I'm trying to have a good concentration of a supernatant from Jurkat cells. I've lysed 5 million cells in 500 ul of RIPA BUFFER (almost standard RIPA I would say) and it was already sticky. Than I sonicated (3 times 30" and ice in between). Still on the same situation. Then I tried to pellet it 12000*g 20 min but I was able to obtain only 200 ul from the total amount.
Any advice?
Thanks
Giuseppe
could be chromosomal DNA. On the other hand, sonication should shear the DNA. Try some DNAse. I'm normally lysing my cells directly in the well (6-well plate). I use a pipett tip to "swirl" the DNA into a clump that can be removed.
Good luck!
u could try homogenization at around 5K rpm for 5 mins.. then spin it at a high speed.. but i dont think u ll b able to recover entire 500 ul of supernatant.. still u should get more than 350 ul..
all the best
Thank you everybody for the suggestions. I'm testing your suggestions to ensure that everything is fine with my protein of interest. Just for information I have tried to use a quick subterfuge suggested in a protocol I've recently read and it looks working good. The use of some ampholyte.
Kind regards
Giuseppe
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