Substrate for AP conjugated secondary antibody - is it the same as Western blot? by NBT and BCIP? (Oct/10/2008 )

Hello all,

I used AP buffer, BCIP and NBT to visualize my AP secondary antibody for ELISA according to Sambruk's handbook (10ml AP buffer, 33ul NBT and 66ul of BCIP) and added 100ul of this solution immediately to my microplate wells and then I read by a microplate reader.

the result wasn't good enough....

I have read that some people use pNPP substrate for AP conjugated antibody\ies.........is it necessary?

ABCAM's protocol is by pNPP.

thanks

bcip and nbt will give a insoluble product. this will make it very difficult to get good readings.

pnpp will give a soluble product and is suitable for elisa.

we use bcip/nbt for our colored westerns and pnpp for our elisas.