Living ColorsTM
User Manual
(PT2040-1)
FOR RESEARCH USE ONLY
(PR74631)
CLONTECH Laboratories, Inc.
Table of Contents
I. Introduction
4
II. Properties of GFP and GFP Variants
A. The GFP Chromophore
B. Red-shifted GFP Variants
C. Blue Emission GFP Variants
D. UV-optimized GFP Variant
E. Acquisition and Stability of GFP Fluorescence
F. Sensitivity
III. Living Colors GFP and GFP Variant Vectors
IV. Expression of GFP and GFP Variants
A. Suitable Host Organisms and Cells
B. Expresion of GFP Fusion Proteins
C. GFP Expression in Mammalian Cells
D. GFP Expression in Plants
E. GFP Expression in Yeast
V. Detection of GFP and GFP Variants
A. Filter Sets
B. Flow Cytometry
C. Fluorescence Microscopy
D. Quantitative Fluorometric Assay
VI. Purified Recombinant GFP and GFP Variants
A. General Information
B. rGFP and rGFP Variants as Standards in Western Blots
C. rGFP and rGFP Variants as a Control for
Fluorescence Microscopy
VII. GFP Antibodies
A. Applications for GFP Antibodies
B. Immunoprecipitation with GFP Monoclonal Antibody
VIII. Troubleshooting Guide
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IX. References
38
X. Summary Reference List
46
XI. Related Products
48
Appendix. Fluorescent Proteins Newsgroup
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I. Introduction & Protocol Overview
List of Figures
Figure 1.
Figure 2.
Figure 3.
Figure 4.
Figure 5.
Figure 6.
The amino acid substitutions of the red-shifted GFP
variants and EBFP
Excitation and emission spectra of wt GFP and red-shifted
GFP variants
Excitation and emission spectra of wt GFP and EBFP
Excitation and emission spectra of wt GFP and GFPuv
Comparison of fluorescence intensity of GFP in sonication
buffer versus GFP in cell lysates
Relative fluorescence intensity of serial dilutions of
GFP-transformed cells
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List of Tables
Table I.
Table II.
Table III.
Living Colors Vectors
Proteins Expressed as Fusions to GFP
Use of GFP in Plants
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I. Introduction
In the bioluminescent jellyfish Aequorea victoria, light is produced when energy
is transferred from the Ca2+-activated photoprotein aequorin to green fluorescent
protein (GFP; Shimomura et al., 1962; Morin & Hastings, 1971; Ward et al.,
1980). The cloning of the wild-type GFP gene (wt GFP; Prasher et al., 1992;
Inouye & Tsuji, 1994a) and its subsequent expression in heterologous systems
(Chalfie et al., 1994; Inouye & Tsuji, 1994a; Wang & Hazelrigg, 1994) has
established GFP as a novel genetic reporter system. When expressed in either
eukaryotic or prokaryotic cells and illuminated by blue or UV light, GFP yields a
bright green fluorescence. Light-stimulated GFP fluorescence is speciesindependent and does not require any cofactors, substrates, or additional gene
products from A. victoria. Additionally, detection of GFP and its variants can be
performed with living tissues instead of fixed samples.
CLONTECH offers many GFP products, including expression vectors, sequencing primers, purified Recombinant GFP Proteins, and GFP-specific Monoclonal
and Polyclonal Antibodies. Also available from CLONTECH are several vectors
encoding GFP variants that were developed to improve the use of GFP as a
reporter for gene expression and protein localization. These variants are ideal for
fluorescence microscopy and flow cytometry. (See Section III for further information on specific vectors and Section XI for a complete list of related products.)
• Red-shifted GFP variants: EGFP (GFPmut1; Cormack et al., 1996) and
GFP-S65T (Heim et al., 1995) have red-shifted excitation spectra and
fluoresce 4–35-fold more brightly than wt GFP when excited with blue light.
• EBFP: The blue fluorescent variant, EBFP, contain four amino acid substitutions that shift the emission from green to blue, enhance the brightness of the
fluorescence, minimize rapid photobleaching, and improve solubility of the
protein (Heim & Tsien, 1996; Cormack et al., 1996).
• GFPuv: When expressed in E. coli, GFPuv is reported to be 18 times brighter
than wt GFP when excited by standard UV light (Crameri et al., 1996) and
should be used in experiments using UV light for excitation. This variant
contains additional amino acid mutations which also increase the translational
efficiency of the protein in E. coli.
In addition to the chromophore mutations mentioned above, the GFP variant
genes hGFP-S65T, EGFP, and EBFP contain more than 190 silent mutations
which create an open reading frame comprised almost entirely of preferred
human codons (Haas, et al., 1996). Furthermore, in the construction of the Living
Colors vectors containing these variant genes, upstream sequences flanking the
coding regions were converted to a Kozak consensus translation initiation site
(Kozak, 1987), and potentially inhibitory flanking sequences in the original cDNA
clones (λgfp10 & pGFP10.1; Chalfie et al., 1994) were removed. All of these
changes increased the translational efficiency of the mRNA—and, consequently, the expression of the GFP variants—in mammalian and plant systems.
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I. Introduction continued
The development of EGFP, GFPuv, and more recently EBFP, opens up a wide
range of exciting new applications that were not possible with GFP alone. The
combined use of EBFP and wt GFP, EGFP, GFPuv, or one of the other green
GFP variants, should permit the following types of double-labeling experiments:
1) microscopy of multiple cell populations in a mixed culture; 2) monitoring gene
expression from two different promoters in the same cell, tissue, or organism;
3) the intracellular localization of two different proteins (Rizzuto et al., 1996);
4) the monitoring of different cell lineages in a single tissue or organism; 5) the
real-time analysis of interactions between two distinct protein fusions (Heim &
Tsien, 1996; Mitra et al., 1996); and 6) FACS® analysis of mixed cell populations
(e.g., a mixture of cells expressing EGFP and EBFP or GFPuv, and nonfluorescent
cells). GFPuv may be used in double-labeling experiments with EGFP by
selective excitation of these two variants.
The GFP Application Notes include information about GFP and GFP variants;
protocols for the expression and detection of GFP and its variants; suggested
applications for the Recombinant GFP Proteins and GFP-specific antibodies;
and a comprehensive list of GFP references.
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II. Properties of GFP and GFP Variants
A. The GFP Chromophore
The GFP chromophore consists of a cyclic tripeptide derived from Ser-TyrGly in the primary protein sequence (Cody et al., 1993) and is only
fluorescent when embedded within the complete GFP protein. (There have
been no reports of a truncated GFP [other than a few amino acids from the
C-terminus] that is still fluorescent.) The crystal structures of GFP and GFPS65T have revealed a tightly packed β-can enclosing an α-helix containing
the chromophore (Ormö et al., 1996; Yang, F. et al., 1996). This structure
provides the proper environment for the chromophore to fluoresce by
excluding solvent and oxygen. Nascent GFP is not fluorescent, since
chromophore formation occurs posttranslationally. The chromophore is
formed by a cyclization reaction and an oxidation step that requires
molecular oxygen (Heim et al., 1994; Davis et al., 1995). These steps are
either autocatalytic or use factors that are ubiquitous, since fluorescent
GFP forms in a broad range of organisms. Chromophore formation may be
the rate-limiting step in generating the fluorescent protein, especially if
oxygen is limiting (Heim et al., 1994; Davis et al., 1995). The wt GFP
absorbs UV and blue light with a maximum peak of absorbance at 395 nm
and a minor peak at 470 nm and emits green light maximally at 509 nm, with
a shoulder at 540 nm (Ward et al., 1980).
wt GFP: Phe 64 Ser Tyr Gly Val Gln 69. . . . Tyr 145
EGFP: Leu 64 Thr Tyr Gly Val Gln 69
EBFP: Leu 64 Thr His Gly Val Gln 69. . . . Phe145
GFP-S65T: Phe 64 Thr Tyr Gly Val Gln 69
Figure 1. The amino acid substitutions (in grey) of the red-shifted GFP variants and EBFP are
shown below the wt GFP sequence.
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II. Properties of GFP and GFP Variants continued
B. Red-Shifted GFP Variants
CLONTECH’s red-shifted GFP variants EGFP and phGFP-S65T contain
different mutations in the chromophore (Figure 1), which shift the maximal
excitation peak to approximately 490 nm (Figure 2). The major excitation
peak of the red-shifted variants encompasses the excitation wavelength of
commonly used filter sets, so the resulting signal from these variants is
much brighter than from wt GFP. Similarly, the argon ion laser used in most
FACS machines and confocal scanning laser microscopes emits at 488 nm,
so excitation of the red-shifted GFP variants is much more efficient than
excitation of wt GFP. In practical terms, this means the detection limits in
both microscopy and FACS are considerably lower with the red-shifted
variants. Although the peak positions in the emission spectra of all the redshifted GFP variants are virtually identical, double-labeling can be performed by selective excitation of wt GFP and red-shifted GFP (Kain et al.,
1995; Yang, T.T. et al., 1996b). It should also be possible to use EGFP and
GFPuv in double-labeling experiments.
Relative Fluorescence
phGFP-S65T contains the Ser-65 to Thr mutation described by Heim et al.
(1995; referred to as S65T in that publication). In addition, the GFP-S65T
gene variant present in phGFP-S65T contains more than 190 silent base
changes that correspond to human codon-usage preferences. These silent
400
500
Wavelength (nm)
Figure 2. Excitation (dashed lines) and emission (solid lines) spectra of wt GFP (grey lines) and
red-shifted GFP variants (black lines). The emission data for wt GFP were obtained with excitation at
475 nm. (The emission peak for wt GFP is several-fold higher when illuminated at 395 nm). The emission
data for the red-shifted GFP were obtained by exciting GFP-S65T at 489 nm (Heim et al., 1995). The other
red-shifted GFP variants have similar spectra.
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II. Properties of GFP and GFP Variants continued
changes increase the translational efficiency and, therefore, the expression of GFP-S65T in mammalian systems (Haas et al., 1996). In side-byside comparisons, GFP-S65T fluorescence is 4–6 times more intense than
wt GFP and has a single, red-shifted excitation peak at 490 nm. GFP-S65T
also acquires fluorescence about four times faster than wt GFP (Heim et al.,
1995).
EGFP contains the double-amino-acid substitutions Phe-64 to Leu and
Ser-65 to Thr (previously published as GFPmut1; Cormack et al. , 1996).
Based on spectral analysis of equal amounts of soluble protein, GFPmut1
fluoresces 35-fold more intensely than wt GFP when excited at 488 nm
(Cormack et al., 1996; Yang, T. T. et al., 1996a), due to an increase in its
extinction coefficient (Em). The Em for EGFP has been measured at
61,000 cm-1 M-1 at 488 nm excitation (D. W. Piston, pers. comm.), compared
with 7,000 cm-1 M-1 for wt GFP, and 39,200 cm-1 M-1 for GFP-S65T (Heim et
al., 1995) under similar conditions. (Data on the rate of formation of an
active fluorophore are not yet available for EGFP.) To ensure maximal
mammalian expression, the EGFP gene also contains the human-codon
optimized silent base changes present in hGFP-S65T (Haas et al., 1996).
