Emsa

CCathyxyz1 · #1 (**permalink**) 03-20-2008, 01:58 PM

Hello;

Would anyone help me answer a question about gel shift? I've been doing 32P labelled shifts. I can get very minor (1%) portion of my probes shifted on the small electrophoresis gels (5cmx7cm). However, when I run my EMSA gels on the big gels (20 cm x10cm) I no longer see the shift. Have you encountered this problem? Thanks for your help.

kmunson779 · #2 (**permalink**) 03-20-2008, 02:52 PM

I suspect your binding is not very good, and the longer time/more electricity it takes to run the longer gels is letting the complex fall apart. Could you try optimizing the EMSA by, for example, reducing the salt concentration, adding cofactors like Zn or Mg, or running a lower percentage gel?

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Electrophoretic Mobility Shift Assay Forum

EMSA Forum. Discuss the Electrophoretic Mobility Shift Assay protocols or technique and the assessment of DNA and RNA binding proteins here. Also post questions and troubleshooting about the shift assay.