Reducing background caused by endogenous alkaline phosphatse in ELISA - (Jun/16/2009 )
Hi, any tips on a good covalent non reversible inhibitor of endogenous alkaline phosphatse from mouse neuroblastoma cells please?
I am performing an ELISPOT in which I adsorb mouse neuroblastoma cells to the membrane of ELISPOT wells before detecting my protein of interest with antibodies. The secondary antibody I use is conjugated to E-coli ALP which I then detect with BCIP/NBT. I am getting very high background, even when I do not use any antibodies, presumbly due to endogenous ALP. I have tried using Levamisole prior to the addition of secondary antibody but it is not having much effect. I think it is getting washed away as it is a revesible inhibitor. When I use it in conjunction with the secondary antibody I get close to 0 spots indicating an inhibitor of ALP will reduce my background, I just need one which I can add prior to my secondary antibody so I can still detect my antigen of interest. Any help would be greatly appreciated.
can you use a different secondary ab?
Is mouse AP different from E coli AP? If so could you use an ab conj against the E coli ab AP conjugate?
Can you conjugate your primary ab with an enzyme other than AP.