Hi Meenu,

we tried non-radioactive labeling and the only one that worked out reasonably was with biotinylated dsDNA probes that are detected by Strep-conjugate detection methods.

We also used 1 μg poly(dI-dC) for EMSA, which gives a comparable signal with 2 ng of 33P-radioactively labelled DNA probe.

My general impression with EMSA is that protein amount is not so important, as this qualitative method is mainly limited by your detection system: Not all of your protein might interact with DNA and you will probably use your DNA-probes in excess. Therefore, the relatively few molecules of DNA that shift need to be detected.

For other methods we used Sybr Green to quantify our DNA-standards and we are able to detect as little as 5 pg (~2pmol) of 25mer dsDNA. Which is in the range of most shifted EMSA-bands.

Anyway, the analysis of complex formation at the DNA was one of the reasons why we started to unite different protocols and proposed the DPI-ELISA for non-radioactive DNA-Protein interactions in vitro.

Hi Meenu,

Did you find some solution for your EMSA. I am using the same kit. I have tried EMSA with total nuclear protein extract plus 20 mer and also with 250mer probe. Proteins remain stuck in the well just I can detect the free probe in the gel.:(

Hey Farah, i'm still facing the same problem, proteins remain stuck in the well.i tried with 4%gel also but no result, though normal proteins(not nuclear extract)stains good wiith sypro ruby but not the emsa sample. Do let me know the modifications if you get the results. Thanks.