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#1 wildtype



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Posted 22 June 2011 - 02:03 PM

I am trying to run an EMSA and transfer the 43 kD protein (possibly bound to other proteins and to an 18 nt oligo) from a 6% native polyacrylamide gel onto a PVDF membrane. The gel was run at 100V for 40 minutes in 0.5X TBE (pH 8.3) for the EMSA. After this, I have been trying to use our lab's standard Western Blotting protocol: Soak PVDF membrane in methanol for 1 minute, soak everything including membrane and gel in CAPS buffer (plus methanol) for a few minutes, load into the cassette (cathode [sponge, filter paper, gel, membrane, filter paper, sponge] anode), run at 100V with ice pack and stir bar for 40 to 60 minutes. I added primary and secondary according to how others have done in my lab but am not seeing anything on the film.

Any suggestions? I was thinking probably lower voltage?

#2 chabraha



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Posted 23 June 2011 - 03:44 PM

You could confirm the presence of your protein of interest by incubating the oligo/protein mixture with an antibody directed against your protein and looking for a "supershift". I wouldn't expect your current method to work since the proteins in your TBE gel are not bound to SDS giving them the negative charge required for transfer. Maybe if you want to continue with your method you could soak your gel in an SDS containing buffer before transfer. You can check to see if you have proteins transferred to your membrane by ponceauS
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