I am using pichia to express protein. After using the bead beater, I found some of my protein has been denatured. I used 1 min break and 3 min cooling for 3 times. I am wondering if I miss anything. Thanks.
Try disruption with a French press, if you have one available. It avoids foaming, which is never good for proteins
generally for Pichia cels bead beater is best. Make sure you perform all the lysis in the cold room, keep the cels and eluate cold on ice after lysis. I do it Thrice or four times and then I check under microscope if the pichia cell are sheared.
For denaturation, you can also try probably by changing the buffer pH or content. Make sure you add PMSF to the lysis buffer. It generally works well.
Good luck!
first after the beating is done collect the beads, autoclave, then wash rigrously in water. if you want you can dip it O/N in 1NHCl (We do it generaööy with fresh eads, when we dont know how clean they are).
Throughly wash with water again. A Lot of water
then air dry the beads, spreading on paper, autoclave the dried beads.
now they are ready to use again
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