Recently l was studying EMSA assay, but l didn't find any detailed information about protein sample. we can use two kinds of protein, including crude protein extracts and recombinant proteins. but l cannot determine which kinds of protein is better? also l am confused how much concentration of protein to be used for EMSA binding reaction? what's more, if l use crude protein extracts, l should transfect how much recombinant plasmid into cells?
Hi,
The concentration depends on your protein of interest (25-200 nM of purified protein). Please, check this publication 10.21769/BioProtoc.838. Best wishes.
Dear Min,
There is no general rule how much you use. In case of the transfection it depends on the method, the size of your plates ...
If you have no idea whether your transfection works you should establish it with an EGFP-epression plasmid. If it works, use e.g. 1 and 4µl and compare your result with an untransfected control. Thats at least a start.
Concerning your recombinant protein a few femtomol should be enough if your protein is active (fully renatured). Give it a try with 1µl of your protein and a few dilutions.
Good luck!
I have performed EMSA Assay using EMSA Kit supplied by Invitrogen where I used Plasmid DNA of about 50 ng and E.coli BL21 expressed and purified protein at a varying concentration ranging from 100 ng to 1 microgram. I think EMSA assay with crude protein will not give the desired result which you are hoping for, However, a purified protein on the other hand is much more recommended for studying DNA-Protein interaction. I have attached the pdf file for you to understand. Hope it helps
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