C. Blue Emission GFP Variants
The EBFP variant, developed at CLONTECH, contains four amino acid
substitutions. The Tyr-66 to His substitution gives EBFP fluorescence
excitation and emission maxima (380 nm and 440 nm, respectively) similar
to other blue emission variants (Figure 3; Heim & Tsien, 1996; Mitra et al,
1996; Heim et al., 1994). The other three substitutions (Phe-64 to Leu; Ser65 to Thr; and Tyr-145 to Phe; Figure 1) enhance the brightness and
solubility of the protein, primarily due to improved protein folding properties
and efficiency of chromophore formation (Heim & Tsien, 1996; Cormack et
al., 1996). In addition, the coding sequence of the EBFP gene contains the
same set of 190 silent base changes present in EGFP and, thus, is human
codon-optimized (Haas et al., 1996). The Em of EBFP is 31,000 cm–1 M–1
for 380-nm excitation, leading to a fluorescent signal that is 2–3-fold
brighter than other blue variants of GFP and roughly equivalent to wt GFP.
D. UV-Optimized GFP Variant
GFPuv is optimized for maximal fluorescence when excited by UV light
(360–400 nm) and for higher bacterial expression. The GFPuv variant is
ideal for experiments in which GFP expression will be detected using UV
light for chromophore excitation (e.g., for visualizing bacteria or yeast
colonies). GFPuv was developed at Maxigene by Crameri et al. (1996)
using an in vitro DNA shuffling technique to introduce point mutations.
GFPuv contains three amino acid substitutions (Phe-99 to Ser, Met-153 to
Thr, and Val-163 to Ala [based on the amino acid numbering of wt GFP]),
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II. Properties of GFP and GFP Variants continued
0.5
Normalized Fluorescence
0.3
0.5
0.4
0.2
0
325
525
425
Wavelength (nm)
Figure 3. Excitation (dashed lines) and emission (solid lines) spectra of wt GFP (black lines) and
EBFP (grey lines). The emission data were obtained with excitation at 390 nm.
Relative Fluorescence
300
200
100
300
400
500
600
Wavelength (nm)
Figure 4. Excitation (dashed lines) and emission (solid lines) spectra of wt GFP (black lines) and
GFPuv (grey lines). The emission data were obtained with excitation at 385 nm. (Data derived from
Crameri et al., 1996.)
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II. Properties of GFP and GFP Variants continued
none of which alter the chromophore sequence. These mutations make
E. coli expressing GFPuv fluoresce 18 times brighter than wt GFP (Crameri
et al., 1996). While these mutations dramatically increase the fluorescence
of GFPuv through their effects on protein folding and chromophore formation, the emission and excitation maxima remain at the same wavelengths
as those of wt GFP (Crameri et al., 1996; Figure 4). However, GFPuv has
a greater propensity to dimerize than wt GFP (see Section II.E.8).
GFPuv expressed in E. coli is a soluble, fluorescent protein even under
conditions in which the majority of wt GFP is expressed in a nonfluorescent
form in inclusion bodies. This GFP variant also appears to have lower
toxicity than wt GFP; hence, the E. coli containing GFPuv grow two to three
times faster than those expressing wt GFP (Crameri et al., 1996). Furthermore, the GFPuv gene is a synthetic GFP gene in which five rarely used Arg
codons from the wt gene were replaced by codons preferred in E. coli.
Consequently, the GFPuv gene is expressed very efficiently in E. coli.
E. Acquisition and Stability of GFP Fluorescence
1. Protein solubility
In bacterial expression systems, much of the expressed wt GFP is found
in an insoluble, nonfluorescent form in inclusion bodies. In contrast, almost
all EGFP and GFPuv is expressed as a soluble, fluorescent protein.
2. Photobleaching
GFP fluorescence is very stable in a fluorometer (Ward, pers. comm.).
Even under the higher intensity illumination of a fluorescence microscope, GFP is more resistant to photobleaching than is fluorescein
(Wang & Hazelrigg, 1994; Niswender et al., 1995). The fluorescence of
wt GFP, EGFP, GFP-S65T, and RSGFP is quite stable when illuminated with 450–490 nm light (the major excitation peak for the redshifted variants, but the minor peak for wt GFP). Some photobleaching
occurs when wt GFP is illuminated near its major excitation peak with
340–390 nm or 395–440 nm light (Chalfie et al., 1994; Niswender et al.,
1995). The rate of photobleaching of wt GFP and its green variants also
varies with the organism being studied; for example, GFP fluorescence
is quite stable in Drosophila (Wang & Hazelrigg, 1994) and zebrafish.
In C. elegans, 10 mM NaN 3 accelerates photobleaching (Chalfie et al.,
1994). Studies of the photobleaching characteristics of GFPuv have
not been performed to date.
Rapid photobleaching has been a problem with previously described
blue variants of GFP; however, our results with EBFP-transfected
CHO-K1 cells indicate that the rate of photobleaching in this variant is
one-half to one-third that of P4-3, a popular predecessor to EBFP which
contains only two amino acid substitutions (Tyr-66 to His and Tyr-145
to Phe; Heim & Tsien, 1996).
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II. Properties of GFP and GFP Variants continued
3. Stability to oxidation/reduction
GFP needs to be in an oxidized state to fluoresce because chromophore formation is dependent upon an oxidation of Tyr-66 (Heim et
al., 1994). Strong reducing agents, such as 5 mM Na2S 2O4 or 2 mM
FeSO4, convert GFP into a nonfluorescent form, but fluorescence is
fully recovered after exposure to atmospheric oxygen (Inouye & Tsuji,
1994b). Weaker reducing agents, such as 2% β-mercaptoethanol,
10 mM dithiothreitol (DTT), 10 mM reduced glutathione, or 10 mM
L-cysteine, do not affect the fluorescence of GFP (Inouye & Tsuji,
1994b). GFP fluorescence is not affected by moderate oxidizing agents
(Ward, pers. comm.).
4. Stability to pH
wt GFP retains fluorescence in the range pH 5.5-12; however, fluorescence intensity decreases between pH 5.5 and pH 4, and drops sharply
above pH 12 (Bokman & Ward, 1981). The EGFP and GFP-S65T
variants exhibit a reduced range of pH stability. For these variants,
fluorescence is stable between pH 7.0 and pH 11.5, drops sharply
above pH 11.5, and decreases between pH.7.0 and pH 4.5, retaining
about 50% of fluorescence at pH 6.0 (Ward, pers. com.).
5. Stability to chemical reagents
GFP fluorescence is retained in mild denaturants, such as 1% SDS or
8 M urea, and after fixation with glutaraldehyde or formaldehyde, but
fully denatured GFP is not fluorescent. GFP is very sensitive to some
nail polishes used to seal coverslips (Chalfie et al., 1994; Wang &
Hazelrigg, 1994); therefore, use molten agarose or rubber cement to
seal coverslips on microscope slides. Fluorescence is also quenched
by the nematode anesthetic phenoxypropanol (Chalfie et al., 1994).
GFP fluorescence is irreversibly destroyed by 1% H2O2 and sulfhydryl
reagents such as 1 mM DTNB (5,5'-dithio-bis-[2-nitrobenzoic acid])
(Inouye & Tsuji, 1994b). Many organic solvents can be used at
moderate concentrations without abolishing fluorescence; however,
the absorption maximum may shift (Robart & Ward, 1990).
6. Protein stability
in vitro:
GFP is exceptionally resistant to heat (T m=70°C), alkaline pH, detergents, chaotropic salts, organic solvents, and most common proteases, except pronase (Bokman & Ward, 1981; Ward, 1981; Roth,
1985; Robart & Ward, 1990). Some GFP fluorescence can be observed
when nanogram amounts of protein are resolved on native or 1% SDS
polyacrylamide gels (Inouye & Tsuji, 1994a).
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II. Properties of GFP and GFP Variants continued
Fluorescence is lost if GFP is denatured by high temperature, extremes
of pH, or guanidinium chloride, but can be partially recovered if the
protein is allowed to renature (Bokman & Ward, 1981; Ward & Bokman,
1982). A thiol compound may be necessary to renature the protein into
the fluorescent form (Surpin & Ward, 1989).
in vivo:
GFP appears to be stable when expressed in various organisms,
however, no measurement of its half-life has been reported.
7. Temperature sensitivity of GFP chromophore formation.
Although fluorescent GFP is highly thermostable (Section II.E.6; Ward,
1981), it appears that the formation of the GFP chromophore is
temperature sensitive. In yeast, GFP fluorescence was strongest when
the cells were grown at 15°C, decreasing to about 25% of this value as
the incubation temperature was raised to 37°C (Lim et al., 1995).
However, GFP and GFP-fusions synthesized in S. cerevisiae, at 23°C
retain fluorescence despite a later shift to 35°C (Lim et al., 1995). It has
also been noted that E. coli expressing GFP show stronger fluorescence when grown at 24°C or 30°C compared to 37°C (Heim et al.,
1994; Ward, pers. comm.). Mammalian cells expressing GFP have
also been seen to exhibit stronger fluorescence when grown at
30–33°C compared to 37°C (Pines, 1995; Ogawa et al., 1995).
EGFP and GFPuv contain mutations that increase the efficiency of
protein folding and chromophore formation (Cormack et al ,1996,
Crameri et al., 1996). The same mutations also suppress the
thermosensitivity of chromophore formation; thus, EGFP and GFPuv
show little difference in fluorescence when expressed at either 25°C or
37°C. One of the GFPuv mutations is also present in other GFP variants
selected for strong fluorescence at 37°C (Siemering et al, 1996).
8. Dimerization of GFP
GFP is fluorescent either as a monomer or as a dimer. The ratio of
monomeric and dimeric forms depends on the protein concentration
and environment. wt GFP dimerizes via hydrophobic interactions at
protein concentrations above 5–10 mg/ml and in high-salt conditions.
Dimerization results in a 4-fold reduction in the absorption at 470 nm
and a concomitant increase in absorption at 395 nm (Ward, pers. com.).
GFPuv has a greater propensity to dimerize than wtGFP; the 470-nm
excitation peak is suppressed at protein concentrations about 5-fold
lower than for wt GFP (Ward, pers. comm.). In most experimental
systems using wt GFP as a reporter, the monomeric form will predominate; however, when GFPuv is used as the reporter, dimerization may
be evident even at moderate expression levels.
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II. Properties of GFP and GFP Variants continued
F. Sensitivity
1. General considerations
GFP, like fluorescein, has a quantum yield of about 80% (Ward, 1981),
although the extinction coefficient for GFP is much lower. Nevertheless, in fluorescence microscopy, GFP fusion proteins have been found
to give greater sensitivity and resolution than staining with fluorescently
labeled antibodies (Wang & Hazelrigg, 1994). GFP fusions have the
advantages of being more resistant to photobleaching and of avoiding
background caused by nonspecific binding of the primary and secondary antibodies to targets other than the antigen (Wang & Hazelrigg,
1994). Although binding of multiple antibody molecules to a single
target offers a potential amplification not available for GFP, this is offset
because neither labeling of the antibody nor binding of the antibody to
the target is 100% efficient.
The mutations present in the green variants GFPuv, EGFP, and GFPS65T significantly increase the sensitivity of GFP as a reporter.
Subcellular localization of GFP can further increase the sensitivity of
GFP by concentrating the signal to a small area within the cell.
However, for some applications, the sensitivity of GFP may be limited
by autofluorescence or limited penetration of light. Studies with wt GFP
expressed in HeLa cells (Niswender et al., 1995) have shown that the
cytoplasmic concentration must be greater than ~1.0 µM to obtain a
signal that is twice the autofluorescence. This threshold for detection
is lower with the red-shifted variants: ~200 nM for GFP-S65T and
~100 nM for EGFP (Piston, pers. comm.).
