ELISA - no saturation - ELISA based protein-protein interaction assay (Dec/04/2008 )
Hello everybody
I´m trying to perform an ELISA based protein-protein binding assay. In detail: I coat protein A at increasing concentrations (o.n. at 4°C), blocking (BSA), then adding the binding partner protein B at a fix concentration. Detection is performed by specific Ab against protein B and 2nd HRP labelled Ab followed by addition of ABTS substrate.
Additionally I coat a fix concentration of protein A, blocking, adding increasing concentrations of protein B and detection is performed as described.
My problem is that in both ways I don´t get a saturation. I thought that at a certain concentration of coated protein A the binding of added proetin B is maximal and saturated, but tthe more protein A is coated or the more protein B i add resp. the higher the OD is. Theoretically i should get a nice sigmoidal curve in both assays, shouldn´t I?
Any suggestions?
Thanx
I´m trying to perform an ELISA based protein-protein binding assay. In detail: I coat protein A at increasing concentrations (o.n. at 4°C), blocking (BSA), then adding the binding partner protein B at a fix concentration. Detection is performed by specific Ab against protein B and 2nd HRP labelled Ab followed by addition of ABTS substrate.
Additionally I coat a fix concentration of protein A, blocking, adding increasing concentrations of protein B and detection is performed as described.
My problem is that in both ways I don´t get a saturation. I thought that at a certain concentration of coated protein A the binding of added proetin B is maximal and saturated, but tthe more protein A is coated or the more protein B i add resp. the higher the OD is. Theoretically i should get a nice sigmoidal curve in both assays, shouldn´t I?
Any suggestions?
Thanx
It could be that your blocking isn't working properly. Try a different protein and see if you can see a difference. What is the make-up of your blocking solution? If you have a look at the R&D Systems website, you will be able to find a variety of blocking solutions in the ELISA Development section.
I block with 3% BSA for 2 hours and this step should be fine. Because in the wells where I don´t coat with protein A, block and add then protein B the signal is just a bit higher than backround signal (blocked wells, no protein A, no protein B,), but much less than in wells which have been coated with protein A.
But trying it with a different protein is a good idea.
You did not optimize both cap and det Abs concentrations for the assay yet, so can't see "$" curve!
Have you ever heard of titration?