I purified a recombinant RNA binding protein from E.coli, I worried that my RNA-binding protein was contaminated with host RNA during purification. I need to remove RNA completely, I have treated with RNase A and use 1M salt in the buffer, and How can I check whether there is still RNA in the protein solution during purification? such as gel electrophoresis or NanoDrop based on 280/260? Thanks a lot.
Hi there,
260/280 ratio is a good reporter. If protein sample is pure, ratio should be 0.57 if contamination exists ratio raises quickly (1.06 if 5% nucleic acid contamination)...
May be PCR with primers, specific to E. coli RNA (16S rRNA, for example). However, it is important to be sure, that PCR reagents itself do not contain E. coli.
I agree with Igor. PCR is the simple and easiest way to confirm contamination. However, it is hard to establish whether or not your protein of interest is bound to RNA. Cross linking of RNA is necessary to stabilize RNA bound to proteins. Therefore one can assume to certain extent that the protein is free. However, I am curious as to how you are proceed on studying your protein since I am not sure if RNAse can be completely inactivated. Thanks.
Hi there,
260/280 ratio is a good reporter. If protein sample is pure, ratio should be 0.57 if contamination exists ratio raises quickly (1.06 if 5% nucleic acid contamination)...
I agree with Dominique.
You can remove your RNA-DNA contamination using cation or anion exchange chromatography (CECH or AECH). If you use anion ECH (MonoQ, Q-sepharose HP )you will bind the RNA contaminants to resin. CECH (MonoS, SP sepharose) will probably bind your protein (if it is basic) and the RNA is in ft. (By choosing right buffers).
I disagree with the PCR approach. Your sample even after RNase treatment does contain a little bit of RNA, but I think that the amounts of it are so little so you shouldn't have any problems in the downstream experiments.
PCR is very sensitive and will give you for sure positive results but that does not mean that your sample is contaminated to the extent that you can't use it for your purposes.
I would measure the 260/280 ration, repeat the RNase treatment and measure again the ratio. If you don't see major changes in the ratio that means that you got rid most of the RNA and you're good to go.
Don't chase after PCR, you'll only get frustrated.
I agree with Dominique.
The 230/260 ratio can also be useful, especially if your protein contain no or very few aromatic amino acids.
Dear Zixun Han, no doubts - your protein preparation does contain RNA. What is a degree of "completeness" that you want to achieve (purity %). Checking the UV absorbance is OK, but depends on the properties of your protein itself: if your protein contains many Trp, Phe and Tyr, than the sensitivity of this approach goes down. I agree with George - PCR is going to be too sensitive.
To get rid of RNA, we utilize hydrophobic chromatography on butyl- or phenyl- sepharose. If you don't mind, than purification under denaturing (7M urea) conditions is your choice.
Hi ,Zixun Han,i agree with Kormal ,you can seperate RNA and protein by chromatography.
If you have indeed degraded all the RNA with the RNAse, the nucleotides remaining should be easily size-separated from the larger protein with gel filtration /desalting. Check 260/280 before and after, but use a more accurate photometer than NanoDrop.
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