I'm extracting protein from highly hazardous bacterial samples.
For the safe of safety, people commonly boil this bacteria prior to full lysis when doing protein extraction.
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?
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.
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.
Some of the protease inhibitors may be inactivated by heat, others not. If there is no strong reason to omit them (cost?), then why not include them?
If the bacteria of interest are not thermostable, probably you can deactivate them below the boiling temperature.
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