Troubleshooting sds-page for high mw protein - SDS-PAGE (Sep/10/2004 )
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.
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.
Probably you know that but somtimes SDS precipitates membrane proteins when boiling samples. Could this be the problem?