I was trying to check phospho-jak2 signals in my mouse cell lines using three different antibodies specific to human and mouse antigens from CST. Lysates were prepared using RIPA and protease inhibitors and I ran the samples on 4-12% Bis-tris gels from Invitrogen. I used invitrogen iblot dry blotting system for transfer and I've tried both standard 7 min protocol and a customized 9 min transfer. Never got any signal. I tried 6% tris-glycine gel and running lysates of other human and mouse cell lines but never saw any signal either. Detecting phospho-jak1 was also a failure. Weird enough, I could detect strong phospho-stat5 from my mouse cell line, which indicates that my extraction method is sufficient for detect phosphorylated proteins. Any tips for detecting phospho-jak1/2? Thanks!
You need a positive control, that you can prepare by treating your cells with a ligand that triggers Jak1/2 phosphorylation. Interferon gamma of the appropriate species will trigger such stimulation to both Jaks and Stats. There might be more appropriate ligands for your cell types.
I agree that you need a positive control. I would call cell signaling and speak with a scientist about your issue. They will send you a positive control for your antibody free of charge.
Oscar L. Sierra Thanks for answering. In fact, I also ran lysate from cells stimulated with TPO after cytokine starvation before but didn't observe any band either. Contacting CST for a positive control might be an alternative.
Check antibodies are working..as suggested you need a positive control
You maybe underloading the sample. STAT5 is potentially more prominent in lysates or antibodies are more sensitive for STAT5.
In your lysate you have protease inhibitors but no phosphatase inhibitors? therefore you maybe losing signal
Can you detect Jak non-phosphorylated: would be good to check