Has anybody done western blot on calmodulin? - (Mar/25/2003 )

Hi everybody,

I'm trying to do a western blot on calmodulin using a monoclonal antibody from
Sigma, but I couldn't get any signal either by our routine western procedure
or by following the protocol from Sigma. Does anybody have experience doing
this? Is there any trick for the transfer or blot of calmodulin? I would
really appreciate it.

HI,
I have been working on CaM western blot for a while now.. But I haven't had any real success. However, I thought maybe we can still help each other.
I was wondering if you are running any positive control, i.e. a purified CaM like Bovine CaM. I do, and it works all the time.. I only have problems with my extracted CaM from the crayfish tissue.
I was also wondering what type of buffer are you using for transfer and what type of membrane?
In addition you might want to go as high as 1:100 dilution of the primary Anti-body to see something.... I have been able to see faint bands at this concentration... but not always.
Hope that this helps.
Thareef.