Can someone please tell me how to make a cell lysis buffer without PMSF I have sodium deoxycholate but no PMSF. I also have NP-40. Please tell me an appropriate recipe one which does not include PMSF!! Thanks
PMSF is a serine protease inhibitor, and it would be helpful to know the reason why you do not want this in your lysis buffer? AESBF is an alternative serine protease inhibitor (less toxic as compared to PMSF), however I suppose that your experimental setup means you will have to avoid this one as well. I agree with Rodrigo above that keeping lysates on ice is a good idea to limit proteolytic activity in your lysates. You may include 250-270 mM sucrose in your lysis buffer in order to stabilise lysosomes, which would decrease the chance of lysosomal proteases acting on your protein(s) of interest during lysis. Depending your the nature of your experiment, you could also include chloroacetamide or NEM which will alkylate reduced cysteines and inhibit cysteine proteases. Best of luck!
PMSF, as well as TPCK or EGTA in a cell lysis buffer, is a protease inhibitor. You can try to do your lysates maintaining the cells at 4`C in order to avoid the lysis of your proteins by lysosomal proteases. Another advise: check the composition of protease inhibitor cocktails (e.g. Roche) to verify if they contain PMSF.
PMSF is a serine protease inhibitor, and it would be helpful to know the reason why you do not want this in your lysis buffer? AESBF is an alternative serine protease inhibitor (less toxic as compared to PMSF), however I suppose that your experimental setup means you will have to avoid this one as well. I agree with Rodrigo above that keeping lysates on ice is a good idea to limit proteolytic activity in your lysates. You may include 250-270 mM sucrose in your lysis buffer in order to stabilise lysosomes, which would decrease the chance of lysosomal proteases acting on your protein(s) of interest during lysis. Depending your the nature of your experiment, you could also include chloroacetamide or NEM which will alkylate reduced cysteines and inhibit cysteine proteases. Best of luck!
Hi Mirza,
I agree with above suggestions and strongly encourage you to check the appropriate protocol to prepare you lysate buffer for your specific goal. The lysate buffer really changes protein extraction and purification results. In case, you can lysis your cells in low temperature and try to finish your experiment in the shortest time.
GOOD LUCK
1% NP40 is probably fine for making a cell lysate. It just depends what you want to accomplish. As many proteinase inhibitors as possible is good to have in the cell lysis buffer, as long as it does not interfere with your procedure and what you want to accomplish. PMSF is not absolutely necessary. But most people use protease inhibitor cocktail mixes such as from Sigma or Roche Diagnostics.
Even with pmsf on ice there is protease activity, minimal, but I have added a cocktail tablet and pmsf before and had a marked difference in the amount and quality protein imaged with Western Blot. RIPA buffer (with NP-40) is the most common lysis buffer. Your cells will lyse without pmsf, but you should try to find a protease inhibitor to use to protect the lysates during the process.
PMSF is necessary only to inhibit the proteases. You can lyse the cells only using Triton x-100, or just homogenize the cells by passing through the syringe. NP-40 is also good, but it is more mild, than Triton. The lysis protocol depends on what material you have and what you want to obtain. Please, be more specific.
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