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Problem in using EMSA to assay protein fractions - (Aug/15/2005 )

Hi everybody,

I am purifying a novel transcriptional factor from a bacteria. What I am doing is to use ammonium sulfate precipitation and then chromatography steps.

In the very first beginning, I tried to use cation exchange chromatography. However, I found it is very difficult to use EMSA to assay the presence of DNA binding activity in the protein fractions. Even though I have already dialysis the fractions, I found that the protein will give a smear in the EMSA gel.

At first, I think that the protein is overloaded. Therefore, I use a smaller volume/mass of the protein during the EMSA. Unfortunately, it did not work.

Conclusively, after cation exchange chromatography and dialysis, a small amount of protein load will give no band, while a higher amound protein load will give a smear.

What can I do? I cannot assay my fractions! I have checked the conductivity of the sample with my buffer. They are the same, which means the sample is perfectly dialysed! Also, I got the same result after gel filtration, which supposes to have no physical environment change, right?!

Please help. Thanks >.<~~~


Maybe try increasing the reaction volume (and therefore the amount of the 5x binding buffer in the reaction) this might compensate for any effect of the sample buffer. Hope that helps.