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!

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 37°C 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.