I am expressing a human RNA binding protein in E. coli and purifying it using ammonium sulfate precipitation followed by heparin, butyl, and size exclusion chromatography. While the first batch of protein purification did not show any RNA contamination, subsequent batches consistently exhibited RNA contamination. I have tried to maintain the same purification protocol but cannot seem to eliminate RNA contamination.
Have other researchers experienced this issue while purifying RNA-binding proteins, and if so, do you have any suggestions for troubleshooting or improving protein purity? I would appreciate any insights or recommendations to help me achieve a pure sample of my protein of interest.
I suspect you have the answer already; since the earliest prep didn't contain RNA you must look carefully for differences in the subsequent preps. If you can reproduce the 1st prep exactly you should see similar material. See also a good discussion here: https://www.researchgate.net/post/How-to-remove-the-RNA-from-the-purified-protein
Edward Michelini Thanks.
I replicated the exact protocol for subsequent batches however, except for the first prep, the rest of the four preps are contaminated.
I found the link helpful. Thank you.
I'd consider including an anion-exchange chromatography step. The highly charged RNA should bind tightly to such a column.
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