My protein is an RNA binding protein which necessitates removal of RNA for its activity.
I assumed by affinity chromatography that RNase A would be removed as it is an affinity-based separation, but I still have residual RNase A activity which is hurting my assay.
If I understand correctly, you are adding RNase A to your protein at some point during the purification to degrade bound RNA, then trying to remove the RNase so that it will not interfere with subsequent experiments. I recommend different approach to stripping the nucleic acid off of your protein, avoiding the addition of RNase. Put your protein through an anion exchange column, such as Q-Sepharose. This resin binds nucleic acids quite strongly. A salt gradient will most likely elute your protein well before the nucleic acid elutes.
If you don't want to do that, add an RNase inhibitor (see one source in the link). You may want to use this in any case.
http://www.lifetechnologies.com/us/en/home/life-science/dna-rna-purification-analysis/rna-extraction/rna-extraction-products/rnase-inhibitors.html?CID=fl-we113374
Hi there
I agree with Adam. However I will suggest you to use an heparin-sepharose column to eliminate any DNA or RNA-binding protein, which is the case of RNAse. This colum works very well, since heparin is a polyanionic polymer that can mimic the RNA. If you are using GE-Healthcare systems, they have already prepacked columns of 1 mL, which are relatively cheap.
Goooood luck. Best
Paco
I agree with Paco and Adam. Avoid RNase A like the plague. It is notoriously sticky and active at homeopathic concentrations! I used to purify recombinant His tagged RNA binding proteins simply over Ni-NTA in the presence of 1M NaCl. The salt stripped most of the RNA and was fully reversible. May work for you too, and is sooo much easier than extra columns.
Good luck!
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