Problem in EMSA

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kashif
kashif's picture
Problem in EMSA

Hi everybody
I am doing EMSA (using pierce Lightshift kit 20148) to study activation of transcription factors (NF-kB and AP-1). Every time I see the shift but the bands are always near the well and I am unable to see the supershift with the addition of antibodies against transcription factor subunits. Firstly I thought that amount of protein is too much but decresing protein changed nothing. I am using BioRad Mini protein 3 Cell electrophoresis system. Could you please suggest something?
Thanks in advance.

samm
samm's picture
If your problem is merely

If your problem is merely bands too near the well, the solution is simple - just decrease the gel percentage, and/or run the gel longer till the dye front and an appropriate size of a prestained marker has run out.
Also, rather than using something like the mini-Protean for supershift assays, you might want to try using a larger gel for supershift assays.