Antibody Optimization - (Jul/30/2011 )

Dear All,

I am stuck in optimizing an antibody which gives a LOT OF background to get the endogenous protein. it is supernatant so I cannot dilute so much. Do you have any idea how to remove all those non specific bindings?

So far, I have tried the following and none of them worked properly:

1-different blocking agents: 5% milk, 3% and 5% BSA, mix of BSA and Milk - the best one was 5% BSA overnight
2- small dilutions with 10% BSA - no work
3- I stripped the blot once, it didnot help

((((((
It drives me crazy. please help

thanks

If you're optimizing you should also try different antibody dilutions, both first and secondary.

Probably you're used o much antibodies in your solutions.

cheers,

I agree with the above.. i have just finished optimising a WB using sera from rabbit against xenopus TFAM, i have ended up using the sera at 1/40,000 dilution for 2 hours, 2 hours in goat anti rabbit HRP 1/20,000 for 2 hours then visualised with ECL, exposed on a Fuji LAS 3000 for 1 minute