRACTION PROCEDURE
Materials
Cork borer #11 (1.5 cm diameter)
Tin foil
Liquid N2
Ice and ice bucket
Glass homogenizer
Extraction Buffer
Centrifuge
Microfuge tubes
4X SDS-PAGE Sample Buffer
Extraction
Pre-prepared solutions:
Buffer (50 mM EPPS, pH 8), 0.5 mM EDTA-Na2, 5 mM MgCl2•6H2O)
0.1 M DIECA
0.1 M ε-aminocaproic acid
0.1 M PMSF
0.1 M benzamidine
5 mM DTT – dissolve 7.7 mg in 50 µL H20 (1M) at time of use
1% (w/v) PVPP – see extraction protocol
At time of use (for one set of 3 leaf disks):
2.5 mL working stock solution
25 µL DIECA solution
50 µL benzamide solution
50 µL ε-aminocaproic acid solution
12.5 µL DTT stock
To 300 μL grinding solution prepared above, add
3 mg PVPP
3 μL PMSF solution at moment of grinding
4X SDS-PAGE sample buffer:
10 mL final - Add to a small amount of H2O (in order):
4.0 mL glycerol
0.682 g Tris Base
0.666 g Tris Hcl
0.006 g EDTA (free acid)
0.8 g SDS
4 mg bromophenol blue
At time of use, add 0.5M DTT stock (7.7 μg in 100 μL water); dilute 1:10
54

For 20 µL sample, add 7.5 µL 4X SDS-PAGE sample buffer and 3 µL 0.5M DTT
stock
 Only add DTT to sample if it’s going to be used at that time; freeze or refrigerate
sample without DTT
To make 1X SDS-PAGE sample buffer, add 1 part 4X sample buffer and 3 parts H2O
Extraction Procedure for Running Gel Immediately (all procedures are performed at 4ºC)
1. Make complete extraction buffer (-PMSF and PVPP).
2. Start the pre-cool program on the Jouan refrigerated centrifuge (15 minute
program).
3. Wrap 3 leaf discs in tin foil. Place foil packet into liquid N2 for 1 minute.
4. Place extraction buffer (100 µL per leaf disc, total 300 μL) into glass
homogenizer (on ice) containing 3 mg PVPP.
5. Add PMSF (dilute 1:100) from 0.1 M stock (1 µL PMSF per 100 µL sample, total
3 μL), followed immediately by leaf discs.
6. Grind leaf on ice until the tissue has been completely disintegrated
7. Place homogenate in eppendorf tube on ice
8. Centrifuge in refrigerated centrifuge (4˚C, 13000 rpm) for 5 minutes
9. Remove supernatant, put in separate eppendorf tube on ice
10. Add sample buffer to proper concentration (stock = 4X) by performing 3:1
dilution (3 part leaf extraction supernatent:1 part buffer)
a. To 20 µL sample, add 7.5 µL 4X SDS-PAGE sample buffer and 3µL 0.5
M DTT stock (or appropriate multiple) and mix thoroughly
b. Freeze unused portion of supernatant
11. Put at 70˚C for 10 minutes
12. Wash pellet by resuspending using pipetman in 1 ml extraction buffer (-PVPP,
add 1:100 dilution of 10 μL PMSF).
13. While resuspended, take off 333 µL (1/3) and place in separate eppendorf. Freeze
remining 2/3 for future use. Spin 1/3 down as described above. Remove and
discard supernatant. Wash 2 times with 0.5 mL extraction buffer, (-PVPP, + 1:100
dilution or 5 μL PMSF), resuspend then spin down as described above each time.
14. Make sure all wash buffer is removed after last wash (let sit 30 sec and take off
any buffer from sides of tube).
15. Resuspend pellet in 180 µL 1X sample buffer. Remove appropriate portion and
add 1:10 dilution of 0.5 M DTT.
16. Put at 70˚C for 10 minutes (mix occasionally); centrifuge all tubes (room
temperature, 13000 rpm) for 30 seconds and remove supernatant.
17. Load supernatant samples on gel in preparation for running.
Extraction Procedure for Freezing (all procedures are performed at 4ºC)
1. Make complete extraction buffer (-PMSF and PVPP).
55
2. Start the pre-cool program on the Jouan refrigerated centrifuge (15 minute
program).
3. Wrap 3 leaf discs in tin foil. Place foil packet into liquid N2 for 1 minute.
4. Place extraction buffer (100 µL per leaf disc, total 300 μL) into glass
homogenizer (on ice) containing 3mg PVPP.
5. Add PMSF (dilute 1:100) from 0.1 M stock (1 µL PMSF per 100 µL sample, total
3 μL), followed immediately by leaf discs.
