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Reducing background caused by endogenous alkaline phosphatse in ELISA


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#1 PG4ever

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Posted 16 June 2009 - 02:24 AM

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.

#2 sgt4boston

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Posted 23 June 2009 - 12:01 PM

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.




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