Hi,
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.
Its very strange phenomenon. After adding cell lysis buffer, how long u incubate cells on ice. You can try one thing, next time add lysis buffer, incubate cells on ice for 10 minutes and then collect everything in 1.5ml eppendorf tube and centrifuge for 15 mintutes at 13000 rpm. Collect supernatant and measure the protein amount in it.
After adding lysis buffer, I keep the cells for 30 min on ice and then centrifuge at 14000 RPM for 15 min.
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Biochemical Techniques