6. Grind leaf on ice until the tissue has been completely disintegrated
7. Place homogenate in eppendorf tube on ice
8. Centrifuge in refrigerated centrifuge (4˚C, 13000 rpm) for 5 minutes
9. Remove supernatant, put in separate eppendorf tube on ice
10. Add sample buffer to proper concentration (stock = 4X) by performing 3:1
dilution (3 part leaf extraction supernatent: 1 part buffer)
a. To 20µL sample, add 7.5µL 4x SDS-PAGE sample buffer and mix
thoroughly
b. Freeze unused portion of supernatant
11. Put at 70˚C for 10 minutes
12. Wash pellet by resuspending using pipetman in 1 mL extraction buffer (-PVPP,
add 1:100 dilution of 10 μL PMSF).
13. While resuspended, take off 333 µL (1/3) and place in separate eppendorf. Freeze
remining 2/3 for future use. Spin 1/3 down as described above. Remove and
discard supernatant. Wash 2 times with 0.5 mL extraction buffer, (-PVPP, + 1:100
dilution or 5 μL PMSF), resuspend then spin down as described above each time.
14. Make sure all wash buffer is removed after last wash (let sit 30 sec and take off
any buffer from sides of tube).
15. Resuspend pellet in 180 µL 1X sample buffer.
16. Freeze all samples at –20˚ C.
17. After thawing, add DTT to sample.
a. To 20 µL supernatant sample in 7.5 µL 4X SDS-PAGE sample buffer
(27.5 µL sample from supernatant tube), add 3 µL 0.5M DTT stock.
b. To 27.5 µL SDS-PAGE treated pellet sample, add 3 µL 0.5M DTT stock.
18. Put samples at 70˚ C for 10 minutes (mix occasionally), then centrifuge (room
temperature, 13000 rpm) for 30 seconds and remove supernatant, before running
on gel.
56
Appendix C
RECIPES FOR MES/MOPS ELECTROPHORESIS AND WESTERN BLOTTING
Electrophoresis
MES Gel-Running Buffer (Upper and Lower Chamber Buffers)
20X Stock buffer: 500 mL
MES: 97.6 g
Trisbase: 60.6 g
SDS: 10.0 g
EDTA free acid: 3.0 g
pH 7.3 (do not adjust)
Dissolve in 400 mL water then make volume to 500 mL
MOPS Gel-Running Buffer (Upper and Lower Chamber Buffers)
20X Stock buffer: 500 mL
MOPS: 104.6 g
Trisbase: 60.6 g
SDS: 10.0 g
EDTA: 3.0 g
pH 7.7 (do not adjust)
Dissolve in 400 mL water then make volume to 500 mL
Lower Buffer: 1L
50 mL 20X MES/MOPS running buffer
950 mL COLD H2O (4˚C)
Upper Buffer
Separate 200 mL of the lower buffer
Add 500 µL NuPAGE Antioxidant immediately prior to the run and mix
thoroughly
Western Blotting
NuPAGE Transfer Buffer (25 mM Bicine, 25 mM Bis-Tris (free base), 1 mM EDTA,
0.05 mM chlorobutanol, pH 7.2)
20X Stock: 125 mL
Bicine: 10.2 g
Bis-Tris: 13.1 g
EDTA free acid: 0.75 g
Chlorobutanol: 0.025 g
pH 7.2 (do not adjust)
Dissolve in 100 mL then make volume to 125 mL
1X (made same day as use): 1L
57
50 mL 20X stock solution
1 mL NuPAGE antioxidant
100 mL MeOH
849 mL COLD H2O
If blotting 2 gels, increase MeOH to 200 mL and reduce COLD H2O to 749 mL
Blocking Buffer
For 25 mL: 25mL TBS
1.25 g 5% non-fat dry milk
TBS - Tris-buffered Saline (100 mM Tris-HCl pH 7.5, 0.9% (w/v) NaCl)
12.11g Tris base
9 g NaCl
900 mL ddH2O
Adjust to pH 7.5 with HCl
Dilute to 1 L with ddH2O
TTBS (100 mM Tris-HCl pH 7.5, 0.9% (w/v) NaCl, 0.1% (v/v) Tween-20)
TBS with 0.1% Tween-20 (Polyoxyethylenesorbitan monolaurate)
(1 ml into 1 L)
Alkaline Phosphate Substrate Solution
Bought from Roche
58
Appendix D
ELECTROPHORESIS AND WESTERN BLOTTING PROCEDURES
Electrophoresis in Invitrogen XCell SureLock Chamber
1. Prepare 1X running buffer using COLD (4˚C) water. Prepare 1X MES buffer by
diluting 20X MES buffer (50 mL buffer, 950 mL H2O from the refrigerator).
