I routinely perform pulldowns in order to purify a protein of interest, and afterwards I digest linear nucleic acids attached to my bait protein using micrococcal nuclease (MNase). So far I have performed digestions after eluting my protein of interest from the resin, with satisfactory results.
I now plan to perform a in-resin MNase digestions, without previous elution of my target protein. Has anyone used MNase in-resin? I wonder how much the digestion efficiency could be affected by the resin itself, which can be critical in order to obtain reproducible results. Any tips regarding how to optimize the reaction?
Thanks.
Hello, did someone contact you to answer you? I could ask you a question, I am trying to degrade an RNA naturally resistant to degradation by RNAses, I wanted to use MNase but it only degraded a little, my sample is in 94uL of RPMI and I added 5uL of Reaction Buffer to a final concentration of 50mM Tris•HCl pH 8, 5mM CaCl2 and heat 30 min at 37 degrees in the water bath. I don't know if I'm doing it right, please could you help me, I don't know anyone who works with this enzyme, I've tried others and it degrades the RNA a bit. I stay tuned, thank you very much
Your buffer composition seems fine, although you could increase the CaCl2 to 10 mM and see whether it increases the degradation rate. How much MNase do you add? I use 1000 units per RNA mg.
I am unfamiliar with RPMI, but I believe it contains a bicarbonate buffering system that keeps physiological pH at 5–10% CO2. Do you run your digestions under these conditions? I wonder whether the pH remains near-physiological in your reactions and whether MNase is more pH-sensitive than other nucleases.
Yes, increasing the buffer sounds to me, and I am putting only 28.9 ug of synthetic RNA, I have tried 5 U and 50 U of MNase. I prepare the buffer at pH8 with the intention of helping me keep it stable because MNase works between 8-10... it could be that the pH is lowering during the reaction. Thank you very much for your recommendations Mikel, thanks for answering me.
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