Base on my understanding, PMSF is an inhibiror for serine protease. I notices that some proteins (eg. ana Fld) requires PMSF in its purification process, usually during the sonication step. Other proteins such as Myoglobin and photolyse do not require PMSF addition in their purification process. I would like to ask why? and when does PMSF needed to be added?
Thank you for reading and helping me with this question !
Myoglobin is proteolytically resistant, sometimes this can be attributed to functionality of a protein.
Binding type proteins, such as myoglobin or streptavidin are remarkably stable under conditions that would result in rapid degradation in other proteins. For example myoglobin needs treatment with both trypsin and Lys-C for to fully digest into peptides, with Streptavidin a recognised purification step is heating to 75'C for 10 minutes.
For the majority of cases when purifying proteins use of protease inhibitors is best practise until you can establish whether it is necessary or not (if you have the luxury of having the time to do so).
You'll need PMSF when you preliminary expression trials show that you have fragmentation and PMSF fixes it. Sorry if this sounds cheeky, but in my mind there's no better way of saying it. You need to do small scale trials, before you have any chance of consistent success in your protein expression experiments.
In my experience, when using BL21, a standard protease knockout strain, you sometimes do see fragmentation of your protein. But protease inhibition almost never helps. Note that this is not particularly surprising in a protease-deficient background.
I don't know what the mechanisms of the fragmentation might be. It happens only to certain proteins, not others. There could be regulated, context-dependent proteases that somehow escape inhibition. Or it might be ribosomal, and not protease-dependent at all.
Dear Xiu
it is difficult to predict which protein require protease inhibitor as PMSF, it depends from the protease susceptibility of the protein. There are some scientists that add protease inhibitors every time while other, like me, do not use it in the first purification trial and add it only in case that this attempt show degradation issue.
When you use pmsf remind that its time life is very short at room temperature in water solvent ( about 30 minutes) therefore it will protect the protein only beetween cell lisys and first purification step and you need to minimize the time beetween cell lysis and coloumn load.
Today more complete mix of protease inhibitor edta free are available on the market ( eg https://www.thermofisher.com/order/catalog/product/A32965#/A32965) and also if it are expensive, in case you face degradation issue, i suggest to use it instead pmsf.
good luck
Manuele
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