We would like to couple a protein of interest to a solid matrix, then pass a lysate over the matrix to see what binds to our protein. What would be a good matrix to use? We have used Affigel from BioRad in the past for coupling antigen in order to affinity-purify antibodies. Would this be the one to use? Obviously, we want to keep background down.
I like the Pierce NHS-activated agarose spin columns, http://www.fisher.co.uk/1/1/10099-pierce-nhs-activated-agarose-spin-columns-333mg.html
http://onlinelibrary.wiley.com/doi/10.1111/jfd.12172/full
It has been a number of years since I did this work, but as I recall, the PEG based matrices are nice because you don't get a lot of H-bonding and the polyether matrix is very water soluble (swelling properties are great) as well as conformationally mobile. Hope this helps!
Directly coupling proteins to a matrix sometimes means that the binding site of interest is damaged or sequestered by the coupling chemistry. This would mean that you would miss any potential interacters simply because the binding site wasn't available. The problem is you may never know this and thereby draw wrong conclusions. An alternative option to overcome this potentially serious problem is to raise a polyclonal antisera (monoclonal will do only if the epitope bound by the antibody doesn't interfere with the binding site of interest), couple the zigzag fraction to a matrix such as Sepharose or similar and then use the antibody to bind to the protein of interest. You can then use this sandwich to go "fishing" for interacters knowing that the relevant binding site is unlikely to be blocked. Using a polyclonal antisera is advantageous here because of the multiple epitopes recognised by the variety of antibody species (I.e. Multiple clones) present some which may block the binding site of interest but others that will not. Good luck fishing!
The tandem affinity purification (TAP) method is well established, have a look at http://en.wikipedia.org/wiki/Tandem_affinity_purification
This strategy overcomes many problems encountered with your approach (aka Ligand fishing), e.g. where complexes assemble cotranslationally and/or with the aid of chaperones but not necessarily when the individual components are mixed. The tagging is site directed as opposed to random coupling to NHS-activated matrices.
In any case, site directed coupling would have advantages over random coupling, The Avitag system may be of particular interest to you.
https://www.avidity.com/
I recommend 'FG beads', which are magnetic nano-beads of 200 nm in diameter and used especially for a purification of drug target proteins. Using this bead, Prof. Handa from Tokyo Institue of Technology identified a thalidomide target protein and revealed that the teratogenicity was caused by the protein (Ref, http://www.ncbi.nlm.nih.gov/pubmed/20223979) .
The beads are also used for the analysis of protein-protein interactions. If you are interested in the beads, please visit our WEB site (http://www.magneticnanoparticle.jp/en/htdocs/index.html). We are providing several kinds of beads (-COOH, -NH2, -NHS, Streptavidin, ProteinA, ProteinG and so on). We are also providing custom services (immobilization of proteins or chemical compounds on the beads, affinity screening of target proteins, identification of target proteins). Please feel free to contact us.
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