Can you suggest me to get rid of this dark background. This is phosho JNK antibody (cat. no: Invitrogen 44682G) I used same antibody previously without any problem so we reorder from same. after I use new ordered antibody I had this problem twice I tried.
antibody dilute 1:1000 in 5% Non Fat Milk. overnight incubation at 4C. after block with 5% Non fat milk with 1:2000 anti rabbit antibody for 1 hour room temperature. Wash with TBST (with Tween 800 mM) for around 2 hours. (change TBST every 5 minutes ) . Developed with pico 80% and femto 20% because 100% pico could not detected. BioRDA chemidoc XRT system used to develop. How can I get rid of this dark background ??
Any suggestion?
Hello Thilini,
My understanding is that milk can be problematic as a blocking agent when using phospho antibodies, because there are phospho proteins in the milk. You might consider trying something in the place of milk, like fish gelatin, for example.
Best regards,
Jon
Thank you so much. But previously it works well. Reordered antibody gave me this problem. I wll block using BSA next time.
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