Keep cold until use.
2. Open gel pouch and rinse cassette with H2O. Dry the cassette before peeling off
its tape. Mark bottom of each well and the bottom of the gel.
3. Pull comb from cassette slowly and smoothly.
4. Use a disposable pipette to rinse the sample wells with 1X running buffer. Invert
the gel and shake vigorously. Repeat two more times. Fill the wells with 1X
running buffer. Make sure to get rid of all air bubbles from wells.
5. Put the buffer core into the lower buffer chamber so the negative electrode fits
into the opening in the gold plate on the lower buffer chamber (Xcell SureLock
Mini-Cell Instruction Manual, Page 9).
6. Insert the “gel tension wedge” into the lower buffer chamber behind the buffer
core. Make sure it is in unlocked position: arm against side of buffer chamber,
not against itself (Illustration on Page 11).
7. Insert gel cassettes into the lower buffer chamber. Place one in front of the core
and one behind, with the shorter well sides facing in to the core. If running only
one gel, put the buffer dam in the rear position.
8. Pull the gel tension lever towards the front of the buffer chamber until it comes to
a firm stop and the gels are snug against the core.
9. The upper buffer chamber (cathode) is the space between the two gel cassettes on
each side of the buffer core.
10. Fill the upper buffer chamber with 200 mL of the upper running buffer. Use
enough to cover the wells.
11. Make sure the upper buffer chamber is not leaking. If it is, the gels are not seated
properly – reassemble the unit.
12. Load the samples. Load 1X sample buffer in any of the wells that does not contain
sample.
13. Fill the lower buffer chamber (anode) by pouring 600 mL of the lower running
buffer through the gap between the gel tension wedge and the back of the lower
buffer chamber.
14. Put the lid on the buffer core (it must be aligned properly to fit.)
15. Put the apparatus inside the refrigerator. Put the power supply out of the
refrigerator.
16. With the power OFF and unit unplugged, connect the electrode cords to the power
supply (red to (+) jack, black to (-) jack).
17. Turn on the power. Set for constant voltage around 170 V. It will run about an
hour.
18. NOTE: if electroblotting next, prepare nitrocellulose membrane ~10 minutes
before gel is finished running.
59
19. At the end of the run, set voltage to zero before turning off the switch, unplug the
power supply, disconnect the cables from the power supply (in that order).
20. Remove the lid and unlock the gel tension lever.
21. Remove the gel cassettes from the mini-cell. Handle cassettes by their edges only.
22. Lay the gel cassettes (well side up) on a flat surface. Separate the three bonded
sides of the cassette by inserting the gel knife into the gap between the cassette’s
two plates.
23. Carefully remove the top plate. If blotting, proceed onto western transfer protocol
without removing the gel from the bottom plate. If staining remove the gel.
Electroblotting in Invitrogen XCell SureLock Blot Module
1. The 1X NuPAGE transfer buffer used during the electroblot transfer MUST be
cold (4˚ C), but the 1X transfer buffer used during setup and for equilibration of
the nitrocellulose and the gel does not need to be made with cold water.
2. While gel is still running, cut a piece of nitrocellulose membrane slightly larger
than the transfer area (use flat-bladed forceps to handle the membrane). Wet the
membrane by slowly introducing the membrane from one corner into a dish of 1X
transfer buffer. Equilibrate for several minutes, along with the blotting pads,
which are soaked and squeezed thoroughly to remove air bubbles.
3. Separate each of the three bonded sides of the gel cassette by inserting the gel
knife into the gap between the cassette’s two plates. At each side of the cassette,
push up and down on the knife handle until the plates are completely separated.
4. Carefully open the cassette by removing and discarding the plate without the gel,
allowing the gel to remain on the other plate. It does not matter which plate the
gel adheres to.
5. Remove wells on the gel with the gel knife by cutting at approximately 5mm
below the bottom of the wells.
6. Place 2 pieces of Whatman 3mm filter paper (soaked briefly immediately before
use) on top of the gel and lay just above the “foot” at the bottom of the gel,
leaving the “foot” of the gel uncovered. Keep the filter paper saturated with the
transfer buffer and remove all trapped air bubbles by gently rolling over the
surface using a glass pipette as a roller.
7. Turn the plate over so the gel and filter paper are facing downwards over a clean
piece of Parafilm.
8. Remove the gel from the plate using the following methods:
a. If the gel rests on the longer (slotted) plate, use the gel knife to push the foot
out of the slot in the plate and the gel will fall off easily.
b. If the gel rests on the shorter (notched) plate, use the gel knife to carefully
loosen the bottom of the gel and allow the gel to peel away from the plate.
