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Troubleshooting sds-page for high mw protein


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#1 Nandinha

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Posted 10 September 2004 - 01:12 AM

Need help separating a membrane protein of approximately 220 kda. I have tried boiling my samples for 3-15 minutes prior to loading as well as reducing the acrylamide % of the resolving gel to 6%. However, the protein of interest (as identified with blotting) does not move out of the well into the resolving gel after running the electrophoresis for over 3 hours at 180-200 volts.

The longer the boil, the greater is the smear at the top of my membrane (after blotting). Anyone has any idea of what the problem could be? I used the Mem-Per kit from Pierce to isolate the proteins and the solution B for this kit to dilute my sample prior to boiling.

Thank you for your suggestions in advance.

#2 mujan

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Posted 23 September 2004 - 11:36 AM

I buy 4-20% gradient gels which work well. There are also other commercially available buffer/gel systems specially designed for high MW proteins. It might save you trouble to by a premade gel. Also, if the pKi of your protein is similar to the pH of your transfer buffer, it won't move.

#3 sarvikvana

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Posted 18 October 2004 - 01:23 AM

Probably you know that but somtimes SDS precipitates membrane proteins when boiling samples. Could this be the problem?




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