The absolute sensitivity attainable with the blue variant EBFP is
approximately equivalent to that of wt GFP. However, relative to other
published blue variants (e.g., P4 and P4-3; Heim & Tsien, 1996), EBFP
is brighter due to its higher extinction coefficient. At this time, we do not
have detailed information regarding the threshold amount of intracellular EBFP protein required to obtain a detectable signal.
2. As a quantitative reporter
The signal from GFP and its variants does not have any enzymatic
amplification; hence, the sensitivity of GFP will probably be lower than
that for enzymatic reporters, such as β-galactosidase, SEAP, and
firefly luciferase. However, GFP signals can be quantified by flow
cytometry, confocal scanning laser microscopy, and fluorometric
assays. Purified GFP and GFP variants can be quantified in a fluorometer-based assay in the low-nanogram range.
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III. Living Colors GFP and GFP Variant Vectors
CLONTECH offers a variety of vectors (described below and in Table I) for
expression of GFP and its variants in many kinds of hosts. (See Section XI for a
complete listing of vectors and related products.) The vector information packet
that accompanies any purchased vector provides a vector map, multiple cloning
site (MCS) sequence, and additional information about the vector. Much of this
information is also available on the CLONTECH Web Site (http://
www.clontech.com).
Living Colors GFP and GFP variant vectors:
• pGFPuv is primarily intended as a source of the GFPuv gene. The gene can
be readily excised using restriction sites in the flanking MCS regions.
Alternatively, the GFPuv coding sequence can be amplified by PCR.
• pBAD-GFPuv expresses the UV-optimized GFP (GFPuv) in bacterial
systems from the regulated arabinose promoter.
• pEGFP is primarily intended as a source of the EGFP gene. The gene can
be readily excised using restriction sites in the flanking MCS regions.
Alternatively, the EGFP coding sequence can be amplified by PCR.
• pEGFP-1 is a transcrption reporter vector used for monitoring the activity
of promoters or promoter/enhancer combinations cloned into the MCS
upstream of the EGFP gene.
• pEGFP-N1, -N2, -N3 express a human codon-optimized, red-shifted GFP
variant and can be used for fusing heterologous proteins to the N-terminus
of EGFP.
• pEGFP-C1, -C2, -C3 express a human codon-optimized, red-shifted GFP
variant and can be used for fusing heterologous proteins to the C-terminus
of EGFP.
• pEBFP is primarily intended as a source of the EBFP gene. The gene can
be readily excised using restriction sites in the flanking MCS regions.
Alternatively, the EBFP coding sequence can be amplified by PCR.
• pEBFP-N1 expresses a human codon-optimized, blue fluorescent GFP
variant and can be used for fusing heterologous proteins to the N-terminus
of EBFP.
• pEBFP-C1 expresses a human codon-optimized, blue fluorescent GFP
variant and can be used for fusing heterologous proteins to the C-terminus
of EBFP.
• pIRES-EGFP is used for the simultaneous expression, from one RNA
transcript, of EGFP and another recombinant protein of interest in transfected mammalian cells.
• phGFP-S65T expresses a human codon-optimized, red-shifted GFP variant and can be used for fusing heterologous proteins to GFP-S65T.
• pGFP is primarily intended as a source of the wt GFP gene. The GFP gene
can be readily excised using restriction sites in the flanking MCS regions.
Alternatively, the GFP coding sequences can be amplified by PCR.
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III. Living Colors GFP and GFP Variant Vectors continued
Sequencing Primers
The GFP-N and -C Sequencing Primers can be used to sequence across
the junction of protein fusions to the N- or C-terminus of GFP, or to confirm
the identity or orientation of promoters inserted upstream of the GFP gene.
Similarly, the EGFP-N and -C Sequencing Primers can be used with EGFP,
EBFP, and hGFP-S65T.
TABLE I. LIVING COLORS VECTORS
Vector
GFP
Variant
pEGFP
EGFP
Fluorescence Excitationa
Intensity Maxima (nm)
35x
Codon
GenBank
Optimized Host Cells Accession #
488
humanb
prokaryoticd
U76561
b
pEGFP-1
EGFP
35x
488
human
mammalian d
U55761
pEGFP-N1,2,3
EGFP
35x
488
humanb
mammalian d
U55762
U57608
U57609
pEGFP-C1,2,3
EGFP
35x
488
humanb
mammalian d
U55763
U57606
U57607
pEBFP
EBFP
n.a.
380
humanb
mammalian d
380
b
mammalian d
b
pEBFP-N1, C1
phGFP-S65T
pGFPuv
pBAD-GFPuv
pGFP
pIRES-EGFP
a
b
c
d
e
EBFP
n.a.
human
GFP-S65T
4–6x
489
human
mammalian d
U43284
GFPuv
18x
395
E. coli c
prokaryotice
U62636
c
e
GFPuv
18x
395
E. coli
prokaryotic
U62637
wild-type
1x
395 (470)
none
prokaryotic
U17997
EGFP
35x
488
humanb
mammalian d
The emission maximum for EBFP is 440 nm; for wt GFP and all other green variants, the
emission maximum is 509 nm, with a shoulder at 540 nm.
The GFP insert in these vectors contains >190 silent base mutations that correspond to
human codon-usage preferences.
Five rarely used arginine codons from the wt GFP gene have been replaced by codons
preferred in E. coli.
Although optimized for mammalian expression, the GFP insert in these vectors can be cloned
into an appropriate vector for high-level expression of GFP in plants.
Although optimized for bacterial expression, the GFP insert in these vectors should be
superior to wt GFP in any expression system using UV light for chromophore excitation.
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CLONTECH Laboratories, Inc.
IV. Expression of GFP and GFP Variants
A. Suitable Host Organisms and Cells
As mentioned in the Introduction (Section I), GFP fluorescence is speciesindependent. Indeed, fluorescence has been reported from many different
types of GFP-expressing hosts, including the following:
1. Microbes
Agrobacterium tumefaciens (Matthysse et al., 1996)
Anabaena (Buikema, 1995)
Candida albicans (Cormack et al., 1997)
Dictyostelium (Hodgkinson, 1995; Maniak et al., 1995; Moores et al.,
1996; Fey et al., 1995)
Escherichia coli (Chalfie et al., 1994; Inouye & Tsuji, 1994a; Burlage
et al., 1995; Kitts et al., 1995; Yu & van den Engh, 1995; Crameri
et al., 1996)
Mycobacterium (Kremer et al., 1995)
Polysphondylium pallidum (Fey et al., 1995)
Pseudomonas putida (Matthysse et al., 1996)
Rhizobium meliloti (Gage et al ., 1996)
Saccharomyces cerevisiae (Flach et al., 1994; Kahana et al., 1995;
Lim et al., 1995; Riles et al., 1995; Schlenstedt et al., 1995;
Stearns, 1995; Halme et al., 1996)
Salmonella (Valdivia & Falco, 1996 )
Schizosaccharomyces pombe (Nabeshima et al., 1995; Atkins &
Izant, 1995)
Ustilago maydis (Spellig et al ., 1996)
Yersinia (Jacobi et al., 1995)
2. Invertebrates
Aedes aegypti (Higgs et al., 1996)
Caenorhabditis elegans (Chalfie et al., 1994; Sengupta et al., 1994;
Treinin & Chalfie, 1995)
Diamondback moth (Chao et al., 1996)
Drosophila melanogaster (Wang & Hazelrigg, 1994; Barthmaier &
Fyrberg, 1995; Brand, 1995; Yeh et al., 1995)
Lytechinus pictus (Seid et al ., 1996)
3. Vertebrates
293 cells (transformed primary embryonal kidney, human) (Marshall et
al., 1995)
A-375 cells (malignant melanoma, human) (Levy et al., 1996)
BHK cells (kidney, hamster) (Olson et al., 1995; Carey et al., 1996)
BOSC23 cells (Cheng et al., 1996)
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Version # PR74631
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IV. Expression of GFP and GFP Variants continued
Chicken embryonic retina cells (Ogawa et al., 1995)
CHO-K1 cells (ovary, Chinese hamster) (Kain et al., 1995; Kitts et al.,
1995; Olson et al., 1995; Yang, T.T. et al., 1996b; Crameri et al.,
1996)
COS-1 cells (kidney, SV40 transformed, African green monkey)
(Ogawa et al., 1995)
COS-7 cells (kidney, SV40 transformed, African green monkey)
(Olson et al., 1995; Pines, 1995; Cheng et al., 1996; Haas et al.,
1996)
EPC cells (epithelial, carp) (Ogawa et al., 1995)
Ferret neurons (Lo et al., 1994)
GH3 cells (pituitary tumor, rat) (Plautz et al., 1996)
HeLa cells (epitheloid carcinoma, cervix, human) (Kaether & Gerdes,
1995; Pines, 1995; Niswender et al., 1995)
Hepatocytes (liver, rat) (Venerando et al., 1996)
JEG cells (placenta, human) (Yu & Dong, 1995)
NIH/3T3 cells (embryo, contact-inhibited, NIH Swiss mouse) (Olson et
al., 1995; Pines, 1995)
PA317 (embryo fibroblast, mouse) (Levy et al., 1996)
PC-12 cells (adrenal pheochromocytoma, rat) (pers. comm)
Pt K1 cells (kidney, kangaroo rat) (Olson et al., 1995)
Mice (Ikawa et al., 1995a; Ikawa et al., 1995b)
Xenopus (Tannahill et al., 1995; Wu et al., 1995)
Zebrafish (Amsterdam et al., 1995)
4. Plants
Arabidopsis thaliana (thale cress) (stable) (Haselhoff & Amos, 1995;
Chiu et al., 1996; Reichel et al., 1996)
Citrus sinensis (L.) Osbeck cv. Hamlin (an embryogenic sweet-orange
cell line, H89) (Niedz et al., 1995)
Glycine max (soybean) (Plautz et al., 1996)
Hordeum vulgar (barley) (Törmäkangas et al., 1995; Reichel et al., 1996)
Nicotiana benthamiana & Nicotiana clevelandii (tobacco) (Baulcombe
et al., 1995)
Onion (Chiu et al., 1996)
Tobacco (Beachy, 1995; Galbraith et al., 1995; Törmäkangas et al., 1995;
Heinlein et al., 1995; Oparka et al., 1995; Chiu et al., 1996; Reichel et al.,
1996)
Zea mays (maize) (Galbraith et al., 1995; Hu & Cheng, 1995; Sheen
et al., 1995; Chiu et al., 1996; Reichel et al., 1996)
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CLONTECH Laboratories, Inc.
IV. Expression of GFP and GFP Variants continued
B. Expression of GFP Fusion Proteins
GFP has been expressed as fusions to many proteins (Table II). In many
cases, chimeric genes encoding either N- or C-terminal fusions to GFP
retain the normal biological activity of the heterologous partner, as well as
maintaining fluorescent properties similar to native GFP (Flach et al., 1994;
Wang & Hazelrigg, 1994; Marshall et al., 1995; Stearns, 1995). The use of
GFP and its variants in this capacity provides a “fluorescent tag” on the
protein, which allows for in vivo localization of the fusion protein. GFP
fusions can provide enhanced sensitivity and resolution in comparison to
standard antibody staining techniques (Wang & Hazelrigg, 1994), and the
GFP tag eliminates the need for fixation, cell permeabilization, and antibody incubation steps normally required when using antibodies tagged with
chemical fluorophores. Lastly, use of the GFP tag permits kinetic studies of
protein localization and trafficking (Flach et al., 1994; Wang & Hazelrigg,
1994). It is also possible to generate recombinant proteins fused to a blue
variant of GFP (such as P4-3; Rizzuto et al., 1996). CLONTECH offers
several vectors designed to generate either C- or N-terminal fusions with
EGFP (in all three reading frames), and C- and N-terminal fusion vectors
for EBFP (Table I and Section III).
