Anyone has run a PAGE with reducing agents in-gel?? - (May/16/2011 )

Hello. I have problems with my samples (drosophila eye homogenizated) and I suspect that some complexes are formed in-gel. That's because in Western BLot the bands appear in stacking gel (even in the top of the well!!). I tried to solubilize with SDS, Tritón and CHAPS, but the result was the same, so I want to try to add an reducing agent directly to the gel and 8M urea.


Someone knows a protocol to do this.

Thks

First of all: how big are the proteins you are looking at?

What percentage gel are you running?

What denaturing conditions are you currently using?

130 KDa and 72 KDa

8% Resolving, 4% Stacking

Denaturing conditions:
Without urea: SDS, BME, Heat
With urea: SDS or Tritón or CHAPS, BME, incubation 30' 30 °C

bob1 on Tue May 17 02:51:22 2011 said:

some aggregates may be physically crosslinked and need more stringent solubilization in guanidine