Hi everybody, recently I performed an RNase A treatment before IP at 37℃ for 30min. But when I take out the tube from the water bath, I found there is protein precipitation in the tube. I add 10ug/ml RNase A in the whole cell lysate. So who can tell the reason? I want to excise the RNA before native IP. Thank you!
Can you tell some more details. What cells or tissue is used for lysis, which lysis buffer is used, did you add other ingredients while adding RNase? In other words, please share your protocol and all steps of the treatment. Did you do this lysis before without this trouble?
The lysis buffer(50 mM Tris-HCL pH 7.4, 150 mM NaCl, 1% TritonX-100, 5% glycerol, 1mM EDTA), 30 min, rotating, 4 ℃, 12,000xg centrifuge, add 3ul RNase A to the supernatant, 37℃, 30min. The control supernetant without RNase A, but no protein precipitaion, it's normal. It's mouse embryonic stem cells.
That is strange indeed. Maybe the concentration of the initial lysate is quite high. You might want to try and dilute the lysate 3 times. Look of the precipitation still occurs. Either add less RNase to the sample. Did you know the NaCl concentration alters the effect of the enzyme: check out the NOTE on page 2 of this leaflet: http://www.thermoscientificbio.com/uploadedfiles/resources/en053-product-information.pdf
Which company makes your RNase? Does thekit provide a buffer? If so, do you use this buffer in the sample with and without RNase?
Young Wu , can you please tell me why you have added 0,5% sodium deoxycholate? did you substitute triton with it or what? I am having the same problem...
Hi Young,
Why don't you add the RNase A directly into the lysis buffer?
Have you tried to add RNase A into the lysis buffer immediately before the cell lysis?
Thank you!
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