PMSF or liquid nitrogen - (May/29/2006 )
i have been guided to grind my protein sample in liquid nitrogen without using PMSF but what about the serine protease that ooze outta cells in the grinding process to cut off my required protein sample..the first thing is why we r using liquid nitrogen instead of PMSF...does it fullfil all the requrement for protein grinding...one of my collegue says that serine proteases require optimum condition to chop proteins..then whats the use of using PMSF ??????might be simple to u protein guys...comeon post a reply.
as long as your sample stays in liquid nitrogen (or at -80C), there is no need to add PMSF. serine proteases need an aqueous environment to do their dirty work, so having your sample frozen by liquid nitrogen will take care of that problem. just be sure not to let your sample thaw while grinding. always keep it cold. if it looks wet, you're in trouble. just keep it as a powder and either on liq N2 or at -80 until you want to extract your proteins. when you do, then use PMSF and some other protease inhibitors in your extraction buffer. also, be sure to note the pH of your buffer when you use PMSF...its half-life is short in buffers w/ pH of 8 or higher, so take note of that.
if, by chance, you're adding buffer to your liquid nitrogen, grinding that and letting it thaw, then yes, use PMSF and other inhibitors, though at that temp, PMSF will precipitate and be useless. you're better off not using this approach.