Dear colleagues,
I would like to ask for optimal sample buffer for IEF electrophoresis. Our attempts with commercially available IEF gels and recommended sample buffer have given poor results.
Many thanks in advance,
Jordan
Dear David
many thanks for your answer and for this useful information.
Best wishes,
J
Dear Jordan,
we commonly use 7M urea, 2M thiourea, 4% CHAPS, 20mM DTT, 0.5% carrier ampholytes 3-10, protease inhibitor (e.g. EDTA-free cOmpleteMini) which you can also use as rehydration buffer and which works quite well for most samples. However, you have to keep in mind that the sample buffer depends on 1.) the sample type (e.g. this buffer works well for soluble samples but might not be the best choice for membrane proteins) and 2.) the labelling/staining method (e.g. for the DIGE-labelling you will need the recommended sample buffer with 7M urea, 2M thiourea, 4% CHAPS, 30mM Tris adusted to pH 8.0 at RT for the saturation DIGE or pH 8.5 on ice for the minimal DIGE).
Best regards,
Steffen
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