This antibody is specific for Myc tagged proteins. The Myc tag epitope (EQKLISEEDL) is located at the dimerization site of c-myc and therefore this antibody does not perform well at recognizing endogenous c-myc. A publication by Baker AM et al. 2016 (PMID: 26826706 DOI: 10.1111/his.12939) shows the IHC staining generated by the 9E10 clone does not correlate with c-myc mRNA expression.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 5 µg/ml.
Use at an assay dependent concentration.
IHC (Methanol fixed)
1/200. PubMed: 17329357
Use 1µg for 106 cells.
ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.
1/500 - 1/1000.
Use at 6 µg/mg of lysate.
Use at an assay dependent concentration.
1/250 - 1/500.
1/1000. See Abreviews.
Use at an assay dependent concentration.
Epitope tags are short peptide sequences that are easily recognized by tag-specific antibodies. Due to their small size, epitope tags do not affect the tagged protein’s biochemical properties. Most often sequences encoding the epitope tag are included with target DNA at the time of cloning to produce fusion proteins containing the epitope tag sequence. This allows anti-epitope tag antibodies to serve as universal detection reagents for any tag containing protein produced by recombinant means. This means that anti-epitope tag antibodies are a useful alternative to generating specific antibodies to identify, immunoprecipitate or immunoaffinity purify a recombinant protein. The anti-epitope tag antibody is usually functional in a variety of antibody-dependent experimental procedures. Expression vectors producing epitope tag fusion proteins are available for a variety of host expression systems including bacteria, yeast, insect and mammalian cells.
c-myc tag antibody
Myc Epitope Tag antibody
Immunocytochemistry/ Immunofluorescence - Anti-Myc tag antibody [9E10] - ChIP Grade (ab32)Image from Harris CJ et al., PLoS Genet. 2016;12(5):e1005998. Fig 5.; doi: 10.1371/journal.pgen.1005998.
(A-D) Representative examples of body forming AtMORC7-MYC, AtMORC4-MYC, At-MORC1-MYC, and AtMORC6-MYC nuclei, respectively. (E) Untransformed wt nucleus subjected to the same antibody staining and imaging procedure. Left panels = anti-MYC channel; middle panels = DAPI channel (gray scaled). DAPI stains DNA, defining the position of dense chromocenters as high intensity white foci; right panels = merged channels (DAPI in blue, MYC in green). White triangles indicate examples of chromocenter adjacent AtMORC localization. Scale bars = 5 μM.
Leaves from three-week old plants were fixed in 4% paraformaldehyde in TRIS buffer (10 mM TRIS pH 7.5, 10 mM EDTA, and 100 mM NaCl) for 20 minutes and washed twice in TRIS buffer. Leaves were chopped in 200–400 microliters lysis buffer (15 mM TRIS pH 7.5, 2 mM EDTA, 0.5 mM spermine, 80 mM KCl, 20 mM NaCl, and 0.1% Triton X-100) and filtered through a 3 μM cell strainer. 5 μL of nuclei suspension was added to 12 μL of sorting buffer (100mM TRIS pH 7.5, 50mM KCl, 2mM MgCl2, 0.05% Tween-20, and 20.5% sucrose) and air dried on chloroform dipped microscope slides for two hours and then post-fixed in 4% paraformaldehyde in PBS for 20 minutes. Slides were washed three times in PBS and incubated in blocking buffer (3% BSA, and 10% horse serum in PBS) for 30 minutes at 37°C. Nuclei were incubated at 4°C overnight in mouse monoclonal antibody against c-Myc (9E10, ab32; 1/200). Slides were washed in PBS and incubated with goat anti-mouse FITC antibody (ab7064; 1/200) for 90 minutes at room temperature. Following PBS washes, nuclei were counterstained and mounted in Vectashield mounting media with DAPI. Nuclei were analyzed with a Zeiss LSM 710 Confocal microscope at 63X or 100X magnification using Zen software.
Western blot - Anti-Myc tag antibody [9E10] - ChIP Grade (ab32)
Anti-Myc tag antibody [9E10] - ChIP Grade (ab32) at 1 µg/ml + E. coli Positive Control (Escherichia coli ) Whole Cell Lysate (ab5395) at 10 µg
Secondary Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution Developed using the ECL technique
Lysate from E. coli recombinantly expressing 11 commonly used tags including myc tag.
