2 replies to this topic

[Ambinlab]

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

[jopa]

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,

[ickypimp]

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