Hi,

I'm tried to ELISA with angiopoietin protein.

But I found nonspecific binding of target protein.

I blocked the well with blocking buffer for 2 h at RT, and incubated with biotinylated angiopoietin protein for 2 h at RT.

Then, I incubated it with streptavidin HRP, TMB. The reaction was stopped with 1 M H2SO4.

I tried various blocking buffers.

1. Two BSA protein with different purity.

2. 1%, 2%, 4% BSA

3. PBS or TBS with pH 7.4

4. Addition 0.05% Tween 20

(I did not tried casien, because of biotin)

But the absorbance was very high (more than 0.8)

Is anyone who can advise for me to prevent the nonspecific binding of angiopoietin protein?

Thank you :)