9. Cut the “foot” off the gel with the gel knife.
10. Wet the gel surface with the 1X transfer buffer and position the pre-soaked
nitrocellulose membrane on the gel. Remove all air bubbles by gently rolling a
glass pipette over the membrane surface.
60
11. Place 2 pieces of pre-soaked Whatman 3mm filter paper on top of the transfer
membrane. Again, remove any trapped air bubbles.
12. Place two soaked blotting pads into the cathode (-) core of the blot module.
Carefully pick up the gel membrane assembly with your gloved hand and place on
the pad in the same sequence such that the gel is closest to the cathode plate. The
assembly should be placed against the bottom of the core.
13. Ensure that the pre-soaked blotting pads (usually 2) rise 0.5 cm over the rim of the
cathode core. Place the anode (+) core on top of the pads. The gel/membrane
sandwich should be held securely between the two halves of the blot module for
complete contact of all components.
14. Holding the blot module together firmly, slide it into the guide rails on the lower
buffer chamber so that the (+) sign can be seen in the upper left hand corner of the
blot module. The inverted gold post on the right hand side of the blot module
should fit into the hole next to the upright gold post on the right side of the lower
buffer chamber. Keep holding the module together firmly.
15. Insert the gel tension wedge and push the lever forward to lock it in place.
16. Fill the blot module with transfer buffer until the gel/membrane sandwich is
covered just enough in transfer buffer.
17. Fill the outer buffer chamber with COLD deionized water by pouring
approximately 650 mL in the gap between the front of the blot module and the
front of the lower buffer chamber until the water level is approximately 2 cm from
the top of the lower buffer chamber.
18. Place the lid on top of the unit
19. Connect the electrode cords to the power supply and set for constant voltage 30 V
for an hour. The starting current should be 170 mA, ending at approximately 110
mA.
Electroblotting in Hoefer Blot Module
1. The 1X transfer buffer used during the electroblot transfer MUST be cold (4˚ C),
but the 1X transfer buffer used during setup and for equilibration of the
nitrocellulose and the gel does not need to be made with cold water.
2. While gel is still running, cut a piece of nitrocellulose membrane slightly larger
than the transfer area (use flat-bladed forceps to handle the membrane). Wet the
membrane by slowly introducing the membrane from one corner into a dish of 1X
transfer buffer. Equilibrate for 15 minutes.
3. Remove stacking gel (wells) and the lip on the bottom of the gel with a sharp,
clean razor blade. Push directly down with the razor blade (not cutting motion) to
prevent gel from tearing. Cut a small piece from the lower left hand corner of the
gel (under lane one) to aid in identifying lanes in subsequent steps.
4. Briefly equilibrate gel in a dish containing ~25 mL of 1X transfer buffer for
appropriate time based on gel thickness (1 mm = 5 minutes).
5. Open the western cassette in a large plastic tray with colored tape on the left. Lay
2 sponges on opposite sides of the open cassette. Fill the tray with 1X transfer
buffer until the sponges are covered. Squeeze air bubbles out of sponges.
61
6. Cut 2 pieces of blotting paper (Sigma, P-4556) slightly larger than the
nitrocellulose membrane and wet in 1X transfer buffer. Lay 1 piece of wet
blotting paper on the sponge on the left hand side of the cassette.
7. Carefully lay the gel on top of the blotting paper. Smooth out gel to remove air
bubbles between the gel and the blotting paper.
8. Carefully place the nitrocellulose membrane on the gel; use a flat-bladed forceps.
Try to let the center contact the gel first, then slowly lower the membrane
outwards to eliminate air bubbles. Remove air bubbles.
9. Make a notch in the membrane corresponding to a notch in the gel. Mark the top
of the resolving gel and the location of the tracking dye (by making holes with a
straight pin).
10. Lay another piece of filter paper on top and smooth out air bubbles. Place the
remaining sponge and right half of the cassette on top of sandwich and close the
latch.
11. Place sandwich into electroblot chamber filled with 1 liter of cold transfer buffer
and magnetic stir bar. Take apparatus to cold room (bring stir plate and power
supply).
12. With the power OFF, connect the black (-) electrode to the side of the chamber to
which the tape on the cassette IS pointing. Connect the red (+) electrode to the
side of the chamber the tape is NOT pointing.
13. Turn on the stir plate. Make sure the magnetic stir bar is spinning appropriately.
14. Connect the base of the electroblot unit to the cold faucet and have a constant cold
water flow throughout the transfer (check periodically to ensure water flow is
adequate).
15. The transfer should be carried out for 90 minutes at 250 mA constant current.
16. After transfer, disassemble the blotting sandwich and remove nitrocellulose
membrane. The membrane can be stained or immunoblotted.
