I recently purchased antibodies for use in Western Blots, and when I found non-specific binding, my tutor suggested that there may be heat-shock proteins present in the antibody stocks I had purchased. I was wondering what else might be present, as I thought these would be mostly pure?
If you are looking for anti HSPs, use mAbs or polyclonals raised using peptides
Exactly what one purchases can sometimes be tricky. Antibodies can be marketed as (1) Crude antiserum (in which only a few percent of the total antibody population is what is listed on the label, along with all of the other plasma proteins); 2 Purified antibody by chemical precipitation;3 Affinity purified by using protein A and/or G to isolate all IgGs in the mix; or 4. targeted affinity purified antibody in which a gel is coupled to an antigen used to generate the antibody in the first place. 2 and 3 are antibody preps but still the total antibody pool, not just what you are interested in. 4 will be the most selective.
Its also important to know what the antigen was (and sometimes commercial vendors are very vague about this) : 1. Off the shelf protein, 2. Purified protein, 3. Recombinant protein, 4. Peptide antigen-protein conjugate. (here it is often not revealed what the peptide sequence was and/or what the carrier protein was. All have varying degrees of purity.
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