use of thioglycolic acid in buffer - electrophoresis prior to N-terminal sequencing (Oct/19/2006 )

Hi everybody!

I want to transfer my protein to a PVDF membrane for N-terminal sequencing.
Consulting protocols on the subject I found out some that mentioned the use of thioglycolic acid in the upper chamber buffer. Some say that we should pre-run the gel in this buffer for 30 min.

Questions:

- do we really have to use thioglycolitic acid?
- if so, in what concentration?
- should we pre-run the gel in this buffer and then run it in normal conditions, using the normal running buffer, or should we do both using thioglycolic acid?
- should we always pre-run the gel or can we just run it with the samples using running buffer containing thioglycolic acid?

Thank you!

when i did this, years ago, i would "age" the gel overnight to ensure that there was no free acrylamide left to modify the protein.

the transfer to PVDF was done using a CAPS buffer.

these conditions worked fine.

well, I am also "aging" the gel overnight and using CAPS as the tranfer buffer... the thing is that some people also mention the pre-run using thioglycolic acid, prior to the "normal" run with CAPS...

I guess it might not be that important!... I hope so! Thanks!