I am purifying protein which has RNA in its bound form. How can I remove this bound RNA from protein without affecting structure of protein. RNase treatment is not much effective.
If you have a tried and tested method for renaturing the protein, you could denature it in urea, pass the solution through an anion exchange column and renature the protein from the pass through (RNA should bind the column, and the protein -being able to bind RNA- is probably rather basic so it shouldn't bind or at least should elute at much lower salt concentrations than the nucleic acid).
Also, if the protein has a catalytic bind/detach cycle fueled by ATP or GTP hydrolysis, you could try adding either ribonucleotide triphosphate in the presence of RNAse. The RNA should detach and then be chewed off. Or you can do the same under conditions unfavorable for RNA binding to the protein; those obviously depend on the specific nature/function thereof. Or do that in combination with AEX chromatography or any other method that can pull the protein and the RNA apart (gel filtration, ultrafiltration, electrophoresis, etc).
Hope it helps!
Have you tried different type of RNAse ? Maybe combination of them could also work.
Anyway, I think situation is a bit complicated and as suggested by Alejandro denaturation and renaturation step is required.
if you protein is his-tagged a high salt wash before elution could remove the RNA
Hi have you used column of Affi-Gel Blue and batchwise elution its a rapid and effective early procedure for removal of nucleic acid.
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