Try higher conc for loading. You should have control cells (non G2-M). You might find Cyclin-B1 degrdaded in G2-M arrested cells (still you'll see degraded band). Control cells should have right molecular weight band. No bands may be due to the following POSSIBILITIES..

1. low protein loaded (check by ponceauS staining)

2. Wrong transfer buffer usage (unless you see marker transferred/ponceau Positivity)

3. Wrong primary Ab

4. Short time primary antibody incubation

5. Wrong secondary antibodies.

I will try to rule out all the possibilites. Thanks Jinesh

Dear Jaya,

Positive control is crucial to find out what is wrong. I would ask the collegues around about not overgrown cells preps were working in their hands in any kind of western. I suppose they should express cyclin B1, since they divide.

Best - jan

Hi Jaya,

if I were you, first of all I would try to fix the western blotting technique as already suggested by Goodwin. However, you can do a time and concentration course to check the expression of your protein in your system. Some cells, if treated with Nocodazole for too long, start to cycle again or undergo apoptosis. These events could interfere with your system. Positive controls are needed (cycling cells should express good amount of Cyclin B1). You can also try to use other marker of G2/M, phospho-cdk1 antibodies for example, to understand if the Nocodazole/Colchicine treatment is working properly. 

Good luck for the experiments,

Alle