TABLE II. PROTEINS EXPRESSED AS FUSIONS TO GFP
Protein
Host Cell or
Organism
CotE
DacF, SpoIVA
Coronin
Myosin
YopE cytotoxin
Histone H2B
Bacillus subtilis
Bacillus subtilis
D. discoideum
D. discoideum
Yersinia
Yeast
Nucleus
Tubulin
Nuf2p
Mitochondrial matrix
targeting signal
Npl3p
Yeast
Yeast
Yeast
Microtubules
Spindle-pole body
Mitochondria
Yeast
Nucleus
Corbett et al., 1995
Nucleoplasmin
Yeast
Nucleus
Lim et al., 1995
Cap2p
Yeast
Waddle et al., 1996
Swi6p
Yeast
Sidorva et al., 1995
HMG-R
Yeast
Hampton et al., 1996
Bud10p
Yeast
Halme et al., 1996
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18
Protocol # PT2040-1
Version # PR74631
Localization
References
Forespore
Prespore/mother cell
Phagocytic cups
Webb et al., 1995
Lewis & Errington, 1996
Maniak et al., 1995
Moores et al., 1996
Jacobi et al., 1995
Flach et al., 1994;
Schlenstedt et al., 1995
Stearns, 1995
Silver, 1995; Kahana et al., 1995
Cox, 1995
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IV. Expression of GFP and GFP Variants continued
TABLE II. PROTEINS EXPRESSED AS FUSIONS TO GFP continued
Protein
Host Cell or
Organism
Pmp47
Yeast
Act1p, Sac6p, Abp1p Yeast
p93dis1
Fission yeast
Exu
Mei-S332
Tau
β-galactosidase
Streptavidin
ObTMV movement
protein
Potato virus X
coat protein
GST
11β-HSD2
MAP4
Mitochondrial
targeting signal
Cyclins
Drosophila
Drosophila
Drosophila
Drosophila
Localization
References
Peroxisome
Cytoskeleton
Spindle-pole body &
microtubules
Oocytes
Centromere
Microtubules
cell movement
Insect cells
Dyer et al., 1996
Doyle et al., 1996
Nabeshima et al., 1995
Wang & Hazelrigg, 1994
Kerrebrock et al., 1995
Brand, 1995
Shiga et al., 1996
Oker-Blom et al., 1996
Tobacco
Filaments
Beachy, 1995;
Heinlein et al., 1995
Potato plant
Zebrafish
Mammalian cells
Mammalian cells
Viral infection
Cytoplasmic(?)
Endoplasmic reticulum
Microtubules
Santa Cruz et al., 1996
Peters et al., 1995
Náray-Fejes-Tóth et al., 1996
Olson et al., 1995
Mammalian cells
Mammalian cells
Mitochondria
Nucleus, microtubules, or vesicles
Rizzuto et al., 1995
Pines, 1995
PML
Mammalian cells
Ogawa et al., 1995
Human glucocorticoid receptor
Mammalian cells
Ogawa et al., 1995
Carey et al., 1996
Rat glucocorticoid
receptor
Mammalian cells
Rizzuto et al., 1995
Htun et al., 1996
Chromogranin B
HeLa cells
Secreted
Kaether et al., 1995
HIV p17 protein
HeLa cells
Nucleus
Bian et al., 1995
NMDAR1
HEK293 cells
Membrane
Marshall et al., 1995
CENP-B
Human cells
Nucleus
Sullivan et al., 1995
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CLONTECH Laboratories, Inc.
IV. Expression of GFP and GFP Variants continued
C.
GFP Expression in Mammalian Cells
Appropriate vectors may be transfected into mammalian cells by a variety
of techniques, including those using calcium phosphate (Chen & Okayama,
1988), DEAE-dextran (Rosenthal, 1987), various liposome-based transfection reagents (Sambrook et al. , 1989), and electroporation (Ausubel et
al., 1994). Any calcium phosphate transfection procedure may be used, but
we recommend using the CalPhos MaximizerTM Transfection Kit (#K20501) for the highest calcium phosphate-mediated transfection efficiencies
with an incubation of only 2–3 hours. Likewise, any liposome-mediated
transfection procedure may be used, but we recommend using CLONfectinTM
(#8020-1) for high, reproducible transfection efficiencies in a variety of cell
types. If you purchase CalPhos Maximizer or CLONfectin, follow the
transfection guidelines provided with those products. For further information on cell culture techniques, we recommend Culture of Animal Cells,
Third Edition, R. I. Freshney (1993, Wiley-Liss; available from CLONTECH
Press, #V2128-1).
The efficiency of a mammalian transfection procedure is primarily dependent on the host cell line. Different cell lines may vary by several orders of
magnitude in their ability to take up and express exogenous DNA. Moreover, a method that works well for one host cell line may be inferior for
another. Therefore, when working with a cell line for the first time, it is
advisable to compare the efficiencies of several transfection protocols. This
can best be accomplished using one of the GFP vectors which has the CMV
immediate early promoter for high-level expression in most cell lines. The
promoterless pEGFP-1 or pGFP-1 vector may be used to determine the
background autofluorescence in the host cell line. After a method of
transfection is chosen, it must be optimized for parameters such as cell
density, the amount and purity of DNA, media conditions, transfection time,
and the posttransfection interval required for GFP detection. Once optimized, these parameters should be kept constant to obtain reproducible
results.
After a calcium phosphate-based or liposome-mediated transfection procedure, wait 24–72 hr before you assay for transient gene expression or
start selection for stable transformants. In most cases, GFP expression
may be detected by fluorescence microscopy, FACS analysis, or fluorometer assays 24–72 hours after transfection, depending on the host cell
line used. If you used electroporation, wait until 48 hr posttransfection to
assay or begin selection. For visualizing GFP-expressing cells by fluorescence microscopy, grow the cells on a sterile glass coverslip placed in a
60-mm culture plate. Alternatively, an inverted fluorescence microscope
may be used for direct observation of fluorescing cells in the culture plate.
We are aware of one report of a stable mammalian cell line expressing GFP
(Olson et al., 1995).
page
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IV. Expression of GFP and GFP Variants continued
D. GFP Expression in Plants
TABLE III. USE OF GFP IN PLANTS
Species (culture)
Method
Reference
Arabidopsis thaliana
(thale cress roots)
Agrobacterium (stable)
Particle bombardment
Haseloff & Amos, 1995
Chiu et al., 1996
Citrus sinensis (L.) Osbeck cv. Hamlin
(embryogenic suspension culture
sweet-orange cells H89)
Electroporation
Niedz et al., 1995
Glycine max
(soybean suspension culture)
Particle bombardment
Plautz et al., 1996
Hordeum vulgar
(barley)
Particle bombardment
Törmäkangas et al., 1995
Nicotiana benthamiana
(tobacco plant leaves)
PVX infection
Baulcombe et al., 1995
Nicotiana clevelandii
(tobacco plant leaves)
PVX infection
Baulcombe et al., 1995
Onion epidermal cells
Particle bombardment
Chiu et al., 1996
family Solanaceae
PVX infection
Santa Cruz et al., 1996
Tobacco
Particle bombardment
Törmäkangas et al., 1995;
Sheen et al., 1995
Galbraith et al., 1995;
Chiu et al., 1996
Beachy, 1995;
Heinlein et al., 1995
Electroporation or PEG
ObTMV infection
Zea mays
(maize protoplast suspension culture)
Electroporation or PEG
Electroporation
Hu & Cheng, 1995;
Galbraith et al., 1995;
Chiu et al., 1996
GFP has been expressed in several plant species by a variety of methods
(see Table III). GFP allows plant scientists to study transformed cells and
tissues in vivo, without sacrificing vital cultures. The wt GFP coding
sequences contain a region recognized in Arabidopsis as a cryptic plant
intron (bases 400 and 483). The intron is efficiently spliced out in Arabidopsis
resulting in a nonfunctional protein (Haseloff & Amos, 1995). Functional
wt GFP has been transiently expressed in several other plant species
indicating that the cryptic intron may not be recognized or is recognized less
efficiently in other plant species. A modified version of wt GFP, in which the
cryptic sequences have been altered, has been used for GFP expression
in Arabidopsis (Haseloff & Amos, 1995). The cryptic intron has also been
removed by the changes to codon usage in hGFP-S65T and EGFP.The
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IV. Expression of GFP and GFP Variants continued
same GFP gene as that contained in the phGFP-S65T Vector has been
successfully used to express GFP in many plant systems using a variety of
transformation techniques (Chiu et al., 1996). EGFP contains only one
additional amino acid mutation (Phe-64 to Leu), but is ~7-fold brighter than
GFP-S65T; therefore, EGFP should also be a useful reporter in plant
expression systems.
E.
GFP Expression in Yeast
wt GFP and GFPuv both express well in yeast. However, for expression in
yeast, the GFP or GFPuv gene contained in CLONTECH's vectors must
first be isolated (by PCR or cut out of the plasmid) and inserted into an
appropriate yeast expression vector by any of several standard methods
(Sambrook et al., 1989). Note that GFP genes with human codon usage
(i.e., hGFP-S65T, EGFP, and EBFP) are not efficiently expressed in yeast
(Cormack et al., 1997). Furthermore, S. cerevisiae strains carrying the
ade2 mitochondrial mutation accumulate a red pigment, which gives the
cells an autofluorescence that interferes with detection of GFP (Smirnov et
al., 1967; Weisman et al., 1987).
For additional information on yeast growth and maintenance, preparation
of yeast competent cells, and transformation of yeast cells, please call our
Literature Request Line at 800–662–2566(CLON) or 415–424–8222,
extension 2, or contact your local distributor, and request a copy of the
CLONTECH Yeast Protocols Handbook (PT3024-1). Additionally, we
recommend Guide to Yeast Genetics and Molecular Biology, C. Guthrie
and G. R. Fink eds. (Vol. 194 in Methods in Enzymology, 1991, Academic
Press; available from CLONTECH Press, #V2010-1).
page
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Protocol # PT2040-1
Version # PR74631
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V. Detection of GFP and GFP Variants
A.
Filter Sets
wt GFP absorbs UV and blue light with a maximum absorbance at 395 nm
and a minor peak of absorbance at 470 nm. The filter sets commonly used
with fluorescein isothiocyanate (FITC) and GFP illuminate at
450–500 nm—well above the major excitation peak of wt GFP. These filter
sets therefore produce a fluorescent signal from wt GFP that is several-fold
less than would be obtained with filter sets that excite at 395 nm. However,
use of the major peak at 395 nm for excitation of the wt GFP is not advisable
due to rapid photoisomerization and loss of the fluorescent signal. For
systems in which standard UV light is used for the detection of fluorescence, GFPuv should be used.
