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Transcription factor assays without radiolabel - Can it be done? (Apr/11/2007 )

I need to measure the activity of E2F1 but I cannot do EMSA because we don't have a radionuclide license. is there any other way to do this?????


There are many non-radioactive EMSA kits. However, they often require additional equipment for the visualization e.g. CCD camera. I've used the lightshift assay from peirce.


Primer/PCR product/Oligo labled with DIG at the 5', 3' or both end work great. You do the EMSA as usual but transfer the DNA to a positively charged nylon membrane and detect the DIG label with anti-dig antibodies conjugated with alkaline phosphotase. The detection can be done with cheminulescence substrate (e.g. CDP-Star) or chromogenic substrate (e.g. BCIP-NBt).