Best way to reduce the back ground colour is to minimize the incubation time in each of the steps.
The possible cause for high back ground colour could be unpure polyclonal preparations so try using only pure poyclonal antibody.....
U could also use normal goat serum for diluting the samples.

Hi
Even I am having this problem of high background.What should I do?
I use 5% BSA in PBS for blocking.

My incubation time is 1hr each.What could I do?
Any suggestions????

Thanks n Cheerio
vvi

hi guys,
i am doing indirect elisa using monoclonal antibody supernatants which was done by myself
surprisingly i am seeing O.D on whatever protein i coat with and also with Tween-20 blocking alone
but there is no colour development with the polyclonal serum on any of these
this is quite surprising as how a monoclonal antibody can bind to so many varieties of proteins like
BSA, milk powder, FCS, goat serum and even Tween-20 alone.
i am using HRP-conjugated secondary antibody
can some one please suggest me
thanking you
leelaram

are you just quantitaing protein? why can;t you use a BCA?

hi,

It will be good if u use 1 % sodium caseinate...
I t is costly but at the same time a very good blocking agent.