Technical Tips: Detecting Myc Epitope Tagging
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1. What product do I use? Top
Cat. No. OP10, our c-Myc (Ab-1), clone 9E10, is commonly used for epitope tagging (Munro et al., 1986. Cell 46, 291-300; Howe et al., 1992. Cell 71, 335-342; Evans et al., 1985. Mol. Cell. Biol. 5, 3610; Johnson and Hersh, 1994. J. Biol. Chem. 269, 7682-7688). OP10 was raised to a 31 amino acid peptide homologous to amino acids 408 - 439 of the human c-Myc protein. This peptide is available from Oncogene Research Products (Cat. No. PP06). The epitope of this antibody is contained in amino acids 410 - 419 (EQKLISEEDL). This sequence can be used for epitope tagging.
2. Where is the Top
The "tag" is generally inserted at the C-terminus of the coding region so that the tag will only detect full-length transcripts. Sometimes several myc sequences are inserted in tandem; this provides more "tags" per protein and thus increases the sensitivity of the detection system.
3. Why does my detection vary with different problems? Top
Antibody performance will vary in each epitope tagging experiment, as it is dependent on variables such as the location of the tag in the protein, the number of tag sequences added, etc. In particular, the epitope may or may not be exposed in the native protein due to constraints imposed by protein folding. It can help to optimize immunoprecipitation and immunofluorescence protocols with respect to the type and concentration of detergents used. This problem can be minimized by mild denaturation and/or using western blot to detect tagged proteins (see Munro et al., 1986. Cell 46, 291-300).
4. Can I detect Myc from other species? Top
OP10 has a weak affinity for rodent c-Myc. There may be some reactivity of OP10 with rodent c-Myc at concentrations of 10 µg/ml or more.
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