Proteomic Analysis of Proteins Secreted by Streptococcus pyogenes - Bacterial Pathogenesis: Methods and Protocols - page 21

high concentration of protein. This is important during isoelectric focusing as
125 L is the maximum volume of sample that can be separated with 7-cm
strips.
17. Usually 15-25 μL of sample will result in optical density values that in the
middle of the standard curve.
18. IPG buffer is supplied in ranges that match the range of the IPG strip being
used. The concentration of IPG buffer can vary from 0.5 to 2.0%, depending on
the sample.
19. To ensure complete sample uptake, do not exceed the 125 μL volume limit for
the strips.
20. Handle the ceramic holders with care, as they are brittle, and with gloves to
avoid contamination. The holder must be clean and completely dry before use.
21. To help coat the entire strip, gently lift and lower the strip and slide it back and
forth along the surface of the solution, tilting the strip holder slightly as needed
to ensure complete and even hydration.
22. Rehydrate for a minimum of 10 h, but overnight is best (10) .
23. If not proceeding to the equilibration step, strips must be frozen immediately
after step 3 is finished.
24. Make sure to start with clean glass plates and chamber equipment. Using lint-free
wipes, clean the glass plates with methanol before assembling chamber.
25. This recipe will make six gels and can be scaled up or down depending on the
number of gels being run.
26. SDS electrophoresis buffer temperature should be maintained at approximately
20 °C. Setting the MultiTemp III at 10 °C will usually keep the buffer in the
recommended temperature range.
27. Adding molten agarose to the sample application piece containing the
SDS-PAGE molecular weight standard solution will prevent the markers from
diffusing out of the sample application piece.
28. Make certain that no air bubbles are trapped between the IPG strip and the gel
surface or between the gel backing and the glass plate.
29. Before placing the gel in fixer, locate the acidic end of the IPG strip, and make
a diagonal cut on the acidic side of the gel at the very top to determine the
proper orientation when imaging the gels.
30. SYPRO Ruby is light sensitive. Always store the gels in a lightproof container.
31. Destaining overnight minimizes background and produces better gel images.
32. Store the gels in water in a covered plastic container at 4 °C.
References
1. Cunningham, M. W. (2000) Pathogenesis of group A streptococcal infections. Clin.
Microbiol. Rev . 13, 470-511.
2. Sumby, P., Barbian, K. D., Gardner, D. J., Whitney, A. R., Welty, D. M.,
Long, R. D., Bailey, J. R., Parnell, M. J., Hoe, N. P., Adams, G. G., DeLeo, F. R.,