Immunodetection
1. Everything is completed on rotary shaker at room temperature. Set the shaker at
the LOWEST possible setting where the entire membrane is still washed.
2. Mark the ladders on the nitrocellulose now using pencil, as they may be washed
off during the next steps.
3. Put the nitrocellulose into a square dish with the side closest to the gel during
transfer face DOWN.
4. The following solution volumes are variable. These are for a gel approximately 8
cm x 7 cm (i.e. the size of the pre-cast gels).
5. Incubate in 25 mL Blocking Buffer for 60 minutes.
a. May store in refrigerator overnight at this point.
6. Thaw out antibodies ON ICE.
7. Remove alkaline phosphate substrate solution from fridge and place it at room
temperature for several hours before use.
8. Incubate in 10 ml blocking buffer (with non-fat milk) with dilution of 1˚ antibody
for 60 minutes.
62
i. DON’T add antibody directly to blot; mix blocking solution and
antibody thoroughly before applying to blot.
9. Rinse FOUR times with 20 mL TTBS for 10 minutes EACH.
10. Incubate in 10 mL blocking buffer (with non-fat milk) with dilution of 2˚
antibody for 60 minutes.
i. DON’T add antibody directly to blot; mix blocking solution and
antibody thoroughly before applying to blot.
11. Rinse FOUR times with 20 mL TTBS for 10 minutes EACH.
12. Rinse once with 20 mL distilled H2O for 15 minutes.
13. Incubate in 8 mL (or less) alkaline phosphate substrate solution, whose bottle is
inverted once before use, until color appears (approximately 30 minutes).
14. Terminate reaction by washing membrane with distilled water. Air dry and
photograph for permanent record. Store at room temperature in the dark.
63
Appendix E
RECIPES FOR TRIS-GLYCINE ELECTROPHORESIS AND WESTERN BLOTTING
Gel
8% Resolving Gel (Upper and Lower Chamber Buffers)
For 35 mL,
7.00 mL 40% acrylamide/bis-acrylamide mix (Sigma A-7802)
8.80 mL resolving buffer with SDS, pH 8.8
18.85 mL water
Then, add
350 µL 10% ammonium persulfate (Fischer BP179-25)
18 µL TEMED (Sigma T-9281)
right before pouring gel.
5% Stacking Gel
For 13.33 mL,
1.67 mL 40% acrylamide/bis-acrylamide mix (Sigma A-7802)
3.33 mL stacking buffer with SDS, pH 6.8
8.20 mL water
Then, add
133 µL 10% ammonium persulfate (Fischer BP179-25)
13 µL TEMED (Sigma T-9281)
right before pouring gel.
Electrophoresis
Tris-Glycine Electrophoresis Buffer (Upper and Lower Chamber Buffers)
5X stock buffer: 1 L
15.1 g Tris Base
94.0 g glycine
Dissolve in 900 mL ddH2O then add 5.0 g SDS and adjust volume to 1 L
1X buffer (on the day of use): 1L (25 mM Tris, 250 mM glycine pH 8.3, 0.1% (w/v) SDS)
200 mL 5X stock solution
800 mL COLD water (4°C)
pH 8.3 (do not adjust)
Western Blotting
Novex Tris-Glycine Transfer Buffer
25X Stock: 500 mL
18.2 g Tris Base
90.0 g glycine
Dissolve in 450 mL ddH2O then adjust volume to 500mL. Buffer pH is 8.3 (do not
adjust). Store at room temperature.
