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Problem with EMSA - Nucleic acid becomes faint, but no shift occurs

Problem with EMSA - Nucleic acid becomes faint, but no shift occurs - Protocols and Methods Forum

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Old 12-20-2012, 07:55 AM
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Default Problem with EMSA - Nucleic acid becomes faint, but no shift occurs



Hello,

This is my first post.

I am trying to perform an RNA/protein EMSA. The nucleic acid is a 20 bp RNA dimer. The RNA binding protein is recombinantely expressed and purified from E. coli, its MW is 38 kDa. The expected Kd should be in the units of nM. Every time I run the gel, I don't observe any change in the mobility of the RNA. I do see a decrease in the intensity of the RNA band at high Protein/RNA ratios, as well as some smearing on the side of these lanes that goes up all the way to well. The protein is nicely folded, and not aggregated in any way, and behaves nicely in solution. I tried running the mixtures on 15% as well as 10% polycarylamide gel with different acrylamide to bis-acrylamide ratios (29:1 and 37.5:1), as well as on a 2.5% agarose gel with variable pH values of the running buffer (8.0 and 6.0) but the problem presists, I keep seeing no shift of the RNA just the band becoming more faint as you add more protein. My binding buffer conditions are 50 mM phosphate, 80 mM KCl, 1 mM DTT, 5% Glycerol, pH 7.2.
I would appreaciate any comments or suggestions.

Thanks a lot in advance,

Alex.
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