Dear colleageus,

I would like to seek your help:

I have performed Western blot, 12% gel, loaded 30 microgram total proteins, for some cell lines. I used 2.5% milk powder for blocking, then apply e.g. anti-tubulin (55 KDa), and MCL-1 (40 kDa) antibody to test.

Yet, I reckon that:

1. The loading is always uneven. I have carefully quantify the proteins using BCA assay, and loaded equal amount, with no overflow during loading.

2. From the manufacturer, they can easily detct MCL-1 in strong signals in my target cell lines. But I have tried a few times and the signal is very weak.

Would you mind pls advise how to improve? (e.g. will using BSA to block, or increase the antibody-to-protein ratio e.g. 1:500 help)? Thank you very much.