protein precipitated - (Mar/14/2007 )
hi all! having spent the past 2months optimising the yield of my protein from BL21 pLYS i have encountered a problem that my protein precipitaes out. after a long day yesterday left my crude protein extract in at 4C o/n ive returned this morning to find it precipated. can i resolubilze it? AND how do i stop this happening again? thanks in advance!
What do you use for solubilisation? If for non native, try to use guanidine HCl. For native, should be imidazole.
And did it make any difference in your results when you re-precipitate it? If not, then no need to worry about that.
proteins are not in true solution. they are in suspension. they can, and do, settle when left sitting. it is especially noticed when the protein suspension is high concentration (as most crude extracts are).
you can usually resuspend the protein with gentle mixing.
however, the precipitation may also be caused by denaturation of protein. so, after mixing, you may want to clarify your suspension.