Production and characterization of antipeptide antibodies against human O6-methylguanine-DNA methyltransferase.
Four synthetic peptides from the sequence of human O6-methylguanine-DNA methyltransferase (MGMT), three corresponding to different hydrophilic regions and one corresponding to the sequence containing the alkyl acceptor residue cysteine 145, were used to immunize rabbits. The antibody against Peptide III (residues 171-184) was highly specific, and MGMT protein could be detected on Western blots of soluble protein extracts containing as little as 1 fmol of active MGMT. Antibodies against all of the peptides were able to immunoprecipitate denatured MGMT, while only the antibody against Peptide III was able to react with active enzyme. The antibody against Peptide III did not cross-react with methyltransferase from mice. The use of synthetic peptides has led to the production of a highly sensitive, specific antibody that recognizes native and denatured human MGMT. This antibody should prove useful in studies involving the detection, purification, and characterization of this enzyme.
Ostrowski, LE; Pegram, CN; Von Wronski, MA; Humphrey, PA; He, XM; Shiota, S; Mitra, S; Brent, TP; Bigner, DD
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