Perhaps the single most widely used tool in the molecular biosciences is the antibody. However, there are serious problems with most affordable polyclonal antibodies. (good 2015 Nature News article on the dilemma)These antibodies are typically generated by injecting a host organism (e.g. rabbit, goat, mouse, etc.) with the antigen, provided by the end user, letting the host natural immune system generate an antibody response, and bleeding the animal to collect anti-sera. The anti-sera are tested for a positive ELISA response to the antigen, and responding anti-sera are usually affinity purified (e.g., protein A or protein G), and then sent to the end user. For smaller animals, it is impractical to bleed those animals multiple times, and large animals are too costly for most academics. So, when the end user needs more antibody, the process must begin again. This leads to extreme batch to batch variability in polyclonal antibodies and irreproducible, sometimes contrary results, as every individual organism in a species has its own unique immune signature. Furthermore, the possibility exists that the polyclonal antibodies were responsive to an impurity in the original end user preparation of antigen and not the intended protein. Non-GMP facilities do not have the QA/QC policies to guarantee identical impurities. It is not logical for the end user to spend time and resources preparing indistinguishable antigen the next time they need antibody. This is in sharp contrast to the exhaustively validated mouse M2 monoclonal anti-FLAG antibody manufactured by Sigma.
The TPG group is TriazolylPhenylGlyoxal and was co-invented by Dr. Thompson at The Scripps Research Institute. It modifies the guanidine functionality of arginine (Arg) amino acids in peptides or proteins chemoselectively with whatever payload is attached to the TPG.
The FLAG tag is the antigen, epitope, or binding partner of the anti-FLAG antibody and is a highly soluble, octapeptide sequence: DYKDDDDK. TPG-FLAG will add the FLAG handle to intrinsic Arg in folded proteins, in most cases not interfering with function of the tagged protein.
To overcome the issues with impure polyclonal antibodies stated above, scientists can add the TPG-FLAG handle to their target protein. Then, by using the M2 anti-FLAG antibody described above, perform western blots, ELISA, immuno-precipitation, etc., and not worry about the quality of their antibody.
In addition, therapeutic peptides and proteins must be non-immunogenic in patients (i.e., they should not elicit an immune response). Adding the TPG-FLAG to a biological therapeutic protein will open up an express lane on the biotech superhighway to all the assays available with antibodies. The primary weakness of this method is the reagent is not specific to just your protein, if you have a mixture of proteins TPG-FLAG will ligate to all of them with exposed arginine.
Now this little beasty is my first solo invention. It turns amines, lysine or the amino terminus, into thioesters. It does so with chemistry pioneered by Phil Dawson.
Dr. Thompson is also adjunct professor of chemistry at University of Idaho Coeur d'Alene. View some of the posters his students have made.