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Western blot problem: not running in gel


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#1 westernbrad

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Posted 05 November 2004 - 01:26 PM

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

#2 Sprag

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Posted 06 November 2004 - 01:14 PM

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,

#3 Mendez

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Posted 06 November 2004 - 01:18 PM

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

#4 aleruiz

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Posted 06 November 2004 - 05:11 PM

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.

#5 westernbrad

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Posted 08 November 2004 - 07:54 AM

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!




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