Low-temperature SDS-PAGE: tips and suggestions - (Jan/08/2013 )

Hi everybody

I'm attempting to check dimer-monomer ratio of eNOS protein using LT-PAGE approach. I've tried it twice and got very confusing and inconsistent results (some control samples showed two bands, while others did not). Here is how I did it. I'll appreciate any tips and suggestions.
I ran unboiled samples diluted in SDS loading buffer w/o DTT or b-mercapto. Both electrophoresis (I used pre-cast 4-12% NuPAGE gels) and the transfer were done in the ice bath, with temps always below 10C. Is there anything else I should be doing?

Thanks in advance!

Yulia

have you run a gel and stained instead of transfer? how did it look?

if the problem was the gel then it may be due to the sds (it will crystallize at refrigerator temperatures). the cure for this is to use lithium dodecyl sulfate (lds) instead of sds (at the same concentrations as you would use the sds). lds will not crystallize at refrigerator temperatures.

Thanks for the reply. I haven't stained the gel, but I've stained the membrane after the transfer and it looked fine

omitting dtt and bme from the loading buffer should suffice to protect the disulfide bonds so you may be able to boil your samples to ensure consistent binding of sds (you could incubate at 60-70C instead of boiling).

Thanks. I'm going to try that