I am planning to digest proteins that might be complexed with DNA with Qiagen protease followed by DNase treatment. As it is a serine protease, I'm thinking of using PMSF to chemically inactivate it. I do not wish to heat inactivate (70C for 15 min.) as I found that high temperature experienced during protease treatment leads to enhance DNase degradation of DNA compared to without heating (kept on ice).
Does PMSF interfere with DNase if stock (dissolved in isopropanol) is diluted 1:20 up to 1:100 dilution?
Thanks in advance!
"""I found that high temperature experienced during protease treatment leads to enhance DNase degradation of DNA compared to without heating""""
If your purpose is to do DNAse treatment, removal of protein will enhance DNAse activity. Why is this abnormal? Also, heating at 70C for 10 mins is the recommended method for QIAGEN enzyme. PMSF may not completely inactivate the protease.
Can you clarify your staments?
AEBSF is a slightly stronger inhibitor. Perhaps best to run some control experiments with a colorimetric or fluorogenic protease substrate to verify that you have fully inhibited the protease for your specific purposes. Also, checking for issues like unexpected interferences are important -- and you should be sure to include such controls in your overall experimental design. (Just because you don't find literature evidence for an interference, or lack of interference, doesn't make it so for your specific case!)
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