I'm working on selecting antibodies against a recombinant protein that has a His-tag. My idea is to first bind the recombinant protein to a HisTRAP column and then use this column for an affinity chromatography to select the antibodies.
However, I haven't found any articles or protocols that describe using this column in this specific context.
Has anyone used the HisTRAP column for this particular purpose? If so, which buffer did you use to elute only the antibodies without eluting the bound protein?
Analogously to what you want to do, HiTrap Protein A HP are prepacked columns for routine preparative purification of monoclonal and polyclonal antibodies such as human IgG. However, to purify the antibody, you first need a good source of it (e.g. hybridoma cell culture supernatant that already contains large quanties of the antibody of interest and it needs to be only purified). If you want to get a specific antibody against your protein, for which you do not have an antibody yet, you will likely not get sufficient amounts of it by using this approach. You will have to raise the antibody first, using ideally a tag-less protein for immunization.
I have used HisTrap HP affinity column (GE Healthcare 71-5027-68 AF) to purify recombinant protein. My colleague helped design the experiment. Then we made a polyclonal antibody from injecting this into the mouse and purified the antibodies using affinity chromatography. I am attaching the reference for the protein purification. I have a preprint for the antibody production and have also included it here. Hope this helps!
Hi Jackelinne,
I don't think it will work well. First, antibodies themselves can bind on NiNTA (generally IMAC was developed for purification of natural non tagged proteins, even before his-tag). Secondly elution, highly acidic conditions will be required to remove antibodies from the antigen. At low pH the recombinant protein itself will elute from the column (firstly histidines are protonated and secondly nickel stripping occurs). Part of the problem with nickel stripping can be circumvented if you use Sepharose Excel. Nickel binds firmly there, but the binding of recombinant protein to the nickel histag will weaken anyway.
It is easier to immobilize the recombinant protein on Sepharose by any suitable method (cyanogen bromide activation, epoxyactivation - there are even ready-made activated resins) and purify the antibodies in this way.
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