In contrast to wt GFP, EGFP has a single, strong, red-shifted excitation
peak at 488 nm, making it well suited for detection in commonly used
equipment which utilize FITC optics. Maximal emission is achieved when
EGFP is excited at 488 nm, which corresponds perfectly to the 488 nm line
of argon ion lasers used in many fluorescence-activated cell sorting (FACS)
machines and confocal scanning laser microscopes. In fact, this variant
was selected specifically on the basis of its increased fluorescence in FACS
assays. Filter sets commonly used in fluorescence microscopy or fluorometry illuminate at 450–500 nm; therefore, EGFP is ideal for use in these
systems as well. The major excitation peak of both GFP-S65T and RSGFP
also encompasses the excitation wavelength commonly used for FACS
analysis, confocal scanning laser microscopes, fluorometry, and fluorescence microscopy. In practical terms, this means the detection limits in both
microscopy and FACS should be considerably lower with EGFP, GFPS65T, or RSGFP. Also, living cells and animals tolerate longer wavelength
light better due to the lower energies; therefore, the fluorescent signal
obtained with illumination of these variants at ~488 nm is more stable and
less toxic than the fluorescence obtained with wt GFP excited at 395 nm.
Chroma Technology Corporation (Tel: 802-257-1800; Fax: 802-257-9400;
72 Cotton Mill Hill, Unit A-9, Brattleboro, VT 05301) has developed several
filter sets designed for use with GFP; they claim the High Q FITC filter set
(#41001) produces the best signal-to-noise ratio for visual work, and the
High Q GFP set (#41014) produces the strongest absolute signal, but with
some background. We have also used a Zeiss filter set (#487909) with a
450–490 nm band-pass excitation filter, 510 nm dichroic reflector and
520–750 nm long-pass emission filter, and the Chroma filter set #31001.
Other filter sets may give better performance, and it is necessary to match
the filter set to the application.
For detection of EBFP, we recommend a filter set with a 380±15-nm
bandpass excitation filter, a 420-nm long-pass dichroic reflector, and a
460±25-nm bandpass filter. DAPI filter sets are not recommended because
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V. Detection of GFP and GFP Variants continued
they have a broad excitation window that lets through shorter wavelength
UV, which results in very rapid photobleaching of EBFP. Again, Chroma
has developed specific sets for blue variants of GFP. Sets used for P4 or
P4-3 (Heim & Tsien, 1996) are suitable for EBFP.
B. Flow Cytometry
Several investigators have sorted GFP-expressing cells by flow cytometry
(Cheng & Kain, 1995; Ropp et al., 1995; Yu & van den Engh, 1995). In
particular, good results have been obtained in FACS analysis with plant
protoplasts, yeast, E. coli , mammalian cells, and mammalian cells infected
with bacteria expressing GFP (Atkins & Izant, 1995; Fey et al., 1995; Sheen
et al., 1995; Cheng et al., 1996). Note that flow cytometry of EBFPexpressing cells requires a light source such as a krypton laser that can
provide excitation in the UV range (Ropp et al., 1996).
C. Fluorescence Microscopy
1. Materials required
• 95% Ethanol
• Phosphate buffered saline (PBS; pH 7.4)
Final Conc.
To prepare 2 L of solution
Na2HPO4
58 mM
16.5 g
NaH2PO4
17 mM
4.1 g
NaCl
68 mM
8.0 g
Dissolve components in 1.8 L of deionized H2O. Adjust to pH 7.4 with
0.1 N NaOH. Add H2O to a final volume of 2 L. Store at room temperature.
• PBS/4% paraformaldehyde (pH 7.4–7.6)
Add 4 g of paraformaldehyde to 100 ml of PBS. Heat to dissolve.
Store at 4°C.
• Rubber cement or molten agarose
2. Fluorescence microscopy procedure
In a tissue culture hood:
a. Sterilize glass coverslip:
i. Place coverslip into 95% ethanol. Shake excess ethanol from
coverslip until it is nearly dry.
ii. Flame-sterilize coverslip and allow to completely air-dry in
sterile tissue-culture dish.
b. Plate and transfect cells in tissue-culture dish containing coverslip(s).
c. At the end of culture period, remove the tissue culture media and
wash cells 3 times with PBS.
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V. Detection of GFP and GFP Variants continued
d. For fixed cells:
i. After cells have been washed with PBS, add 2.0 ml of freshly
made PBS/4% paraformaldehyde directly on coverslip.
ii. Incubate cells in solution at room temperature for 30 min.
iii. Wash cells twice with PBS.
iv. Proceed with steps for unfixed cells.
e. For unfixed cells:
i. Carefully remove the coverslip from the plate with forceps. (Pay
close attention to which side of the coverslip contains the cells.)
ii. Mount the coverslip onto a glass microscope slide.
a) Place a tiny drop of PBS on the slide, and allow the coverslip
to slowly contact the solution and lie down on the slide.
b) Carefully aspirate the excess solution around the edge of the
coverslip using a Pasteur pipette connected to a vacuum pump.
c) Attach coverslip to the microscope slides with either molten
agarose or rubber cement.
Note: Do not use nail polish, as this reagent inhibits GFP fluorescence.
d) Allow to dry for 30 min.
iii. Immediately examine slides by fluorescence microscopy, or
store them at 4°C for up to 2–3 weeks .
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CLONTECH Laboratories, Inc.
V. Detection of GFP and GFP Variants continued
D. Quantitative Fluorometric Assay
GFP can also be used in fluorometric assays to confirm and measure GFP
fluorescence. Furthermore, using known amounts of purified recombinant
GFP or GFP variant (rEGFP, rGFP-S65T, rGFPuv, or rGFP), fluorometric
assays can be used to quantify the expression of GFP or GFP variant. After
generating a standard curve using known amounts of recombinant protein,
the fluorescence intensity of experimental samples can be measured and
compared to the standard curve to determine the amount of GFP expressed
in the sample. A detailed method for quantitation of wt GFP levels using the
TD-700 fluorometer is available from Turner Designs, Inc. (408-749-0994;
http://www.turnerdesigns.com). When generating the standard curve, the
recombinant protein should be diluted in the same buffer as the experimental samples. In some expression systems, GFP can be detected directly in
the fluorometer; in other cases, cells will need to be disrupted in order to
optimally detect GFP fluorescence. Figure 5 shows a comparison of the
fluorescence intensity of GFP detected in sonication buffer and in a clarified
bacterial cell lysate. These results illustrate that the sensitivity and the
linear range of detection of GFP are relatively unaffected by the cell lysate
background.
GFP in 6.25 µg JM109 lysate
4
GFP in S.B.
Log RFU
3
2
1
0
0
1
2
3
4
Log GFP (ng)
Figure 5. Comparison of fluorescence intensity of GFP in sonication buffer (S.B.) versus GFP
in cell lysates. LB was inoculated with a culture of untransformed JM109 cells and incubated
overnight at 30°C. Cell lysates were prepared as described in the protocol. Two-fold serial dilutions
of rGFP ranging from 2.5 µg to 9.5 ng were prepared in both sonication buffer and JM109 cell lysate.
Fluorescence intensity was measured in a modified Hoefer Pharmacia Biotech, Inc. DyNA QuantTM
200 Fluorometer using an excitation filter of 365 nm and an emission filter of 510 nm. Similar results
can also be obtained using rGFP-S65T. (RFU = relative fluorescence units.)
page
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V. Detection of GFP and GFP Variants continued
The following procedures have been developed using CLONTECH’s
Recombinant GFP Protein (rGFP; see Section VI; #8360-1, -2). Use an
excitation filter of 365 nm and an emission filter of 510 nm when assaying
wt GFP or GFPuv, and an excitation filter of 450–490 nm and an emission
filter of 510 nm when assaying EGFP and GFP-S65T.
Note: “rGFP” refers to recombinant wt GFP or one of the recombinant GFP variants.
1. Materials required
• Sonication buffer (pH 8.0)
50 mM NaH2PO4
10 mM Tris-HCl (pH 8.0)
200 mM NaCl
Adjust pH to 8.0. Store at room temperature.
• PBS (pH 7.4; see Section V.C.1)
2. Generating a GFP standard curve:
a. Prepare serial dilutions of rGFP in sonication buffer, deionized H2O,
or 10 mM Tris-HCl (pH 8.0).
Note: Samples can be stored at –20°C.
b. Assay dilutions in a fluorometer.
c. Plot the log of the relative fluorescence units (RFU) as a function of
the log of fold-dilution of each sample such as the standard curve
shown in Figure 6.
3. Measuring fluorescence intensity of bacterial samples:
a. Pellet 20 ml of bacterial cell culture.
b. Remove the supernatant and freeze pellet at –70°C.
c. Wash pellet once with 1.6 ml of PBS.
d. Resuspend pellet in 1.6 ml of sonication buffer.
e. Prepare 2-fold serial dilutions in sonication buffer.
f. Assay dilutions in a fluorometer.
g. Compare results with those of the standard curve to determine the
amount of GFP protein expressed in the experimental samples.
4. Preparation of cell lysates:
a. Pellet 100 ml of bacterial cell culture and remove the supernatant.
Note: Cell pellets may be stored at –70°C for later use.
b.
c.
d.
e.
Wash pellet twice with 4 ml of PBS.
Resuspend pellet in 4 ml of sonication buffer.
Freeze/thaw sample in a dry ice/ethanol bath five times.
Vortex sample for 1 min, and then incubate on ice for 1 min.
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V. Detection of GFP and GFP Variants continued
f. Repeat Step e two more times.
g. Run sample through an 18-gauge needle several times.
h. Transfer cell lysate to a 1.5-ml microcentrigue tube and centrifuge
at 12,000 rpm for 5 min at 4°C.
i. Collect the supernatant and determine the protein concentration
using a standard assay (e. g., Bradford, Lowry, etc.; Scopes, 1987).
j. Assay the dilutions in a fluorometer.
k. Compare the results with the standard curve to determine the
amount of GFP expressed in the experimental samples.
Log RFU
2
1
0
0
1
2
Log Fold Dilution
Figure 6. Relative fluorescence intensity of two-fold serial dilutions of a suspension of GFPtransformed cells in sonication buffer. LB was inoculated with JM109 cells transformed with a
plasmid encoding wt GFP and incubated overnight at 30°C. Fluorescence intensity was measured
by reading 5-µl samples of 2-fold dilutions in a modified Hoefer Pharmacia Biotech, Inc. DyNA Quant
200 Fluorometer using an excitation filter of 365 nm and an emission filter of 510 nm.
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VI. Purified Recombinant GFP and GFP Variants
A. General Information
CLONTECH’s Recombinant Green Fluorescent Protein (rGFP; #8360-1),
Recombinant GFP-S65T (rGFP-S65T; #8364-1), Recombinant EGFP
(rEGFP; #8365-1), and Recombinant GFPuv (rGFPuv; #8366-1) are purified from transformed E. coli using a method which ensures optimal purity
of the recombinant protein and maintenance of GFP fluorescence. All
proteins are 27-kDa monomers with 239 amino acids. The excitation and
fluorescence emission spectra for the rGFP is identical to GFP purified from
Aequorea victoria (Chalfie et al., 1994). The purified proteins retain their
fluorescence capability under many harsh conditions (Section II.E) and are
suitable as control reagents for GFP expression studies using the Living
Colors line of GFP reporter vectors.
Applications of purified rGFP and rGFP variant proteins include use as
standards for SDS or two-dimensional PAGE, isoelectric focusing, Western blot analysis, calibration of fluorometers and FACS machines, fluorescence microscopy, and microinjection of GFP into cells and tissues.
B. rGFP and rGFP Variants as Standards in Western Blots
When used as a standard for Western blotting applications in conjunction
with GFP Monoclonal Antibody (#8362-1) or GFP Polyclonal Antibody (IgG
Fraction; #8363-1), the recombinant proteins can be used to correlate GFP
expression levels to fluorescence intensity or to differentiate problems with
detection of GFP fluorescence from expression of GFP protein.
