Rapid Detection System for Stress Responses
- Use in Proteomic Analysis
Introduction
Nicola J. Holden (1), Maurice P. Gallagher (1), and Frank Fox (2).
(1) Institute of Cell and Molecular Biology, University of Edinburgh, Edinburgh EH9 3JR, Scotland, UK.
(2) EA Biotech Ltd., Unit 4, Strathclyde Business Centre, 416 Hamilton Road, Flemington G72 7XR, Scotland, UK.
The recent availability of the nucleic acid sequences for entire genomes of
bacteria has provided an opportunity to link gene expression with complete
cellular protein profiles. Such an approach which involves analysis of the
cellular protein composition by two-dimensional gel electrophoresis, is referred
to as proteomics. Potentially, this strategy has great importance for the
pharmaceutical and food processing industries. It can be used to explore the
global effects of toxic drugs, of sanitising agents and sanitising procedures or,
of antibiotics on cells. Such applications may provide important information on
drug design and specificity, cellular survival mechanisms, or strategies used
by cells for host infection, for immune evasion or for combating therapeutic
agents.
The ability to analyse complex protein mixtures has benefited from improved
procedures for performing two-dimensional electrophoresis (2-DE) under
highly reproducible conditions and in particular, from the commercial
availability of IEF strips with chemically fixed pH gradients. In addition, these
improvements have been complemented by the development of highly
sensitive methods for protein characterisation using tandem mass
spectroscopy. Together this combination of approaches permit the
identification of proteins by comparison of polypeptide fragments, revealed by
tandem mass spectroscopy, with all the predicted coding regions from the
genome of the organism examined. In essence then, the complement of
proteins in the cell at any one time can be determined.
Analysis of the dynamics of cellular responses, as opposed to the
determination of the static protein composition of cells, requires evaluation of
de novo protein synthesis and generally require pulse labelling the cells with
radioactive amino acids, before and after exposure to the stimulus of interest.
One of the limitations of this strategy is the time required for data acquisition,
as the signals from 35S-labelled proteins commonly takes several weeks to
detect using X-ray film.
In the present report, we describe the application a novel waxed-based
system for enhanced autoradiography which is simple to apply and
enhances both detection sensitivity and speed. Our studies show that
application of EA-WaxTM enhances detection sensitivity to a level which
equates with direct detection using a Molecular Dynamics phosphoimager. In
contrast however, application of EA-WaxTM does not require costly
phosphoimaging machinery. An outline of the basic procedure is described
and a comparison of the results obtained by phosphoimaging and the EAWaxTM system is illustrated. The cost and ease of use of this strategy makes it
readily applicable as a standard approach for use in both academic and
industrial environments.
polyacrylamide IPG strips (Amersham-Pharamacia), pH 3-10. IPG strips were
pre-run at 300V, 1Vhr followed by 14,000V for 20Vhr. After separation, IPG
strips were bonded onto a 1.5 x 150 x 180mm acrylamide slab gel for the
second dimension and electrophoresed at 30mA for 16h at 4oC. The gels
were subsequently blotted onto PVDF membrane at 20V for 16h, 4oC.
Membranes were then imaged for 24h, using a Molecular Dynamic
phosphoimager or were processed using the EA-Wax system described
below.
Results
Enhanced Autoradiography
The EA-Wax system is a simple process which dramatically enhances the
detection efficiencey of low energy radionuclides. In the current context it is
used to enhance detection of samples which have been electroblotted onto
PVDF membrane. However, it can be used with a variety of other flat
matrices, such as paper, polyester-backed TLC plates, or cellulose nitrate
filters. It is supplied in a simple to use kit format, containing sheets of EA-Wax
and silicone release membrane (Figure 1).
Figure 2 Application of EA-Wax to PVDF.
EA-Wax is shown adjacent to a sheet of PVDF membrane which
has been placed on a sheet of silicon release membrane (panel
A). Several strips of wax are removed and layered on the PVDF
membrane (panel B). The number of strips required varies with
the size of the membrane.
S35 labelled cells of Salmonella were fractionated by 2-D
electrophoresis using IPG strips (pI 3-10; left to right) from
Amersham-Pharmacia, prior to electroblotting onto a PVDF
membrane. The membrane was imaged for 24h using a
Molecular
Dynamics Phosphoimager
(panel A,
left).
Subsequently, the membrane was processed using the EA-Wax
Conclusion
Figure 4. Compression of the wax.
The completed assembly is shown, with the PVDF/wax assembly
clamped between the sheets of Silicon Release Membrane and
heated glass plates.
