Smear formed in western blots while using tubulin as control - (Aug/12/2013 )

Hello friends,

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.

Thanks.

How long do you do the secondary incubation for?  What species are the primaries and secondaries for both proteins?

bob1 on Tue Aug 13 22:21:27 2013 said:

How long do you do the secondary incubation for?  What species are the primaries and secondaries for both proteins?

its for 1 hr. And they are mice tissues so primary is Rb anti Ms and secondary is Goat anti Rb

Ok, that should be fine.  Have you tried doing the tubulin alone on the same samples, this will tell you if there is some antibody cross-reactivity, which is what I suspect is happening.

well here is the update. I tried doing with different concentrations and had changed my secAb as well....didnt worked. So finally i borrowed tubulin from adjacent lab and it worked pretty good. Dang!! never had a clue that our stock for tubulin is messed up. Thanks a lot bob1 for bearing with me. Have to order a new one now.