Hello Researchers,
I have been looking for a proper primary antibody against p-JNK and JNK for mouse macrophage lysate (±LPS treatment) in western blot. We tried 4 antibodies against p-JNK; 3 from Cell signaling (4668, 9255, 9251); 1 from Abcam (ab124956). None of them is working. I couldn't detect the band even using 1:100 with 60ug/lane. Also, we failed to detect the total JNK with two antibodies (both from Cell Signaling, 9252 and 9258). I searched the literature and found that most of them used Cell signaling antibodies! The thing is only JNK I have difficulties detecting (p-p38 or p-ERK are fine in my hand with the same membrane). I am desperate and have time pressure now. Any idea about p-JNK or JNK antibodies?
Sharing experiences and suggestions are all welcomed.
Thank you in advance!
We previously used Cell Signaling antibodies (including JNK) and except for membrane proteins they always gave a good band at 1:1,000. Have you checked the epitope of the antibodies you have used vs. your host cell line? Possibly look for a polyclonal antibodies since they have less specific binding. E.g. polyclonal antibodies from ThermoFisher.
I have not used Thermo's JNK antibodies, so overall I'm not sure how they'll work. But some possible ones:
- https://www.thermofisher.com/antibody/product/Phospho-JNK1-JNK2-Thr183-Tyr185-Antibody-Polyclonal/44-682G
- https://www.thermofisher.com/antibody/product/JNK1-Antibody-Polyclonal/PA5-17889
If you contact Cell Signaling's technical team, they have good customer service and will typically help you troubleshoot your protocol and provide you with control samples. If all these don't work, possibly you do not have enough protein expression to begin with - you can try qPCR to confirm?
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