Secondary antibody - need to strip blot? - (Oct/05/2006 )

Hi,

I have a blot that I'll work on first with Primary Ab that is monoclonal raised from goat. Then as secondary Ab, I'll use "rabbit anti-goat Ab".

Next I'm planning to work on the same blot, this time with Primary Ab that is monoclonal raised from rabbit. Then secondary Ab, I'll use "Goat anti-rabbit Ab".

Do you think I should strip blot for the latter before proceeding? What worries me is that the secondary "Goat anti-rabbit" will interact with residual of secondary "Rabbit anti-goat". Do you think the protein interaction of secondary to primary Ab will break during the reprobing steps or that it's better to just STRIP?

Ha...
Thanks in advance.

Cheers

I would definitely strip the membrane before re-probing

good luck

you may succeed without stripping but you should inactivate rest activity of peroxidae (if you use HRP-labeld 2nd Ab) of first round by incubation with NaN3; thereafter, wash extensively

the goat anti-rabbit will bind to the rabbit anti-goat.
you have to strip

Appreciate the reply.

I think I'll strip the membrane as suggested. Thanks.

Cheers