Hi there.
I'm trying to optimize my protocol for HRP detection in western blot, but so far the results haven't been producing bands. This is even though I used a positive control which worked for AP-conjugated antibodies.
I've tried using 0.05% DAB in TBS + 10 uL 30% hydrogen peroxide. I've also used protocol using Nickel and Cobalt ions. But so far, none of these have worked.
Can someone provide an alternative protocol or a way to troubleshoot these problems?
Thank you so much for any help you may be able to provide.
The easiest way would be to use a commercial kit. Try peroxidase as a positive control.
Hi!
You can try these protocols:
1)TrisHCl pH7,5 10mM
DAB 0,6mg/ml
CoCl2 0,3%
H2O2 1000x
H2O to 10ml or
2) It always works:
TrisHCl pH7,5 2M - 500mkl
Imidazol 0,1M - 500mkl
Tween20 - 50mkl
DAB - some granules
H2O2 - 3mkl
H2O to 10ml
Good luck!
Jason Tan Liwag did you figure out a good method? I am encountering the same problems as you were 2 years ago. I would really appreciate a good protocol.
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