Protocol Online logo
Top : New Forum Archives (2009-): : SDS-PAGE and Western Blotting

Can add new secondary Ab? - (May/06/2009 )


I have PVDF membrane which I probed with my primary antibody & then my secondary antibody. Then realised my secondary antibody is not working so have an aliquot of another secondary. Can I add new secondary directly to same blot or do I need to strip it to remove any of the old, not working, secondary antibody first? Primary antibody quite expensive & only get a tiny amount so hoping I dont have to strip it to conserve the primary antibody!



If your secondary didnt work and you are certain it is the problem, then it shouldnt hurt to use your other secondary antibody without stripping. You can always strip it after the second time if you find that it doesnt work.