problem with cell synchronization - (Sep/19/2014 )

Hi,

Iam using nocodozole (20-100ng) and colchicine (0.5-2.5uM) on C3H10 cell to arrest the cells in G2/M phase for 18-24 hrs. Then I do a western for cyclin B1. What I found was that there was no cyclin B1 in the western. Does this mean that the concentration that I have used is not enough. This concentration I saw some papers and then fixed. What should I do now. Help needed.

Thanks,

jaya

Okm- you've been here long enough to know this... please don't double post. Bob.

Tell us more about what you did, especially with regards the western. Have you used the antibody to sucessfully detect cyclin B before?

I used the same antibody that we use for immunofluorescence, which gave us a positive result.

So you got some signal on IF - how do you know that that signal was specific?  Proper validation of antibodies is very very important,especially for IF/IHC!

i dont know if the signal was specific. this ab was used  in the lab earlier. so assumed that it would work. i also got a new ab from Abcam,  but didnt get signal even in that.