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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.

-NovelProtein-

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

-bob1-

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

-NovelProtein-

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.

-bob1-

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. 

-NovelProtein-