Odd secondary pellet formed after lysis with B-Per or BugBuster - (Mar/01/2013 )
I've got an odd problem. I'm evaluating multiple lysis methods (sonication, Lysozyme w/Benzonase, SoluLyse, B-Per, and Bug Buster) on induced and non-induced BL21 (DE3) cells. The soluble lysates were withdrawn without disturbing the cell pellets after centrifugation at 14,000 x g for 20 minutes and stored at -80C overnight. With the B-Per and Bug Buster samples (which both lysed very well) when the samples were thawed and warmed to RT, a sizeable loose pellet formed at the bottom of the tube. I ran SDS-PAGE on it's sup and resuspended and purified the resuspended material and am confident it doesn't contain my protein of interest - and the pellet formed in both induced and non-induced samples. I centrifuged the 'soluble' lysate again (14,000 x g for 5 min) and the diffuse pellet became very small and smeared to the side of the tube. I withdrew the lysate and attempted to resuspend the pellets both in TBS and in SDS Sample buffer and it won't resuspend. The pellet dislodges from the side of the tube but stays flat and breaks into flat flakes when mixing. I've never seen a pellet that looks like these. Can anyone tell me what this pellet could be?
for those two methods, did you collect the supernatant as well? are you sure what you see is not genomic DNA?
I did collect the supernatant (if by that you mean the soluble lysate). How would I know if it were genomic DNA? I added DNAse (Benzonase Nuclease) to the lysis buffer so I would have expected the genomic DNA to be chopped up. Would small fragments of DNA still form a pellet and would that pellet be expected to basically be insoluble?
Since I wrote the initial post, I took the soluble supernatant from the second time it was spun (so the secondary pellet was removed) and froze it again. Upon thawing the same type of pellet formed again. If the pellet resuspended normally, I would say it is a new precipitation reaction of host proteins not liking freeze/thaw cycles but the pellet is so odd.....