64
0.5X Towbin buffer: 1 L (12 mM Tris Base, 96 mM glycine pH 8.3)
40 mL 25X stock solution
200 mL methanol
760 mL ddH2O
Blocking Buffer
For 25ml,
25 mL TBS
1.25 g 5% non-fat dry milk
TBS - Tris-Buffered Saline (100 mM Tris-HCl pH 7.5, 0.9% (w/v) NaCl)
12.11 g Tris Base
9 g NaCl
900 ml ddH2O
Adjust with HCl to pH 7.5
Dilute to 1 L with ddH2O
TTBS (100 mM Tris-HCl pH7.5, 0.9% (w/v) NaCl, 0.1% (v/v) Tween-20)
TBS with 0.1% Tween-20 (Polyoxyethylenesorbitan monolaurate)
(1 ml into 1 L)
Alkaline Phosphate Substrate Solution
Bought from Roche
65
Appendix F
RECEIPES FOR TWO-DIMENSIONAL ELECTROPHORESIS
Apparatus/Materials
Bio-Rad PROTEAN IEF cell
ReadyStrip IPG strips, pH 4-7 (7 cm, 11 cm or 17 cm), stored at -20C
IEF focusing tray with lid, same size as IPG strips
Disposable rehydration/equilibrium trays with lid, same size as IPG strips
Electrode wicks, precut
Blotting filter papers
Mineral oil
Forceps
IPG protein stains and destain, use either
 BioRad IEF stain; Coomassie Brilliant Blue R-250 Destaining Solution, catalog
#1610438 or destain solution containing 10% acetic acid/40% methanol in water
 Bio-Safe Coomassie stain; 20 mM Tris-HCl, pH 8.8 destain
8-16% SDS-PAGE gels (Ready Gel, Criterion, or PROTEAN II Ready Gel precast
gels)
SDS-PAGE electrophoresis cell (Mini-PROTEAN 3, Ready Gel, Criterion, or
PROTEAN II XL cell)
SDS-PAGE protein stain, use either
 BioRad Bio-Safe Coomassie stain; water destain
 Coomassie Brilliant Blue R-250 stain, catalog #161-0436; destain solution
containing 10% acetic acid/40% methanol in water)
Coomassie Brilliant Blue R-250 stain (0.1% Coomassie Blue R-250 in 40%
methanol, 10% HOAc)
Coomassie Blue R-250
1.0 g
Water
500 mL
Acetic acid
100 mL
Methanol
400 mL
ReadyPrep 2-D starter kit, stored at 4C, which includes:
ReadyPrep Rehydration/Sample Buffer (provided with kit)
When reconstituted,
8 M urea
10 mL
CHAPS
2%
DTT
50 mM
®
Bio-Lyte 3/10 ampholytes
0.2% (w/v)
Bromophenol Blue
trace
First time before use: reconstitute with 6.1 mL nanopure water provided. Then,
use to treat E. coli protein sample.
Nanopure Water (one bottle)
15 mL sterile nanopure water
Equilibrium Buffer I (two vials with stirbars; WHITE or SILVER caps)
66
When reconstituted,
6 M urea
20 mL
SDS
2%
Tris-HCl (pH 8.8)
0.375 M
Glycerol
20%
DTT
2% (w/v)
First-time before use: reconstitute with 13.35 mL of the supplied 30% glycerol
solution and mix until all solids dissolved. To expedite the process, bottle can be
warmed slightly in the palm of the hand or placed into a 25-30°C water bath
during stirring. Do not heat above 30°C.
Equilibrium Buffer II (two vials with stirbars; RED caps)
When reconstituted,
6 M urea
20 mL
SDS
2%
Tris-HCl (pH 8.8)
0.375 M
Glycerol
20%
First-time before use: perform as above for Equilibrium Buffer I. Then add one
vial of iodoacetamide provided to each bottle of Equilibrium Buffer II and stir
until fully dissolved.
30% glycerol solution (one bottle; CLEAR cap)
70 mL sterile 30% (v/v) glycerol
Iodoacetamide (two vials; RED caps)
0.5 g / vial of an ultrapure grade of iodoacetamide
Overlay Agarose (one bottle)
50 mL of 0.5% low melting point agarose in 25 mM Tris, 192 mM glycine, 0.1%
SDS and a trace of Bromophenol Blue
Second dimension
1X Tris/glycine/SDS (TGS) electrophoresis running buffer (25 mM Tris, 192
mM glycine, 0.1% SDS, pH 8.3)
Tris base
3.03 g
Glycine
14.4 g
SDS
1.0 g
Water
to 1 L
pH 8.3 (do NOT adjust)
Towbin Transfer Buffer with 20% MeOH (25 mM Tris, 192 mM glycine, 20%
v/v MeOH, pH 8.3)
Tris
3.03 g
Glycine
14.4 g
ddH2O
800 mL
67
MeOH
200 mL
For 10% MeOH, use 900 mL ddH2O and 100 mL MeOH instead.
Footnote:
CHAPS is 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, a zwitterionic
detergent.
Bio-Lyte® 3/10 ampholytes is a mixture of carrier ampholytes, pH 3-10
68
Appendix G
TWO-DIMENSIONAL GEL ELECTROPHORESIS PROCEDURE
Rehydration of IPG strips (DAY 1)
1. Remove the desired number of pH 4-7 ReadyStrip IPG strips from the -20°C
freezer, including two extras for post-IEF staining.
2. Place one disposable rehydration/equilibration tray of the same size as the IPG
strips to be run onto the bench with the sloped end facing to the right. Mark
electrodes polarities and channels.
3. The recommended amount of protein for loading per IPG strips is as followed
(based on BioSafe E. coli protein sample):
IPG strip length
Sample Volume
Protein Loaded
7 cm
125 L
169 g
11 cm
185 L
250 g
17 cm
300 L
405 g
4. Pipet the appropriate volume of protein sample as a line along the back edge of
channel #1, extending along the whole length of the channel except for about 1
cm from each end. Avoid introducing any bubbles which may interfere with the
even distribution of sample along the length of the strip.
