Western blot problem: not running in gel - western blot troble (Nov/05/2004 )

Hi - if someone can help I will be forever in debt! I work with rhodopsin (GPCR) it's 40kd and seems to be getting stuck up in the gel. I use a 10% gel, and have tried using biorad precast gels too. it does not migrate well but all other proteins i have tried do (BSA, C-MYC etc, cell lysate) Is it aggregating? Am i getting dimers/trimers? It appears to be barely running at all...maybe 10% of the way in the gel (corresponding to 200kd on the ladder) the ladder rund fine but my proteins do NOT! I have made all reagents fresh several times and keep getting the same problem....I am not boiling the protein...(tried that too - but that leads to aggregations of membrane proteins)...nothing seems to work! ANY SUGGESTIONS? PLease post here and email if you feel like it... thanks!!!!

brad.chewpoy@utoronto.ca

If you are not to boil your sampes (?!) then try to dissolve in 8M urea... You need to completely denature your protein otherwise you'll see these dimers/trimers, and you can't really trust your markers etc etc...

good luck,

I have had simialr problems with membrane proteins aggregating after boiling. our current protocols now includes 1 hour incubations at 30 degrees celcuis and increasing the MeSH to 10% from 5%. It solved the problem for us. Good luck

I`m agree with Sprag about the urea; other option is using the urea in the gel (prepare gel with urea 8%). I suggest boil your sample too.

good luck.

hey everyone! thanks...for the input, I will try these things out soon...but I am under the impression that you are NOT supposed to boil membrane proteins...I have tried that before and had the same problem I will increase MeSH and or urea. Thanks again!