Western Blot Stripping Blots

[isabelle]

Hi, ive 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 am 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".

Also do you think the protein interaction of secondary to primary Ab will break during the re-probing steps or that it's better to just STRIP?

[admin]

Hi again, I would definitely strip the western blot membrane before re-probing with another antibody

[oBWhat]

Hi, you may succeed without stripping but you should inactivate rest activity of peroxidae (if you use HRP-labeled 2nd Ab) of first round by incubation with NaN3; then wash extensively. However I think your goat anti-rabbit will bind to the rabbit anti-goat... im getting confused...