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13. Ensure the tray is fl at using the spirit level on the tray. Apply
the solution evenly across the entire slot without bubbles.
14. Fill the cup with 2× sample buffer and observe that the level of
buffer does not diminish, which would signify a leak. If there is
a leak, remove the cup and reposition again.
15. Gels can be stored after IEF in 10 mL of equilibration buffer
at −20°C.
16. Acrylamide, TEMED, APS, and SDS are extremely hazardous.
Please consult the manufacturer's material safety data sheets
for correct handling of these chemicals. The 10% APS solution
should be made freshly prior to use.
17. Pay special attention to stop air entering the feed tube. Once
all the acrylamide solution is out of the maker, clean it by fi ll-
ing 2 L of water into the chambers, and pump the water
through the feed tube to waste.
18. Lubricate the cassettes with SDS running buffer to facilitate
loading into the tank.
19. After second dimensional SDS-PAGE is complete, the gels
should be scanned as soon as possible, to minimize the diffu-
sion of protein spots.
20. Avoid saturation, and adjust the PMT voltage accordingly. The
PMT used is usually between 500 and 600 V.
21. It is advisable to run an aliquot of the internal standard on the
preparative gels to assist with correct selection of spots for pro-
tein identifi cation.
Acknowledgments
This work was supported, in part, by the National Health &
Medical Research Council of Australia (program grant #487922
(R.J.S)), and funds from the Operational Infrastructure Support
Program provided by the Victorian Government of Australia.
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teomics by quantitative shotgun proteomics.
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3. Chambers AF, Groom AC, and MacDonald IC
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4. Woodhouse EC, Chuaqui RF, and Liotta LA
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