cell lysis problem - (Oct/03/2014 )

Iam using MC3T3 E1 cells for my experiment. For an experiment I was growing the cells for 10 days. Daily I used to collect the cells for western. On the 5th day, after I added the lysis buffer, there was a precipitate formed in the tube, which was not till the day 4. I repeated the expt with different plate thinking that I made some mistake, but the answer was the same. When I asked my boss, he said its just the ECM being formed. Is this true. If this is true, why didnt I see it in the culture flask, when the cells are grown for more than 5 days. Has someone faced the some problem.

Precipitate forming when? Before adding to the cells, or after lysis is complete?  If the latter - assuming you are using a fixed volume of lysis buffer per plate, the cells are proliferating so there will be more cells present at the later times than earlier, hence the cells won't dissolve so well. You may have to modify the amount of buffer you use or think about lysing based on cell number (lift and count the cells) rather than using a defined volume.

Thank you.