We are currently trying to do western blot by using the rabbit polyclonal antibody specific to our protein of interest. The result of the western blot showed a lot of backgrounds that also appear in the negative control. We used cell lysate protein from the bacteria without expression our protein of interest. Therefore we suspect that the antibody binds unspecifically to other proteins coming from the bacteria itself.
If this is the case, you could deplete the polyclonal antibody of undesired reactivities with the negative bacteria (or bacterial lysate). For insance if you pre-incubate the polyclonal antibody with bacteria, you can remove both bacteria and antibodies recognizing them by centrifugation.
But you have to take into account that the problem can at least be partially caused by non-specific background, not depending on specific reactivity to bacteria. Its important to try several dilutions of the polyclonal antibody to determine the optimal signal to noise ratio. Diluting both primary and secondary antibodies in a suitable blocking solution (for instance PBS containing 4% skim powder milk) also reduces the background. Any antibody (if its too concentrated) can produce non-specific signals to many unrelated proteins in any immunochemical technique. You have to find conditions in which the antibody specific reactivity can be distinguished from that non-specific background.
Alternatively you can do a 'positive' purification of your antibody with recombinant protein (if you get it to near single band purity) bound to NHS agarose beads, or even the combination of both the depletion suggested by Gertrudis and the positive purification.
Thermo Fisher offers a good handbook describing different techniques.
https://tools.thermofisher.com/content/sfs/brochures/1601975-Antibody-Production-Purification-Guide.pdf
Thank you for the suggestions. I would like to start with diluting more my primary antibody and maybe also with the secondary as well. Is there any optimum duration time for the primary antibody incubation? I am afraid that the duration will also affect the unspecificity of the antibody.
1-2h are common incubation times. Usually reducing/increasing the incubation time does not result in enhanced specificity. Concentration changes and blocking conditions are likely to have a bigger impact for sure. But some people do overnight incubation at 4 degrees to reduce background.
Gertrudis Rojas in an answer above, you mentioned to clean up antibody with a negative bacterial lysate. Can you provide me detailed protocol or any reference for that? I need to do ChIP but my antibodies have some non-specific reactivity which I want to remove before the actual experiment.
Thank you in advance
I had similar problems before and solved by diluting the antibody in TBST 1x with 1% skimmed milk and 100 micrograms of the extract of your negative control. Hopefully that can titrate all the inespecific antibodies available. I am not sure exactly what you are using as negative control. In my case I had two options:
1) When looking for a specific protein for which I had a mutant strain available (with the gene enconding the protein of interest delected) I added cell extract of this mutant strain to the diluted antibody before incubating with my blot membrane
2) In case you do not have corresponding deleted strain...in my case the antibodies were raised aganis recombinant proteins that were produced in E. coli BL21(DE3). So I used the extract of E. coli BL21(DE3) instead
good luck
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