I'm doing a western blot with biotinylated antibodies. Everything worked fine until 2 months, a black background appeared on handling with strepta-poly-HRP.
My typical protocol: PVDF membrane, saturation 1 at 2 hours with blocker, hybridization with my biotinylated Ac 2h at 0.5 μg / mL, washes, hybridization with strepta poly hrp at 1:40 000 for 20 min, ECL western blot reagent .
We have done many tests with many different parameters: 7 different blockers, buy a new ECL, new batch of Strepta-poly-HRP + new reference + new concentration, new membrane including nitrocellulose test, with / without antibodies...
I have no idea to solve my problem (I don't have it with direct couplings), If you have hypothesis, don't hesitate.
Thank you very much,
Hi Victoria Dolange ,
I had this problem with NC membranes and using "normal antibodies". I think it could be your chemiluminescent system. Remove well the reagents before imaging blots.
Did you test transfer, your proteins are there?
Best,
Jacó
I agree with answer above. The ECL reagents are too concentrated, so you can try a 1:2 dilution or further diluted, it depends on how saturated your membrane is.
Hi,
Thank you for your answers,
Jacó Joaquim Mattos , indeed my proteins are there. As far as ECL Select is concerned, I try to better eliminate a surplus and I'll try to dilute it, Lady López. But I am afraid of losing sensitivity.
Especially that before everything worked well... Do you think it could be one of my buffers contaminated by something? Kind regards,
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