Nuclear extract problems - (Dec/22/2011 )
May be someone could spare a bit of time to share experience on nuclear extract preparation. I tried to get it from primary leukocytes according to a published protocol that in principle involves 2 buffers. A brief incubation with a hypotonic buffer and NP40 to lyse the cells and then a longer lysis of the nuclear pellet. In the end however I got very little protein. The Bradford assay estimated only about 2ug per ml from 5 million cells. I think that I lost proteins in the course of the extraction. The protocol calls for resuspension of the nuclear pellet after the first lysis. I found it very difficult to do as the pellet was extremely sticky. I pipetted it up and down for 15 min, incubated on ice 20 min and then continued for to pipette for another 10 min, but there were still insoluble clumps present. Your advice is most appreciated! Any links to good protocols to get nuclear extracts from limited quantity of cells for emsa? Thank you! Merry Christmas!
It is very likely that you are loosing quite a few nuclei at the first lysis step - the stickiness is DNA which has been released from the nuclei during lysis. You may have to play with the detergent concentration and buffer components for your cells, as these can vary a bit with cell type.
You will only get a tiny fraction of the total protein content of the cell when you are doing a nuclear prep anyway - think about the size of the nucleus relative to the cell overall!
2 ug/mL is indeed really small.. I wonder what is the volume of your resuspension buffer to lyse your nuclear extract? I wonder if you are using hypertonic buffer to lyse your nuclear extract? 3
Thank you for advice! The nuclear lysis volume was 50 ul. Both first and second lysis buffers were based on hepes, edta, nacl and kcl. To the first I added np40 and to the second glycerol. I don't have at the moment the protocol with me so can't check the exact concentrations. I start to think that the first lysis (cell) could have been too harsh. May be I should try with Tris and succrose based first buffer or use less detergent?