The c-Myc protein is an oncogenic transcription factor, encoded by the c-Myc gene on human chromosome 8q24. The c-Myc protein is implicated in several physiological processes including proliferation, differentiation, apoptosis, cell cycle progression and cellular metabolism [4]. Deregulated expression of the c-myc has been reported in a variety of human tumors including breast carcinomas, colorectal cancer and hepatocellular carcinoma [5, 6].

The c-Myc gene encodes a polypeptide with a predicted molecular weight of 49 kDa but also demonstrates aberrant electrophoretic mobility in western blotting to manifest an apparent molecular weight of around 62 kDa (p62c-myc) [7]. The anti-c-Myc antibody recognizes an epitope located within the amino acid sequence (Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu-) of the product of the human oncogene c-myc, also known as the c-Myc tag. The antibody detects the c-Myc tag sequence when it is placed at either the amino or the carboxyl terminus of the fusion protein [1]. The c-Myc epitope tag is used to elucidate the topology, movement, and modification of target proteins and protein complexes [8]. Myc-tagged expression vectors and proteins are often available from suppliers. For example, Choi JH et al employed full-length mouse Znrf3/RNF43 expression vector with a Myc–DDK epitope from OriGene to investigate its interaction with other proteins [9]. Figure 1 illustrates subcellular localization (present on the plasma membrane and intracellular punctate structures) of constructs encoding N-terminal Myc epitope-tagged PRL-1, -2, and -3 (Prenylation-dependent Association of Protein-Tyrosine Phosphatases) [1].

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Figure 1. Localization of N-terminal Myc Epitope-Tagged PRL-1, -2, and -3. Association of PRL phosphatases (PRL-1, -2, and -3) with the plasma membrane and intracellular punctate structures. Constructs encoding N-terminal Myc epitope-tagged PRL-1, -2, and -3 were transiently transfected into CHO cells, and the cells were processed for indirect immunofluorescence microscopy to detect the expressed myc-PRL-1 (A), myc-PRL-2 (B), and myc-PRL-3 (C), respectively. Bar, 10 mm. Arrows indicate the labeling on the plasma membrane. Abbreviations: PRL., prenylation-dependent association of protein-tyrosine phosphatases. Reproduced from Figure 2 of [1].

Furthermore, the c-Myc epitope tag proffers the detection of poorly expressed or newly discovered proteins when specific antibodies are not attainable. The myc epitope tag is generally used to determine the expression levels of recombinant proteins in bacterial, yeast, and mammalian systems [10], sometimes in a tandem 6x format (6xMyc) [11]. Figure 2A illustrates western blotting of anti-c-Myc monoclonal antibodies to detect both endogenous c-Myc and a c-Myc protein fused to the ligand binding domain of the estrogen receptor (MycER). c-Myc and c-MycER are distinguished by the higher molecular weight of c-MycER fusion protein. In Figure 2B, immunofluorescence microscopy depicts the nucleus accumulation of c-MycER [2].

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Figure 2. c-Myc ER Expression and Localization in NHF-MycER. (A) Cell lysates from Normal Human Fibroblasts (NHF-MycER and NHF-BABE) were analyzed by Western blot using anti-c-Myc monoclonal antibodies to detect both endogenous c-Myc and c-MycER protein. c-Myc and c-MycER are distinguished by the higher molecular weight of c-MycER fusion protein. (B) Immunofluorescence microscopy of c-MycER. c-MycER was visualized in NHF-MycER cells in the absence or presence of ligand (top and lower panels, respectively) using anti-Myc monoclonal anti-bodies and FITC-conjugated secondary antibodies. c-MycER rap-idly (30 min) accumulates in the nucleus after ligand addition. Actin was detected using Texas red-X phalloidin. Reproduced from Figure 1of [2].

To facilitate Labome visitors to locate the most suited antibodies, including Myc tag antibody, Labome surveyed randomly selected publications. Table 1 lists the major suppliers.

Recently surveyed literature cite Santa Cruz's anti-myc antibodies in immunohistochemistry [22], immunohistochemistry [23], immunoprecipitation [24], and Western blots [25, 26]. Kulkarni S et al used Thermo Fisher anti-myc 6E10 for western blot and immunoprecipitation [20]. Ling Q et al used MilliporeSigma myc antibody for both western blot and immunoprecipitation from plant lysates [11].

