I am interested in using an antibody against a hamster heat shock protein to detect a Drosophila homolog in Western blot analysis. It has already been shown that the antibody I am interested in using reacts with the yeast homolog. The antibody I am using was made against a 15 AA conserved region in the hamster sequence which has 60% identity with my target. I am curious to know if there are acceptable ways to improve cross-species reaction without increasing background.
If you have a lot of background, you can try using a higher salt concentration (0.5 M NaCl added) and 5% milk (to counter the milk striping by the salt). This could result in higher specificity.
I use higher Tween 20 concentrations (up to 0.3%) without adding milk or BSA. It works pretty well as it avoids any non-specific interactions which may occur with milk proteins or BSA. This is surprising but very effective!
The above suggestions are methods to reduce non-specific background. I have not heard of a particular method to somehow enhance recognition of the antibody epitope (without a concomitant increase in non-specific binding).
In these cases, it's probably just as well to go ahead and use it and hope for the best! Good luck!
You can modify the affinity / specificity of the first antibody to some extent by playing with the incubation conditions. Optimizing the temperature is fairly common, but changing the pH and salt concentration should also have significant effects.
If it is a polyclonal antibody, you can affinity purify the species cross-reactive antibodies, elute them and re-use for your assays, thus enriching for the fraction of IgG that is active for your antigen of interest.
If you are using a monoclonal Ab, then you simply have to hope for good conservation of the relevant epitope.
Hyperimmunisation: in other words, multiply immunise whichever animal was used (eg: 6x over 3-6 mo) so that you elicit the highest binding affinity antibodies the animal is capable of making. I am sure it will work with a peptide immunogen as I have done it years ago for a similar reason - however, you don't generally get particularly high titres unless this is fused to something as a carrier.
Might it not be easier to compare the homology across your entire protein to species against which antibodies are more commonly made (human, mouse, rat etc)? You can then search for antibodies generated against epitopes that map to more highly conserved regions.
All of these are great suggestions. I am using a polyclonal. We made it many years ago to a short 18 AA highly conserved segment of the protein. The affinity purification trick is elegant and probably would work very well. However making the column would be difficult since we have not expressed the Drosophila heat shock protein. It might end up that we would do that for other reasons anyhow.
Temperature seems to be a good variable to try (easy). I presume that low temperatures and longer incubation would work better? The removal of protein from the blocking solution sounds like another fairly simple thing to try.
Thanks to everyone.
Hi Douglas. Depending on the scale of your purification (or if you just want to try proof-of-principle first), you can try to adsorb some of your antigen to PVDF membrane and see if you can elute off in a microfuge tube. But yes, you'll need to purify the Drosophila heat shock protein. A small amount of bacterially expressed material or a tagged version in HEK293s might give enough antigen to pilot these experiments.
Affinity purification does not need a column - and in fact does not even need purified protein: here is a simple technique which worked well for me some 25 years ago.
http://www.mcb.uct.ac.za/Manual/monospec_antibodies.htm
Hi Ed
Great Idea, this would be a way of reducing background by selecting for cross reacting antibodies. I think I will try this if there isn't anything that I can do by adjusting the washing or the conditions for reaction with primary antibody. Thanks again. I printed out your online protocol.
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