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I have to perform a hydrolysis of BSA with the enzyme trypsin. As a control I want to inactivate the trypsin enzyme. Can I inactivate it permanently by boiling (100oC) for 10 minutes, or does it it require 1 hour of boiling? After inactivation, I have to do a test (with an active enzyme) and a control with BSA at 37oC). At this point the inactivated trypsin (the control) should not be re-activated. So my questions are (1) whether I can irreversibly inactivate trypsin by boiling, (2) how long I have to boil it, and (3) if the boiling process will irreversibly inactivate the trypsin.

AliceD
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kt123
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2 Answers2

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In my experience 10min at 100°C are sufficient, this is also supported by the paper linked below. Heat inactivation works by denaturing the protein and is therefore final. Depending on what you want to do with you sample, it might be problematic, as this is probably also denaturated at least to some degree.

You can also chemically inactivate trypsin, either by adding fetal bovine serum (which is done in cell culture), which contains protease inhibitors as α1-antitrypsin and α2-macroglobulin or by using commercially available inhibitors. Trypsin is also inhibited by calcium and magnesium ions, so your reaction buffers shouldn't contain any of them.

Chris
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  • The loading buffer for SDS-PAGE + heat(without dye) can also be used if little bit of SDS is okay. Or simply 2-mercaptoethanol + heat will also do [ref]. – WYSIWYG Nov 14 '14 at 18:25
  • Since the purpose of this buffer is the denaturation of proteins, this will definitively work. The color helps during the loading and also forms the "running front" in the gel later. – Chris Nov 14 '14 at 19:04
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You can consider these also:

  • Add 2-mercaptoethanol and heat at 95⁰ for 10 min [ref]
  • Add PMSF, incubate for some time and then heat for some time (lets say 70⁰ for 10min) to inactivate residual PMSF.
WYSIWYG
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