ELISA background - (Jul/24/2006 )

I was doing protein DNA interaction on the ELISA plate, DNA is biotin labelled and ELISA plate is streptavidin coated, the protein DNA is interacted in the tube and then transferred to the plate, then primary and then secondary antibody added.

Earlier experiment was going fine, but after changing the secondary antibody(earlier it was AP conjugated now HRP conjugated) I am getting very high background, close to the signal. But again the well where there in no protein added (BSA blocked, primary and secondary treated ) there is no background.

Can anybody explain how to remove the background..

Is your protein positively charged? That may be one source for nonspecific interaction that causes high background. You may have to try higher concentrations of BSA see if that will counter act charge-related effect.

hello,
i got confused ,
in ur statement below u mentioned all conditions which r necessary for a nagitive control...., and u donot see any back ground.
when u add protein u see signal, in this situation which one ur assuming as back ground,
please clarify my doubt.

i m sorry for not getting ur explanation.

thanks
payeli