I’m performing western blotting to detect anaplastic lymphoma kinase (ALK) in SH-SY5Y cells, and I'm also probing for beta-actin on the same blot. While I’m getting bands for beta-actin, I’m not seeing any bands for ALK. I’ve used a 1:1000 dilution of the primary antibody for ALK and even tried 1:500. I also increased the protein loading to 60 µg, but still no bands for ALK. Any suggestions on what might help?"
It may be for two reasons:
1. This protein may not be present in that particular cell type or may not exert particular functions assumed. Please check the paper citing this Ab.
2. You can use the same Ab from another supplier. Sometimes a particular Ab may not work in specific samples.
That's a suggestion from me!
Best,
Dr. Nayeem
Thank you for your reply, Junayed Nayeem sir. SHSY5Y is a well-known cell line for active ALK, so I am sure that ALK expression exists.
The antibody I am using is from Thermo.
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