Does the absence of cyclin B1 in western mean that the concentrations used were too low?

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Goodwin G. Jinesh

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.

Jaya Aseervatham

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

Jan Piwowarski

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