Clone 9E10 is the most commonly cited Myc antibody. Clone 9E10, a mouse monoclonal antibody (IgG1, kappa), was raised against a synthetic peptide [AEEQKLISEEDLLRKRREQLKHKLEQLRNSCA] comprising residues 408-439 of human c-myc [27]. The binding epitope is EQKLISEEDLN, and this is, of course, the myc tag, widely used in protein expression. The clone does not bind to rodent or avian myc proteins [27]. The clone is one of most researched antibody. Figures 3 and 4 display both its sequence information and the 3D structure with its epitope peptide [28]. A unique binding mode, antigen clasping, where two antigen-binding sites clasp the same epitope, contributes to its high affinity and specificity [29]. A variant of 9E10, Hyper-Myc, with improved affinity and thermal stability has been generated through in vitro evolution [30].

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Figure 3. Myc antibody 9E10 clone sequences. A: IgG1 heavy chain (GenBank: CAN87018.1) with 470 aa; B: kappa light chain (GenBank: CAN87019.1) with 238 aa. Data from NCBI.

Clone 9E10 has been extensively studied. Its epitope was investigated through substitutional analysis and length variation using peptide spot synthesis on cellulose [31], indicating the residues LISE are crucial for antibody binding, and the shortest peptide with a strong binding is KLISEEDL. The binding of 9E10 Fab fragment with its epitope peptide complex was investigated through x-ray structural analysis [28]. Interestingly, clone 9E10 failed to recognize two out of four recombinant proteins with the myc-tag in Western blot, while detecting all four proteins by immunofluorescence and immunoprecipitation assays [32]. Clone 9E10 exhibits high context-dependent affinity and users of this clone should be keenly aware of the ramification of such a dependency [33]. There are more recent exchanges between researchers about the specificity of clone 9E10 [34, 35].

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Figure 4. Myc antibody 9E10 3D structure (MMDB ID: 62345PDB ID: 2OR9), with its epitope peptide (11 amino acids, in silver color), including the whole length kappa light chain (218 aa, in purple color), and heavy chain variable region with the CH1 region (228 aa, in blue color). Both image and Data from NCBI.

Researchers have used clone 9E10 from MilliporeSigma for western blots [15], from Synaptic Systems for dot blots and Western blots [36], from Thermo Fisher for immunoprecipitation and western blot [20], from Santa Cruz Biotechnology (e.g., sc-40) to perform western blot [13], immunoprecipitation [24], and ChIP-qPCR [37], from Abcam ( ab32) for immunopurifying myc-tagged DROSHA [19], from other suppliers to perform immunohistochemistry [38].

The other cited clones include D84C12 [9] and 9B11 [39, 40] from Cell Signaling Technology. Schüchner S et al evaluated the binding affinities of monoclonal myc tag antibodies (clone 9E10, 4A6, 9B11 and 71D10) and two polyclonal antibodies with different neigboring amino acids and N- or C-terminal placements and found clone 4A6 and 9B11 to be less variable than others in Western blotting [33]. The most cited polyclonal Myc antibody is sc-764 from Santa Cruz Biotechnology, which has been discontinued, see the example in Figure 5. Frottin F et al immunoprecipitated fusion proteins with Miltenyi biotech µMACS c-myc Isolation kits to study nucleolus as a phase-separated protein quality control entity [12]. Steichen JM et al screened a protein surface display library with a C-terminal myc tag using an FITC-conjugated chicken anti-myc polyclonal antibody from ICL [21].

Please note that there are antibodies against endogenous c-Myc, not the myc tag. For example, Abcam ab32072 is a rabbit monoclonal antibody against the N-terminal of human c-myc, and has been used [41]. It can not be used to detect the myc tag. So are others like Abcam ab39688 [42].

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Figure 5. ERα and c-MYC Co-regulate Estrogen-Responsive Genes. Estrogen stimulation enhances the interaction between estrogen receptor (ERα) and c-MYC in the nucleus of MCF7 cells. MCF7 cells were hormone-starved for 3 days and treated with 10 nM 17β-estradiol (E2) for the indicated time periods. Nuclear extracts were prepared and immunoprecipitated with the indicated antibodies or rabbit IgG (negative control). Precipitates were subjected to immunoblotting with antibodies against ERα or c-MYC as indicated. Abbreviations are as follows: I.P., immunoprecipitation; I.B., immunoblot. Reproduced from Figure 7D of [3].

c-Myc fusion proteins can be detected by western blotting, immunofluorescence microscopy, immunoprecipitation, and affinity chromatography.

The small size of the c-myc epitope tag (EQKLISEEDL; ten amino acids long) compared to other commonly used tags [i.e., GST (Glutathione S-transferase; 211 amino acids) or GFP (Green Fluorescent Protein; 238 amino acids] makes it less likely to disrupt the target protein's properties (conformation, function, and localization).

The c-myc epitope can be placed at either the amino or carboxy terminus of the targeted protein in transfected cells.

A cocktail of different protease inhibitors should be included in purification and analysis buffers to ensure an intact tagged protein.

No precise answer can be given since distinct applications request different amounts of tag-specific antibody reagents.