A Comparison of EA-Wax with Detection by
Phosphoimaging.
S. typhimurium SL1344 was used for pulse labelling experiments. All cultures
were grown aerobically, in Luria Broth (LB), at 37oC.
Figure 5. Comparison of 35S detection using EA-Wax and
Phosphoimaging.
kit and exposed to X-ray film for 24 h (panel B, right).
Application of the EA-Wax System
Materials and Methods
Strain SL1344 (Hosieth & Stocker, 1981) was grown at 37oC to midexponential phase in Spitzizen minimal media, supplemented with histidine.
Samples were then removed at specified times and labelled with 5mCi [35S]
methionine for 5 minutes at 37oC. Cell were then lysed by sonication and the
samples were processed using 2-DE, essentially as described by O’Farrell
(1975). For the first dimension samples were run on pre-cast 110mm
Figure 3. Assembling the Sample with Wax
Figure 1. The EA-Wax Starter Kit
The key components of the EA-Starter Kit are shown (panel A,
left panel alongside the sheets of silicone release membrane and
strips of EA-Wax (panel B, right).
EA-Wax contains a wax-embedded fluor which enhance autoradiography.
Following 2-D electrophoresis, samples are electroblotted onto a suitable
matrix such as PVDF membrane. After draining, the PVDF is then placed on
top of a clean sheet of silicon release membrane. Strips of wax containing the
fluor are then layered on top of the sheet of PVDF in a fairly even manner
(Figure 2).
In order to determine the value of the EA-Wax system, we compared the
results of direct image analysis, using a Molecular Dynamics Phosphoimager,
with data obtained after wax impregnation. In order to achieve this, cells of
Salmonella were grown at 37oC and radiolablelled with 35S-methionine (as
described in Materials and Methods). Samples were then sonicated and
analysed by 2-D gel electrophoresis, prior to electroblotting onto PVDF
membrane. The resulting membrane was then phosphimaged directly for 24
h. Subsequently the membrane was impregnated in EA-Wax as described
above and was then exposed to autoradiography film for 24 h. The results of
both treatments are shown (Figure 5). It can clearly be seen that wax
treatment resulted in a major enhancement in the speed and sensitivity of
detection by autoradiography and indeed, the images obtained using both
systems were very similar.
A pre-heated glass plate is placed on a level stand (panel A,
left). Shown also, is the Silicon Release Membrane/ PVDF/wax
assembly which is layered on the glass plate. A second sheet of
Silicon Release Membrane can then be applied to the top of the
stack (panel B, right).
Finally, a second pre-heated glass plate is placed on top of the stack and the
sample is clamped with even compression, in order to spread the wax over
the PVDF membrane (Figure 4). After a few minutes when the sample has
cooled and the wax has solidified, the clamping system can be removed and
the sample can be exposed to X-ray film in an autoradiography cassette
which has been lined with aluminium foil to aid reflection.
Bacterial Strains and Growth Conditions
Pulse labelling and 2-D gel electrophoresis
Following layering of the wax, the Silicon release membrane, PVDF and wax
assembly are placed on a square glass plated which has been pre-heated to
60oC and a second sheet of silicon release membrane is then layered on top
of the assembly (Figure 3).
The dynamics of cellular protein synthesis can be recorded by combining
radioactive pulse labelling with 35S-methionine together with 2-D gel
electrophoresis. However, the time required for detection using
autoradiography represent a rate-limiting step in the process. Whilst samples
can generally be imaged in approximately 24h with a phosphoimager, image
capture on X-ray film commonly takes 2-3 weeks. Nevertheless, this still
represents a useful process as X-ray film images are generally sharper and
so can be compared more readily using analysis software, such as the
Phoretix package.
Analysing cellular response mechanisms using proteomics is a very powerful
tool and has a wide range of application of relevance to the pharmaceutical
and food industries. However, the time required for detection of 35S labelled
samples using autoradiography has been a limitation to the speed of analysis
and a constraint for high throughout processing. Our studies have shown that
use of EA-Wax can greatly improve this situation and can reduce exposure
time from several weeks to less than 24h. EA-Wax therefore represents a
relatively inexpensive and user friendly method for enhancing autoradiograph
sensitivity, allowing detection to be performed with equivalent sensitivity to a
phosphoimage detection system.
References
Hosieth, S. K., & B.A.D. Stoker. (1981) Nature. 291: 238-239.
O’Farrel, P.H. (1975). J. Biol. Chem. 4007-4021.