I would try using 5% milk in TBS-T and incubate your primary overnight at 4C with shaking. This is the standard protocol we used in our lab imaging with HRP and Chemi. However, in order to rule out the secondary and detection steps I would try first with a very clean antibody. Personally, I use complex II (Invitrogen Cat Number: 459200).  I use 1.4ul of this antibody in 7ml of 5%millk TBS-T and incubate ON at 4C. This is the cleanest antibody I have ever come across. If you already have a very clean antibody you could try with that instead. Best, JH

Thanks for the help, I am thinking that my antibody is probably not so clean. I have tried that standard protocol and it was a mess. This might be more of an antibody specific problem. There was tons of background. The best western I have gotten out of this one was with 5% BSA overnight blocking at 4 degrees and then a 1 hour incubation at RT with the primary. But I really cant figure out why that would work. 

There are significant differences in the blocking steps between HRP-based systems and the Odyssey.  In my experience, some antibodies and blockers that work very well for the Odyssey (and therefore near-infrared fluorescence) don't work well at all for chemiluminescence, and vice versa.  I would switch the blocker of choice: I've never had low background when using BSA as a blocker (either system), but I have performed some western blots with sera that matched the secondary antibody (Odyssey worked well with sera but not milk; chemi worked well with milk and sera).  

You mention that blocking overnight gave you the best results; that makes me think that your antibody has a lot of nonspecific binding, which would be affected by both overnight blocking and shorter incubation.   I think switching your blocker or increasing its % would help with your background noise.  Has anyone else published with this antibody?  You could contact that lab and ask for their protocol.

Good luck.

Thanks a bunch, people have used it and published with it. But many people use the odyssey (all the people I have contacted) I will try with the matching sera. That is a good idea. 

Troubleshooting primary antibody issue is difficult. In my experience, usually incubating the primary ON at 4C provides better specificity (someone explained why this is to me before but I don't remember their reason.) Also, sometimes when I change the actual protein prep I get better specificity. In some cases I have increased the denaturing boiling time and this has helped. Best bet is to get some first-hand advice from someone that uses that specific antibody, but it sounds like you are already going that route. Good luck!

Julie-

The primary antibody incubation at 4 degrees C overnight is more specific because 4 degrees C limits the activity of the antibody.  Using an antibody at room temperature allows antibodies and proteins to react/bind together much more often and more vigorously than at lower temperatures, which is why room temperature incubations are limited to an hour or so (and have higher background).  By decreasing the temperature, only the strongest binding events will still have enough energy to happen (which should, hopefully, be the binding of your antibody to the right target, instead of a similar-but-not-right target), and weaker events won't happen.

You are right about the prep-you don't want to boil the sample (that leads to protein aggregates, which the antibody may not recognize), but you do want the protein to be denatured (or the antibody might not recognize the right epitope).

Makes sense! Thanks for the explanation John- I knew there was some sort of good reason for this :)