I'm extracting protein from highly hazardous bacterial samples.
For samples like this, people commonly boil the bacteria prior to full lysis.
Because the bacteria is boiled, do I still have to add protease inhibitors to my lysis buffer to prevent protein degradation?
Or does the heat inactivate all the proteases?
It depends on the type of strain you are working on. Many thermotolerant strains have also thermostable proteases.
Are you sure that your protein are stable after boiling process?
As proteins begin to denature when heated, they become more susceptible to proteolysis by whatever proteases are more thermostable than the proteins. I think you should add the protease inhibitors.
Thank you very much for the answers.
Leilei Zhang
It is not a thermotolerant strain. I'm not sure of the stability as this is all new stuff I've never tried before. I'm trying to copy published protocols, and none mention any protease inhibitors.
Adam B Shapiro
I don't think I can add protease inhibitors prior to boiling as the inhibitors are thermo-sensitive. Is that correct?
But I should still add some inhibitors after boiling in case some proteases survive?
Thank you again.
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