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Sample reduction/alkylation in urea before SDS-PAGE - (Jul/14/2010 )

I am having a tough time finding a protocol for the reduction and alkylation in the presence of urea prior to SDS-PAGE.

1) Is it OK to adjust sample to 7M urea, 2% SDS, 100mM Tris pH 8, 10% glycerol, 0.001% Bromophenol blue, 50mM DTT for reduction?

2) How long should I incubate, and at what temperature?

3) Should I then bring to ~166mM iodoacetamide RT for 15 min, and directly load sample on gel without desalting or adjusting pH?

Will the pH be too high for SDS-PAGE since alkylation pH optimum is ~8, while SDS-PAGE pH optimum is 6.8?

I am trying to keep volumes as low as possible, and want to avoid any precipitation/filtration concentration step.

Thanks in advance for your help/advice!

-shmaggregates-

Your protocol sounds very much like the 2nd dimension in 2d gels. In that case:

1) This recipe looks good.

2) Generally, equilibration of IEF strips in 2DE (in the above reduction buffer) takes between 15-30 (or more... it doesn't really hurt) minutes at room temperature. Never heat buffers containing urea above 37C because carbamylation of proteins may occur.

3) You should be able to load the sample without desalting or pH'ing. This works fine for 2D gels.

-braincow-