5. Repeat the above procedure for all samples using channels from both sides of the
rehydration/equilibration tray for even weight distribution and especially
consistency for transfer later to the focusing tray.
6. When all the protein samples have been loaded, use forceps to peel the coversheet
from one of the pH 4-7 ReadyStrip IPG strips before gently placing the strip gel
side DOWN onto the sample contained within the channel, with its “+” and “pH
4-7” label positioned at the left side of the tray. Avoid trapping air bubbles
beneath the strip and seeping of the sample onto the plastic backing of the strips.
If the former happen, use forceps to lift the strip up and down from one end until
the air bubbles move to the end and out from under the strip. Repeat for all filled
channels.
7. IPG strips at this stage can be left to rehydrate up to 1 hour before addition of
mineral oil. Overlay each of the strips with 2-3 mL of mineral oil by carefully
pipetting the oil onto the plastic backing of the strip along its length to prevent
evaporation during the rehydration process.
8. Cover the rehydration/equilibration tray with the plastic lid and leave the tray
sitting on a level bench overnight for 11-16 hr to rehydrate the IPG strips and load
the protein sample.
First Dimension-Isoelectric Focusing (DAY 2), duration apprx. 7.5 hours
1. Place a clean, dry PROTEAN IEF focusing tray the same size as the rehydrating
IPG strips onto the lab bench.
69
2. Using forceps, place a paper wick at both ends of the channels covering the wire
electrodes. For consistency, use channels with the same numbers as those used
during rehydration.
3. Pipet 8 L of nanopure water onto each wick to wet them. Readjust their position
if necessary.
4. Remove the cover from the rehydration/equilibration tray containing the IPG
strips. Carefully hold the strips vertically with forceps for about 7-8 seconds to
allow the mineral oil to drain, before transferring them to the corresponding
channels in the focusing tray while maintaining the gel side DOWN and avoiding
the trapping of air bubbles beneath strips.
5. The disposable rehydration/equilibration tray can be cleaned and dried at this
point, which then can be used later to store strips after completion of the firstdimension run.
6. Cover each IPG strips with 2-3 mL of fresh mineral oil. Check (and removed) any
trapped air bubbles beneath the strips. Place the lid onto the tray (positive “+” to
the left when the inclined portion of the tray is on the right).
7. Place the focusing tray into the PROTEAN IEF cell and close the cover
8. Choose the relevant program based on the length of IPG strips used. For all strip
lengths, used the default cell temperature of 20°C, with a maximum current of 50
A/strip and No Rehydration.
7 cm
Step 1
Step 2
Step 3
Total
Voltage
250
4000
4000
Time
20 min
2 hr
-5 hr
Volt-Hours
--10000
14000
Ramp
Linear
Linear
Rapid
11 cm
Step 1
Step 2
Step 3
Total
250
8000
8000
20 min
2.5 hr
-5.3 hr
--20000
~30000
Linear
Linear
Rapid
17 cm
Step 1
Step 2
Step 3
Total
250
10000
10000
20 min
2.5 hr
-7 hr
--40000
~50000
Linear
Linear
Rapid
9. Press START to initiate the electrophoresis run.
Completion of IEF
1. Remove all IPG strips from the focusing tray and place them gel side UP into a
new or clean, dry disposable rehydration/equilibration tray. Hold the strips
70
vertically with forceps and allow the mineral oil to drain from strips for ~5 sec
before transfer. Maintain IPG strips in the same order as in the focusing tray.
Staining of IPG strips (optional)
1. Transfer 2 of the IPG strips from rehydration/equilibration tray to a clean, dry
piece of blotting filer paper with the gel side up.
2. Thoroughly wet a second filter paper of the same size with nanopure water before
laying it onto the IPG strip. Press firmly over the entire length of the strip but
avoid squishing the gel, before “peeling” back the top filter paper again. This
blotting step removes mineral oil on the IPG surface, thereby reducing
background staining and generally improving the staining of the IPG strips.
Repeat for second strip.
3. Transfer the two IPG strips to a staining tray containing approximately 50 mL of
Bio-Safe Coomassie stain or Bio-Rad’s IEF stain and shake it on the belly dancer
for 1 hr. Proceed to second-dimension SDS-PAGE for remaining IPG strips.
4. Destain the IPG strips twice for 10 min each. For Bio-Safe stain use 20 mM TrisHCl, pH 8.8. For IEF stain, use Coomassie Brilliant Blue R-250 Destaining
Solution (catalog #161-0438) or destain solution (10% acetic acid/40% methanol
in water). Complete destaining of the IPG strips may take several hours. Changing
the destain solution several times can accelerate this process.
