Am doing westerns for a novel protein and is getting expected bands. However, while quantifying it relatively with tubulin...blots give smear/background when i do it for tubulin.
I run gel, transfer and then follow usual protocol for detecting my new protein. Once i get those bands, i strip the blot wash it properly and redo the same stuff but using tubulin as primary anitbody which though shows tubulin bands but then there is a smear/background starting from the top of the gel and ends just approx 1 cm before tubulin band.
I use 3% BSA for blocking and 1:10,000 dilution for 1 & 2 antibody. My protein concentration is 30 micro gram. I cannot reduce the protein loading concentration as my protein band gets fade out with less concentration.
Practically, I can cut the strip/section where tubulin bands are and make up my presentation. However, am annoyed why those ugly things always appear.
Please help me out.