Results (continued)

A Comparison of EA-Wax with Detection by Phosphoimaging.
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.

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.

Figure 5. Comparison of 35S detection using EA-Wax and Phosphoimaging.

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 kit and exposed to X-ray film for 24 h (panel B, right).

Conclusion
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 enhan