Use a standard procedure for discontinuous polyacrylamide gel electrophoresis (PAGE) to resolve the proteins on a one-dimensional gel (Laemmli,
1970). If another electrophoresis system is employed, the sample preparation should be modified accordingly. Just prior to use, allow rGFP protein
to thaw at room temperature, mix gently until solution is clear, and then
place tube on ice.
On a minigel apparatus, 25–75 µg of lysate protein per lane is typically
needed for satisfactory separation (i.e., discrete banding throughout the
molecular weight range) of a protein mixture derived from a whole cell or
tissue homogenate. If rGFP is to be used as an internal standard in a
Coomassie blue-stained minigel, we recommend loading 500 ng of rGFP
per lane. If rGFP is added to a total cell/tissue lysate or other crude sample,
the amount of total protein loaded per lane must be optimized for the
particular application.
For Western blotting applications, we recommend loading 40–400 pg of
rGFP (or rGFP variant) per lane for a strong positive signal using
CLONTECH’s GFP Monoclonal or Polyclonal Antibody in conjunction with
a chemiluminescent detection system (Kain et al., 1994). Typically, the
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VI. Purified Recombinant GFP and GFP Variants continued
GFP Monoclonal and Polyclonal Antibodies can be used at 1:500 and
1:1000 dilutions, respectively. Please refer to Section VII and the Product
Analysis Certificate (PAC) provided with each antibody for further information on using CLONTECH's GFP Antibodies.
Some GFP fluorescence may be observed when the protein is resolved on
an SDS-PAGE gel if nanogram quantities of rGFP are present. Generally,
rGFP will not fluoresce on a Western blot.
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VI. Purified Recombinant GFP and GFP Variants continued
C. rGFP and rGFP Variants as a Control for Fluorescence Microscopy
The following two protocols are for use of rGFP or rGFP variants as a control
on microscope slides in fluorescence microscopy. The purified proteins
may be used to optimize lamp and filter set conditions for detection of GFP
fluorescence, or as a qualitative means to correlate GFP fluorescence with
the amount of protein in transfected cells.
1. Unfixed samples
Use this method for live cell fluorescence or other cases where a
fixation step is not desired.
a. Perform 1:10 serial dilutions of the 1.0 mg/ml rGFP or rGFP variant
stock solution with 10 mM Tris-HCl (pH 8.0) to yield concentrations
of 0.1 mg/ml and 0.01 mg/ml.
Notes:
• These dilutions should suffice as a positive control. The 1.0 mg/ml solution will
give a very bright fluorescent signal by microscopy.
• The diluted samples can be aliquoted and stored frozen at –70°C for up to
1 yr with no loss of fluorescence intensity. Avoid repeated freeze-thaw cycles
with the same aliquot.
b. Using a micropipette, spot 1–2 µl of diluted protein onto the
microscope slide. If using a slide that contains a mounted coverslip,
position the spot several millimeters away from the sample such
that a second coverslip can be added over the protein spot.
c. Allow the protein to air-dry for a few seconds, and mark the position
of the spot on the other side of the slide to aid in focusing.
d. Add a coverslip over the spot using a 90% glycerol solution in
100 mM Tris-HCl (pH 7.5).
e. Fluorescence from the spot is best viewed at low magnification,
using either a 10X or 20X objective.
2. Fixed samples
In some cases it may be necessary to fix the recombinant protein to the
microscope slide prior to microscopy. This can be done by dipping the
section of the microscope slide containing the air-dried protein spot
(after Step 1.c above) into 100% methanol for 1 min. Allow the slide to
dry completely and place a coverslip over the sample as in Step 1.d
above.
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VII. GFP Antibodies
A. Applications for GFP Antibodies
CLONTECH offers two antibodies, GFP Monoclonal Antibody (#8362-1)
and GFP Polyclonal Antibody (IgG Fraction; #8363-1, -2). Both antibodies
specifically recognize wt GFP, each of the red-shifted GFP variants, EBFP,
and GFPuv. GFP Monoclonal Antibody is purified from the serum-free
media of mouse hybridoma cultures. Since the GFP Monoclonal Antibody
only recognizes a single epitope of GFP, the signals are highly specific and
have minimal background. GFP Polyclonal Antibody is a purified fraction of
an Anti-rGFP Antiserum that has been purified by passage through a
protein G column to remove nonspecific immunoglobulins from the serum.
Both antibodies can be used to confirm GFP protein expression by Western
blot analysis and to correlate levels of GFP protein expression with
fluorescence intensity (detected with microscopy, flow cytometry, or fluorometry). Typically, the GFP Monoclonal and Polyclonal Antibodies can be
used at 1:500 and 1:1000 dilutions, respectively, for Western blotting
applications. Please refer to the Product Analysis Certificate (PAC) enclosed with each antibody for recommended dilutions. Avoid repeatedly
freezing and thawing the GFP Antibodies.
In addition to detection of GFP and its variants on Western blots, the GFP
antibodies can be used to immunoprecipitate GFP-fusion proteins (Silver,
pers. comm.) and for in situ detection of GFP (Lauderdale, pers. comm.).
An immunoprecipitation procedure, modified for use with GFP Monoclonal
Antibody, is provided below. This procedure has been optimized using
mammalian cells expressing GFP, but should work with lysates prepared
from any cell type. However, if you are using a bacterial or yeast host, you
will need to perform additional steps to break down the cell wall.
B. Immunoprecipitation with GFP Monoclonal Antibody
1. Solutions Required
• Lysis buffer (100 mM Potassium Phosphate [pH 7.8] + 0.2% Triton
X-100) To make 10 ml of Lysis Buffer, mix together:
9.15 ml of 100 mM
K 2HPO4
0.85 ml of 100 mM
KH2PO4
20 µl of Triton X-100 (0.2% final)
If necessary, adjust pH to 7.8. Store buffer at room temperature.
Just prior to use, add 10 µl of a 1 M stock solution of DTT (1 mM final
concentration) and an appropriate protease inhibitor or combination of inhibitors, such as the Protease Inhibitor cocktail from
Boehringer Mannheim (#1697 498).
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VII. GFP Antibodies continued
• 1.0 M Dithiothreitol
Add 1.54 g of dithiothreitol to 8 ml of deionized H2O and mix
gently until dissolved. Adjust final volume to 10 ml with deionized
H2O. Aliquot into microcentrifuge tubes and freeze at –20°C.
• GFP Monoclonal Antibody, diluted to 1 mg/ml in PBS (pH ~7).
• Protein A Sepharose beads,
Spin down beads to remove the ethanol; then wash beads once
in PBS and resuspend them at 1 mg/ml in PBS.
• PBS (see Section IV.C.1 for recipe)
2. Procedure
a. Spin down ~ 1 x 106 cells and resuspend the cell pellet in 20 ml of
cold (4°C) Lysis Buffer containing 1 mM DTT and protease inhibitors.
b. Place cells on ice for 45 min.
c. Clear the lysate by pelleting the cells at 12,000 x g for 10 min at 4°C.
d. Transfer 500 µl of cleared lysate to a 1.5-ml microcentrifuge tube.
e. Add the diluted mAb to a final concentration of 2–10 µg/ml.
f. Place the tube at 4°C for 2 hr with continuous gentle inversion.
g. Add 40 µl of Protein A Sepharose beads to the antibody-antigen
mixture in a 1:1 suspension in PBS.
h. Vortex and incubate at 4°C for 1 hr with continuous gentle inversion.
i. Centrifuge at 10,000 x g for 10 min at 4°C.
3. Analysis of sample
a. Wash the beads with 1 ml of Lysis Buffer.
b. Repeat this wash procedure twice.
c. Add 50 µl of 1X SDS-PAGE sample buffer to beads.
d. Vortex, then boil for 10 min.
e. Centrifuge at 12,000 x g for 5 min at room temperature.
f. Electrophorese the supernatant on a polyacrylamide gel and analyze the resolved proteins on a Western blot.
Notes:
• Load 10–20 µl of supernatant per well (mini-gel)
• We recommend the Western ExposureTM Chemiluminescent Detection System
(#K2030-1, #K2031-1) for detection of positive signals.
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VIII. Troubleshooting Guide
A. Potential Difficulties in Using GFP Fluorescence
• Variability in the intensity of GFP fluorescence has been noted. This
may be due in part to the relatively slow formation of the GFP
chromophore and the requirement for molecular oxygen (Heim et al.,
1994).
• The slow rate of chromophore formation and the apparent stability of
wt GFP may preclude the use of GFP as a reporter to monitor fast
changes in promoter activity (Heim et al., 1994, Davis et al., 1995). This
limitation is reduced by use of EGFP or GFP-S65T, which acquire
fluorescence faster than wt GFP (Heim et al., 1994).
• The wt GFP coding sequences contain a cryptic plant intron (between
bases 400 and 483) that is efficiently spliced out in Arabidopsis and
results in a nonfunctional protein (Haseloff & Amos, 1995). Functional
wt GFP has been transiently expressed in several other plant species
indicating that the cryptic intron may not be recognized or recognized
less efficiently in these species. hGFP-S65T and EGFP do not contain
the cryptic intron and can be used for expression in plants.
• Some people have put GFP expression constructs into their system
and failed to detect fluorescence. There can be numerous reasons for
failure, including use of an inappropriate filter set, expression of GFP
below the limit of detection, and failure of GFP to form the chromophore. Recombinant GFP Protein and GFP Monoclonal or Polyclonal
Antibody may be used to troubleshoot GFP expression in these cases.
Since GFPuv and EGFP fluoresce brighter than wt GFP, they are better
alternatives for expression of GFP.
• GFP targeted to a low pH environment may lose fluorescence (see
Section II.E.4).
B. Requirements for GFP Chromophore Formation
• Formation of the chromophore in wt GFP and GFP-S65T appears to be
temperature sensitive; however, the mutations in EGFP and GFPuv
suppress this thermosensitivity (see Section II.E.7). In some cases,
E. coli, yeast, and mammalian cells expressing wt GFP or GFP-S65T
have shown stronger fluorescence when grown at lower temperatures
(Heim et al., 1994; Ward, pers. comm.; Lim et al., 1995; Pines, 1995;
Ogawa et al., 1995). Hence, incubation at a lower temperature may
increase the fluorescence signal obtained when using wt GFP and
GFP-S65T.
• GFP chromophore formation requires molecular oxygen (Heim et al.,
1994; Davis, D. F. et al., 1995); therefore, cells must be grown under
aerobic conditions.
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VIII. Troubleshooting Guide continued
C. Photobleaching or Photodestruction of Chromophore
• Excite at 470 nm for wt GFP, 360–400 nm for GFPuv, 360–400 nm for
EBFP, and 488 nm for the red-shifted GFP variants. Excitation at the
395-nm peak for wt GFP may result in rapid loss of signal. For GFPuv
and EBFP, use the longest possible excitation wavelength to minimize
the rate of photobleaching.
• A tungsten-QTH or argon light source is preferable. Mercury and xenon
lamps produce significant UV radiation which will rapidly destroy the
chromophore unless strongly blocked by appropriate filters.
D. Autofluorescence
• Some samples may have a significant background autofluorescence,
e.g., worm guts (Chalfie et al., 1994; Niswender et al., 1995). A
bandpass emission filter may make the autofluorescence appear the
same color as GFP; using a long-pass emission filter may allow the
color of the GFP and autofluorescence to be distinguished. Use of
DAPI filters may also allow autofluorescence to be distinguished
(Brand,1995; Pines, 1995).
