The human c-myc gene has been favored over many other tags most likely for historical reasons dating back to the 1982 discovery of c-myc in library screening. In 1985 the continuing identification of c-myc as an important oncogene stimulated a vigorous program to generate higher quality antibodies for its identification, assay development, and the exploration of its role in oncogenesis. Hence, antibodies to the fusion-tags were readily available to monitor fusion protein expression, co-localization, and purification.
Among these, clones 4A6 and 9E10 have proven of particular value. They have been used in western blotting, immunoprecipitation, and flow cytometry. The 4A6 antibody, raised to a smaller consensus myc tag sequence, recognizes myc tag on either the amino- or carboxyl-terminus of a recombinant protein. Its ability to recognize myc tag is independent of the sequence following or preceding the tag.
The 4A6 clone has been successfully employed in immunofluorescence protocols. C-myc-tagged proteins can be affinity purified by coupling the Mab 4A6 cross-linked to protein G agarose by dimethyl-pimelidate. So over the years myc has acquired a role as one of the universal tags much like secondary antibodies.
Clone 9E10 has long been valued as a tool for the detection of the myc-epitope tag during analysis, expression, and purification of recombinant proteins. However, its ability to recognize N-terminally myc-tagged proteins is dependent on the amino acid sequence following the myc-epitope tag sequence. This may be because the 9E10 antibody was raised against a larger peptide from human c-myc (residues 408-432), and therefore not as specific to the primary myc sequence.
Upstate offers an exclusively licensed anti-myc tag, clone 4A6 monoclonal antibody, which provides better results than the monoclonal 9E10. Clone 4A6 detects myc-tagged recombinant protein in sequence contexts not well recognized by anti-myc tag, clone 9E10. These reagents offer investigators a range of experimental possibilities.
EMD Biosciences (www.emdbiosciences.com) handles a range of epitope-tagging products. Among these is the His•Tag Antibody Plate, a 96-well, ELISA-compatible plate containing an immobilized His tag monoclonal antibody, covalently immobilized to the surface to retain maximal binding activity. The antibody specifically recognizes five consecutive histidines, and so will bind with high affinity to virtually any His•Tag fusion protein in which the tag is exposed, according to the company. Well-to-well variability is less than 5% and the plates are stable when stored dry at 4C. The His•Tag Antibody Plate can be used in a variety of binding assays where immobilization of His•Tag fusion proteins is required.