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!!!!
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...
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.
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!