• Most autofluorescence in mammalian cells is due to flavin coenzymes
(FAD and FMN; Aubin, 1979) which have absorption and emission
maxima at 450 and 515 nm respectively. These values are very similar
to those for wt GFP and the red-shifted GFP variants, so
autofluorescence may obscure the GFP signal. The use of DAPI filters
when using 450/515 nm light for excitation may make autofluorescence
appear blue while the GFP signal remains green. In addition, some
growth media can cause autofluorescence. Since GFPuv uses excitation of 360–400 nm, autofluorescence of flavins should not be a
problem. When possible, perform microscopy in a clear buffer such as
PBS, or medium lacking phenol red.
• Some cell types can produce a speckled autofluorescence pattern
which is likely due to mitochondrially bound NADH (Aubin, 1979). This
problem is minimized with excitatory light around 488 nm, as opposed
to UV excitation (Niswender et al., 1995). Therefore, we recommend
using EGFP for expression in these systems. Such problems are
unavoidable with GFPuv and EBFP, whose excitation requires light in
the UV range (360–400 nm).
• For mammalian cells, autofluorescence can increase with time in
culture. For example, when CHO or SCI cells were removed from
frozen stocks and reintroduced into culture, the observed autofluorescence (emission at 520 nm) increased with time until a plateau was
reached around 48 hours (Aubin, 1979). Therefore, in some cases it
may be preferable to work with freshly plated cells.
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VIII. Troubleshooting Guide continued
• Always use a mock-transfected control or cells transfected with a
promoterless vector such as pEGFP-1 to gauge the extent of
autofluorescence.
• For fixed cells, autofluorescence can be reduced by washing with
0.1% sodium borohydride in PBS for 30 min after fixation.
E. Considerations for Mammalian Expression
• If you have not been able to detect wt GFP in a mammalian expression
system, try using EGFP or EBFP instead. The brighter fluorescence
and improved translation should make detection of the expression of
these variants easier than expression of wt GFP.
• Have you verified your GFP plasmid construct and concentration with
a restriction digest? Verify that all subcloning steps have been done
correctly, keeping in mind specific restriction sites in some vectors
which are inactivated by methylation. CLONTECH’s GFP and EGFP
Sequencing Primers may be used to verify sequence junctions.
• Is the vector compatible with your cell type? Has the CMV promoter
been used previously for transient expression in your cells?
• Do you have another assay to estimate transfection efficiency? This
can be accomplished by transfection with a second reporter plasmid
which contains the same promoter element, like pCMVβ (#6177-1),
and employing standard assays or stains to detect β-galactosidase
activity. Alternatively, use a secreted reporter such as secreted alkaline
phosphatase (SEAP), which can be quantified using the culture medium, leaving the cells intact for analysis of GFP expression.
• Expression of GFP protein can be verified by Western analysis using
either the GFP Monoclonal or Polyclonal Antibody.
F. Optimizing Microscope/FACS Applications
• In general, optimal visual detection of GFP fluorescence by microscopy
is achieved in a darkened room, after your eyes have adapted for
10–20 minutes.
• In microscopy, the primary issue is the choice of filter sets. Choose the
filter set that is optimal for the GFP variant that you are using. In
general, conditions used for fluorescein should give some signal with
all variants except GFPuv. Autofluorescence may be a problem in
some cell types or organisms. For more information on the filter sets
recommended for GFP and its variants, see Section V.A.
• A simple control for the microscope setup is to spot a small volume of
purified recombinant GFP protein (e.g., rGFP, rGFP-S65T, rEGFP, or
rGFPuv) on a microscope slide. As a crude substitute, any source of
fluorescein can be used, such as a fluorescein-conjugated antibody or
avidin-fluorescein.
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VIII. Troubleshooting Guide continued
• GFP fluorescence is very sensitive to some nail polishes used to seal
coverslips (Chalfie et al., 1994; Wang & Hazelrigg, 1994). In place of
nail polish for mounting coverslips, we recommend molten agarose or
rubber cement.
• Exciting GFP intensely for extended periods may generate free radicals that are toxic to the cell. This problem can be minimized by
excitation at 450–490 nm.
G. Anomalous Band in some GFP Plasmid Preparations
We have occasionally observed a band running at approximately 500 bp in
some (not all) plasmid preparations of the GFP vectors that have the
backbone carrying the kanamycin resistance gene and the f1 origin (i.e.,
pEGFP-1, pEGFP-N1, -N2, -N3, -C1, -C2, -C3, and pEBFP-N1, -C1). The
presence and level of this band varies greatly from one plasmid DNA
preparation to another. We have not thoroughly characterized this band;
however, it appears to be a small circular DNA, possibly single-stranded,
that is generated as a by-product of plasmid replication. As far as we can
ascertain, this band does not interfere with ligating inserts into the vectors,
does not affect transformation or transfection efficiencies, and does not
have any deleterious effect on mammalian cells.
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IX. References
Amsterdam, A., Lin, S. & Hopkins, N. (1995) The Aequorea victoria green fluorescent protein can
be used as a reporter in live zebrafish embryos. Devel. Biol. 171:123–129.
Atkins, D. & Izant, J. G. (1995) Expression and analysis of the green fluorescent protein gene in the
fission yeast Schizosaccharomyces pombe. Curr. Genet. 28:585–588.
Aubin, J. E., (1979) Autofluorescence of viable cultured mammalian cells. J. Histochem. Cytochem.
27:36–43.
Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A. & Struhl, K.
(1994) In Current Protocols in Molecular Biology (John Wiley and Sons, NY), Vol. 1, Ch. 5 and 9.
Barthmaier, P. & Fyrberg, E. (1995) Monitoring development and pathology of Drosophila indirect
flight muscles using green fluorescent protein. Devel. Biol. 169: 770–774.
Baulcombe, D. C., Chapman, S. & Santa Cruz, S. (1995) Jellyfish green fluorescent protein as a
reporter for virus infections. Plant J. 7:1045–1053.
Beachy, R. (1995) Presentation at Fluorescent Proteins and Applications Meeting, Palo Alto, CA.
Bian, J., Lin, X. & Tang, J. (1995) Nuclear localization of HIV-1 matrix protein P17: The use of A.
victoria GFP in protein tagging and tracing. FASEB J. 9:A1279 #132.
Bokman, S. H. & Ward, W. W. (1981) Renaturation of Aequorea green-fluorescent protein. Biochem.
Biophys. Res. Comm. 101:1372–1380.
Bonner, J. T., Compton, K. B., Cox, E. C., Fey, P. & Gregg, K. Y. (1995) Development in one
dimension: The rapid differentiation of Dictyostelium discoideum in glass capillaries. Proc. Natl.
Acad. Sci. USA 92:8249–8253.
Brand, A. (1995) GFP in Drosophila. Trends Genet. 11:324–325.
Buikema, W. (1995) Presentation at Fluorescent Proteins and Applications Meeting, Palo Alto, CA.
Burlage, R., Yang, Z. & Mehlhorn, T. (1995) Fluorescent microorganisms are detected in bacterial
transport experiments. Poster presentation at Fluorescent Proteins and Applications Meeting, Palo
Alto, CA.
Carey, K. L., Richards, S. A., Lounsbury, K. M. & Macara, I. G. (1996) Evidence using a green
fluorescent protein-glucocorticoid receptor that the RAN/TC4 GTPase mediates an essential
function independent of nuclear protein import. J. Cell Biol. 133(5):985–996.
Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W. & Prasher, D. C. (1994) Green fluorescent protein
as a marker for gene expression. Science 263:802–805.
Chalfie, M. (1995) Green fluorescent protein. Photochem. Photobiol. 62(4):651–656.
Chao, Y. -C., Chen, S. -L. & Li, C.-F. (1996) Pest control by fluorescence. Nature 380:396–397.
Chattoraj, M., King, B. A., Bublitz, G. U. & Boxer, S. G. (1996) Ultra-fast excited state dynamics in
green fluorescent protein: Multiple states and proton transfer. Proc. Natl. Acad. Sci. USA 93:
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Chen, C. & Okayama, H. (1988) Calcium phosphate-mediated gene transfer: a highly efficient
transfection system for stably transforming cells with plasmid DNA. BioTechniques 6:632–638.
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cytometry. CLONTECHniques X(4):20.
Cheng, L., Fu, J., Tsukamoto, A. & Hawley, R. G. (1996) Use of green fluorescent protein variants
to monitor gene transfer and expression in mammalian cells. Nature Biotechnol. 14:606–609.
Chiu, W.-L., Niwa, Y., Zeng, W., Hirano, T., Kobayashi, H. & Sheen, J. (1996) Engineered GFP as
a vital reporter in plants. Curr. Biol. 6(3):325–330.
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IX. References continued
Cody, C. W., Prasher, D. C., Westler, W. M., Prendergast, F. G. & Ward, W. W. (1993) Chemical
structure of the hexapeptide chromophore of Aequorea green-fluorescent protein. Biochemistry
32:1212–1218.
Coghlan, A. (1995) Bright future for glowing gene. New Scientist, March 4th issue, pp. 23.
Corbett, A. H., Koepp, D. M., Schlenstedt, G., Lee, M. S., Hopper, A. K. & Silver, P. A. (1995) Rna1p,
a Ran/TC4 GTPase activating protein, is required for nuclear import. J. Cell Biol. 130(5):1017–1026.
Cormack, B. P., Valdivia, R. & Falkow, S. (1996) FACS-optimized mutants of the green fluorescent
protein (GFP). Gene 173:33–38.
Cox, J. (1995) Presentation at Fluorescent Proteins and Applications Meeting, Palo Alto, CA.
Coxon, A. & Bestor, T. H. (1995) Proteins that glow in green and blue. Chemistry & Biology 2:
119–121.
Crameri, A., Whitehorn, E. A., Tate, E. & Stemmer, W. P. C. (1996) Improved green fluorescent
protein by molecular evolution using DNA shuffling. Nature Biotechnol. 14:315–319.
Cubitt, A. B., Heim, R., Adams, S. R., Boyd, A. E., Gross, L. A. & Tsien, R. Y. (1995) Understanding,
improving and using green fluorescent proteins. Trends Biochem. 20:448–455.
