FLAG immunoreactivity in ICC - (Oct/13/2005 )

I'm using the Sigma M2 FLAG monoclonal antibody for immunocytochemistry, but I seem to get nuclear staining when the antibody shouldn't.

Has anyone else had this problem and if so have you remedied it?

Any help would be appreciated.

Moz

-Moz-

QUOTE (Moz @ Oct 13 2005, 08:40 AM)

I have used the FLAG antibody (M2 clone) from Stratagene extensively. I don't get any nuclear staining. However, are you seeing nuclear, OR are you really seeing Golgi/ER labeling??

It would help to know what your samples are, etc etc

-viper-

Hi
I also am using that M2-FLAG monoclonal antibody from Sigma for immunocytochemistry.
I didn't have problems but actually the protein I was looking for is expressed in the nucleus so I also saw it in the nucleus but is was a specific signal.

Can it be an overexpressing problem?
Maybe the protein localization changed due to overexpression?!

which concentration ab/dilution are you using?

-macedo-

QUOTE (macedo @ Dec 14 2005, 06:04 AM)

I use 1:100 for case....every protein is different...so every dilution is different at times.

-viper-

I am sorry viper for the misunderstanding.
I was asking what dilution MOZ was doing because I could then compare the amount I use and give some suggestion on that in case he was using to much.
I didn't do a titration For ICC I dilute 1ul(4.3ug) in 200 ml PBS+ (PBS+ : PBS+0.5%BSA+0.15%glycine).

I didn't do a titration of the Ab but this amount and even less ( 1ul in 250ul) worked well for me.

-macedo-