5. Check for optimum isoelectric focusing pattern.
IPG equilibrium
1. Due to time constraints, proceed to second-dimension SDS-PAGE gel while
staining the IPG strips (see above). Alternatively, tray holding the IPG strips can
be saran-wrapped and stored at -70°C. Thaw them out for 10-15 min before use
the next day. Frozen IPG strips containing sample are opaque white and turn to
clear after thawing and redissolving of the urea present inside each strip.
2. Add the appropriate volume of equilibration buffers to each channel containing an
IPG strip.
Strip Length
Equilibration Buffer I
Equilibration Buffer II
7 cm
2.5 mL
2.5 mL
11 cm
4 mL
4 mL
17 cm
6 mL
6 mL
3. Place the tray with Equilibration Buffer I at low shaking on the belly dancer for
10 min.
4. Decant the liquid from the square side of the rehydration/equilibration tray after
incubation, until the tray is positioned vertically. When most of the liquid has
been decanted, flick the tray a couple of times to remove the remaining drops of
Equilibration Buffer I.
5. Repeat the above three steps for Equilibration Buffer II.
6. During second incubation, melt the overlay agarose solution in microwave oven.
- Remove the bottle cap and insert a piece of cotton or Kimwipes
71
-
Microwave on high for 45-60 secs until agarose liquefies. If desired, a
stirbar can be added and the bottle set to stir slowly.
Second Dimension-SDS-PAGE
1. Fill a 100 mL graduated cylinder or a tube that is the same length or longer than
the IPG strip length with 1X Tris-glycine-SDS running buffer. Use a Pasteur
pipette to remove any bubbles on the buffer surface.
2. Blot away any excess water remaining inside the IPG well using Whatman 3 MM
or similar blotting paper. Lay the gels onto the bench with the top of the gel
facing you and the back (bigger) plate on the bottom.
3. Remove an IPG strip from the disposable rehydration/equilibration tray and dip
briefly into the graduated cylinder containing the 1X Tris/Glycine/SDS running
buffer. Lay the strip gel side UP onto the back plate of the SDS-PAGE gel above
the IPG well. Repeat same for all IPG strips.
4. Hold the SDS-PAGE gel with IPG strip vertically in rack with the short plate up
and facing towards you. Use a Pasteur pipette and pipet overlay agarose solution
into the IPG well of the gel.
5. Using forceps, carefully push the strip into the well, taking care not to trap any air
bubbles beneath the strip. Push on the plastic backing to the strip and not the gel
matrix. Repeat same for all SDS-PAGE gels.
6. Allow the agarose to solidify for 5 min by standing the gels vertically in a test
tube rack.
7. Mount the gel per the instruction manual provided with the apparatus.
8. Fill the reservoirs with 1X Tris/glycine/SDS running buffer and run the
electrophoresis according to the appropriate conditions. The migration of
Bromophenol Blue present in the overlay agarose solution is used to monitor the
progress of electrophoresis.
Strip Length
Electrophoresis
cell
Conditions
Approximate
run time
7 cm
Mini-PROTEAN
11 cm
Criterion
17 cm
PROTEAN II XL
200 V, constant
200 V, constant
40 min
65 min
16 mA/gel for 30
min, then 24
mA/gel for ~5 hr
5.5 hr
SDS-PAGE gel staining
1. For Bio-Safe Coomassie stain,
- Fill an appropriate number of staining trays with nanopure water and set
aside
Ready Gel and Criterion
200 mL / gel
PROTEAN II Ready Gel 400 mL / gel
72
-
Slide gels into tray with water after removing them from gel cassettes
upon completion of electrophoresis.
- Wash gels 3 times for 5 min each. Add fresh water for each wash.
- Add enough Bio-Safe stain to completely cover each gel.
Ready Gel and Criterion
50 mL / gel
PROTEAN II Ready Gel 100 mL / gel
- Incubate gels on belly dancer for at least 60 min or overnight if desired.
- Discard the stain and wash gels twice for 15-30 min with water. Longer
water washes may be needed to remove remaining background. Gels can
be stored in water for several days.
2. For Coomassie Brilliant Blue R-250 stain,
- Add enough Coomassie Brilliant Blue R-250 stain to one or more staining
trays to completely cover each gel.
- Slide gels into tray with stain after removing them from gel cassettes upon
completion of electrophoresis.
- Incubate gels on belly dancer for at least 60 min.
- Destain gels with destain solution (10% acetic acid/40% methanol in water)
until the background staining is acceptable. For best results, change the
destain solution several times. Gels can be stored for several days in a
solution of 10% acetic acid.
- For overnight destaining, use 7% acetic acid/5% methanol in water.
73