Davis, D. F., Ward, W. W. & Cutler, M. W. (1995) Posttranslational chromophore formation in
recombinant GFP from E. coli requires oxygen. Proceedings of the 8th international symposium on
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X. Summary Reference List
Background
Brand, A. (1995)
Chalfie, M., et al. (1994)
Haseloff, J. & Amos, B. (1995)
Hassler, S. (1995)
Hodgkinson, S. (1995)
Kolberg, R. (1994)
Morin, J. G. & Hastings, J. W. (1971)
Morise, H., et al. (1974)
Pines, J. (1995)
Prasher, D. C., et al. (1992)
Prasher, D.C. (1995)
Shimomura, O., et al. (1962)
Reviews on GFP technology
Chalfie, M. (1995)
Coghlan, A. (1995)
Coxon, A. & Bestor, T. H. (1995)
Cubitt, A. B., et al. (1995)
Hassler, S. (1995)
Kolberg, R. (1994)
Prasher, D.C. (1995)
Stearns, T. (1995)
Youvan, D. C. (1995)
Properties of GFP
Bokman, S. H. & Ward, W. W. (1981)
Chattoraj, M. et al. (1996)
Cody, C. W., et al. (1993)
Davis, D. F., et al. (1995)
Inouye, S. & Tsuji, F. I. (1994)
Niswender, K. D., et al. (1995)
Niwa, H., et al. (1996)
Ormö, M. et al. (1996)
Robart, F. D. & Ward, W. W. (1990)
Roth, A. (1985)
Surpin, M. A. & Ward, W. W. (1989)
Ward, W. W. (1979)
Ward, W. W., et al. (1980)
Ward, W. W. (1981)
Ward, W. W. & Bokman, S. H. (1982)
Yang, F., et al. (1996)
Mutagenesis of GFP
Cormack, B., et al. (1996)
Crameri, A., et al. (1996)
Delagrave, S., et al. (1995)
Ehrig, T., et al. (1995)
Heim, R., et al. (1994)
Heim, R., et al. (1995)
Heim, R. & Tsien, R. Y. (1996)
Reichel, C., et al. (1996)
Siemering, K. R., et al. (1996)
Yang, T. T., et al. (1996a)
page
46
Protocol # PT2040-1
Version # PR74631
Expression of GFP in Prokaryotes
Burlage, R., et al. (1995)
Chalfie, M., et al. (1994)
Crameri, A., et al. (1996)
Dhandayuthapani, S. et al. (1995)
Inouye, S. & Tsuji, F. I. (1994a)
Inouye, S. & Tsuji, F. I. (1994b)
Kahana, J. A., et al. (1995)
Kain, S. R., et al. (1995)
Kitts, P., et al. (1995)
Kremer, L. et al. (1995)
Yu, J. & van den Engh, G. (1995)
Expression of GFP in Eukaryotes
Atkins, D. & Izantz, J. G. (1995)
Baulcombe, D. C., et al. (1995)
Bonner, J. T., et al. (1995)
Brand, A. (1995)
Chalfie, M., et al. (1994)
Chao, Y.-C., et al. (1996)
Chiu, W.-L., et al. (1996)
Davis, I., et al. (1995)
Galbraith, D. W., et al. (1995)
Halme, A., et al. (1996)
Haselhoff, J. & Amos, B. (1995)
Higgs, S., et al . (1996)
Hodgkinson, S. (1995)
Kahana, J. A. et al. (1995)
Kain, S. R., et al. (1995)
Kitts, P., et al. (1995)
Lo, D. C., et al. (1994)
Moss, J. B. & Rosenthal, N. (1994)
Murphy, S. M. & Stearns, T. (1996)
Niedz, R. P., et al. (1995)
Ogawa, H., et al. (1995)
Pines, J. (1995)
Plautz, J. D., et al. (1996)
Riles, L., et al. (1995)
Schlenstedt, G., et al. (1995)
Tannahill, D., et al. (1995)
Törmäkangas, K., et al. (1995)
Troemel, E. R., et al. (1995)
Waddle, J. A., et al. (1996)
Wu, G.-I., et al. (1995)
Yang, T. T., et al. (1996a, b)
Yu, K. L. & Dong, K. W. (1995)
Transgenic expression of GFP
Amsterdam, A., et al. (1995) [Zebrafish]
Barthmaier, P. & Fyrberg, E. (1995) [Drosophila]
Chalfie, M., et al. (1994) [ C. elegans]
Chiu, W.-L., et al. (1996) [Tobacco]
Davis, I. et al. (1995) [Drosophila]
Haseloff, J. & Amos, B. (1995) [Arabidopsis]
Higgs, S. et al. (1996) [Mosquito]
Ikawa, M., et al. (1995a & 1995b) [Mouse]
Technical Service
TEL:415-424-8222 or 800-662-CLON
FAX: 415-424-1064 or 800-424-1350
CLONTECH Laboratories, Inc.
X. Summary Reference List continued
Reichel, C. et al. (1996) [Various plants]
Ogawa, H., et al. (1995)
Santa Cruz, S. et al. (1996) [Potato plant]
Oker-Blom, C. et al. (1996)
Shiga, Y. et al. (1996) [Drosophila]
Olson, K. R. et al. (1995)
Wang, S. & Hazelrigg, T. (1994) [Drosophila] Oparka, K. J., et al. (1995)
Yeh, E. et al. (1995) [Drosophila]
Peters, K. G., et al. (1995)
Rizzuto, R., et al. (1995)
Expression of GFP-fusion proteins
Rizzuto, R., et al. (1996)
Bian, J., et al. (1995)
Rutter, G. A. et al. (1996)
Carey, K. L. et al. (1996)
Sengupta, P., et al. (1994)
Corbett, A. H., et al. (1995)
Sidorova, J. M., et al. (1995)
Doyle, T. & Botstein, D. (1996)
Stearns, T. (1995)
Dyer, J. M. et al. (1996)
Sullivan, K. F. et al. (1995)
Flach, J., et al. (1994)
Treinin, M. & Chalfie, M. (1995)
Halme, A., et al. (1996)
Venerando, R., et al. (1996)
Hampton, R. Y., et al. (1996)
Waddle, J. A., et al. (1996)
Heinlein, M., et al. (1995)
Wang, S. & Hazelrigg, T. (1994)
Htun, H. et al. (1996)
Webb, C. D., et al. (1995)
Jacobi, C. A., et al. (1995)
Kaether, C. & Gerdes, H.-H.(1995)
Retroviral expression of GFP
Kahana, J. A., et al. (1995)
Cheng, L., et al. (1996)
Kerrebrock, A. W., et al. (1995)
Levy, J. P., et al. (1996)
Lewis, P. J. & Errington, J. (1996)
Muldoon, R. R., et al. (1997)
Lim, C. R., et al. (1995)
Flow cytometry of GFP-expressing cells
Maniak, M., et al. (1995)
Atkins, D. & Izant, J. G. (1995)
Marshall, J., et al. (1995)
Cheng, L. & Kain, S. (1995)
Moores, S. L., et al. (1996)
Cheng, L., et al. (1996)
Nabeshima, K., et al. (1995)
Cormack, B. P., et al. (1996)
Náray-Fejes-Tóth, A. & Fejes-Toth, G. (1996) Fey, P., et al. (1995)
Galbraith, D. W., et al. (1995)
Levy, J. P., et al. (1996)
Ropp, J. D., et al. (1995)
Ropp, J. D., et al. (1996)
Sheen, J., et al. (1995)
Subramanian, S. & Srienc, F. (1996)
TEL:415-424-8222 or 800-662-CLON
FAX:415-424-1064 or 800-424-1350
Technical Service
Protocol # PT2040-1
Version # PR74631
page
47
CLONTECH Laboratories, Inc.
XI. Related Products
Product
Cat. #
Wild-Type GFP Vectors
• pGFP Vector
• p6xHis-GFP Vector
6097-1
6087-1
GFP Variant Vectors
• pGFPuv Vector
• pBAD-GFPuv Vector
• pEGFP Vector
• pEGFP-1 Vector
• pEGFP-N1 Vector
• pEGFP-N2 Vector
• pEGFP-N3 Vector
• pEGFP-C1 Vector
• pEGFP-C2 Vector
• pEGFP-C3 Vector
• phGFP-S65T Vector
• pEBFP Vector
• pEBFP-N1 Vector
• pEBFP-C1 Vector
• pIRES-EGFP Vector
6079-1
6078-1
6077-1
6086-1
6085-1
6081-1
6080-1
6084-1
6083-1
6082-1
6088-1
6068-1
6069-1
6070-1
6064-1
Other Products
• GFP-N Sequencing Primer
• GFP-C Sequencing Primer
• EGFP-N Sequencing Primer
• EGFP-C Sequencing Primer
• Recombinant GFP Protein
• Recombinant GFP-S65T Protein
• Recombinant EGFP Protein
• Recombinant GFPuv Protein
• GFP Monoclonal Antibody
6476-1
6477-1
6479-1
6478-1
8360-1, -2
8364-1, -2
8365-1
8366-1
8362-1
page
48
Protocol # PT2040-1
Version # PR74631
Technical Service
TEL:415-424-8222 or 800-662-CLON
FAX: 415-424-1064 or 800-424-1350
CLONTECH Laboratories, Inc.
XI. Related Products continued
Product
Cat. #
•
•
•
•
•
•
•
•
8363-1, -2
8021-1, -2
K2050-1
8020-1
K2040-1
K2041-1
6177-1
V2010-1
•
•
GFP Polyclonal Antibody (IgG Fraction)
CalPhos Maximizer™
CalPhos MaximizerTM Transfection Kit
CLONfectinTM
Great EscAPeTM SEAP Reporter System
Great EscAPeTM SEAP Detection Kit
pCMVβ Reporter Vector
Guide to Yeast Genetics and Molecular Biology
(Guthrie, C. & Fink, G. R., eds.)
Culture of Animal Cells, Third Edition
(Freshney, R. I.)
Western ExposureTM Chemiluminescent Detection System
with membranes
without membranes
TEL:415-424-8222 or 800-662-CLON
FAX:415-424-1064 or 800-424-1350
Technical Service
V2128-1
K2030-1
K2031-1
Protocol # PT2040-1
Version # PR74631
page
49
CLONTECH Laboratories, Inc.
Appendix. Fluorescent Proteins Newsgroup
A newsgroup for the discussion of fluorescent proteins has been created within
the bionet hierarchy of Newsgroups. This newsgroup is intended to provide a
forum for discussion of bioluminescence, to promote further development of
reporter proteins obtained from bioluminescent organisms (e.g., GFP, luciferase,
and Aequorin), and to facilitate their application to interesting biological questions. (A full copy of the newsgroup charter can be found in the BIOSCI archives.)
We hope you will find this newsgroup useful and encourage you to participate in
the discussions.
Discussion Leaders: Paul Kitts & Steve Kain, CLONTECH Laboratories, Inc.
Administration: BIOSCI International Newsgroups for Biology
Newsgroup Name: bionet.molbio.proteins.fluorescent
To subscribe:
If you use USENET news, you can participate in this newsgroup using your
newsreader.
You can also access the newsgroup on the World Wide Web using the URL http:/
/www.bio.net/hypermail/FLUORESCENT-PROTEINS/
To receive “The BIOSCI electronic newsgroup information sheet” which describes the BIOSCI newsgroups and gives instructions on how to subscribe via
e-mail:
If you are located in the Americas or the Pacific Rim:
Send a mail message to the Internet address:
biosci-server@net.bio.net
Leave the subject line of the message blank and enter the following line in
the mail message:
info usinfo
This message will be automatically read by the computer and you will be
sent the latest copy of the information sheet.
If you are located in Europe, Africa, or central Asia:
Send a mail message to the Internet address:
biosci-server@net.bio.net
Leave the subject line of the message blank and enter the following line in
the mail message:
info ukinfo
This message will be automatically read by the computer and you will be
sent the latest copy of the information sheet.
page
50
Protocol # PT2040-1
Version # PR74631
Technical Service
TEL:415-424-8222 or 800-662-CLON
FAX: 415-424-1064 or 800-424-1350
CLONTECH Laboratories, Inc.
Notes:
CalPhos Maximizer™, CLONfectin™, Great EscAPe ™, Living Colors ™, Transformer™ , and
Western Exposure™ are trademarks of CLONTECH Laboratories, Inc.
DyNA QunatTM is a trademark of Hoefer Pharmacia Biotech, Inc.
FACS® is a registered trademark of Becton Dickinson Immunocytometry Systems.
© 1997, CLONTECH Laboratories, Inc.
TEL:415-424-8222 or 800-662-CLON
FAX:415-424-1064 or 800-424-1350
Technical Service
Protocol # PT2040-1
Version # PR74631
page
51