2 replies to this topic

[Nandinha]

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.

[mujan]

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.

[sarvikvana]

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