Immunocytochemistry/ Immunofluorescence - Anti-Myc tag antibody [9E10] - ChIP Grade (ab32)This image is courtesy of an anonymous Abreview
Ab32 staining a Myc tagged protein in HeLa cells by ICC/IF (Immunocytochemistry/Immunofluorescence). Endogenous c-myc was not detected under these conditions. Cells were fixed with paraformaldehyde, permeabilized with 0.5% Triton and blocked with 5% Serum for 30 minutes at 25°C. Samples were incubated with primary antibody (1/1000 in 5% Serum) for 1 hour at 25°C. An Alexa Fluor® 488 conjugated Goat anti-Mouse was used as a secondary antibody.
Immunocytochemistry/ Immunofluorescence - Anti-Myc tag antibody [9E10] - ChIP Grade (ab32)Image from Molla-Herman A et al., PLoS One. 2008;3(11):e3728. Fig 8(B).; doi: 10.1371/journal.pone.0003728.
RPE1 cells grown on coverslips were transfected with βarr2-myc, grown in low serum and then fixed and stained for Kif3A (red) and ab32 (green). Insets show higher magnifications of a representative PC. Kif3A was found in the cytoplasm and at the tip of the axoneme where it was colocalized with βarr2.
Cells were incubated with primary antibodies in permeabilization buffer (PBS with 1 mg/mL bovine serum albumin (PBS-BSA) and 0.1% triton-X-100) for 45 minutes at room temperature. After two washes with PBS-BSA, cells were incubated for 30 minutes at room temperature in PBS-BSA containing secondary antibodies. After one wash with PBS-BSA and two washes in PBS, cells were laid down on microscope slides in a PBS–glycerol mix (50/50) with DAPI.
Western blot - Anti-Myc tag antibody [9E10] - ChIP Grade (ab32)Menssen R et al., (2001) Curr Biol. Mar 6;11(5):345-50.
Predicted band size : 41 kDa
Menssen R et al., (2001) Curr Biol. Mar 6;11(5):345-50.
Phosphorylation of Cdc15 changes during the cell cycle. Exponentially growing cells (cyc) of CDC15-MYC9 (W1114) were arrested in G1 with a factor pheromone and released into fresh medium at 25°C. Cells were harvested at the indicated times, the percentage of divided nuclei was determined by DAPI staining of fixed cells, and proteins were analyzed by western blotting with 9E10 (ab32).
ChIP - Anti-Myc tag antibody [9E10] - ChIP Grade (ab32)Image from Durand-Dubief M et al., PLoS Genet. 2012;8(9):e1002974. Fig 7(A).; doi: 10.1371/journal.pgen.1002974.
Cse4 histone variant occupancy at the endogenous CEN3 locus (eCEN3) and at a conditional CEN3 locus (cCEN3) when it is repressed (in the presence of galactose) or induced (glucose) by ChIP. Control (SC138) and Δfun30 (SC140) cells are grown in YP Gal until≈1 OD, then shifted in YP Glu or YP Gal containing 15 µg/ml of nocodazole and incubated for 4 hours. Cse4-Myc associated chromatin was immunoprecipitated and the immunoprecipitated DNA was analyzed by PCR followed by agarose gel electrophorsis and ethidium bromide staining to visualize the DNA fragments. Shown are the relevant, cropped out bands from a single, representative gel, input was 1/243th of the immunoprecipitated material.
Overnight cultures grown in YPD at 30°C were diluted to 0.2 OD595, then grown to 0.7 OD595 at 30°C before crosslinking. Samples were crosslinked 15 min for H3, 3Myc-Htz1 and Cse4-Myc or 30 min for Fun30 detection with 1% final formaldehyde and chromatin extracts were sonicated to ∼500 bp. Triplicates or duplicate ChIP samples were validated by qPCR. Chromatin extracts were then immunoprecitated with 2 µg of mouse monoclonal anti-myc (9E10, ab32) for Cse4-Myc.
Jiang H et al. The regulator of calcineurin 1 increases adenine nucleotide translocator 1 and leads to mitochondrial dysfunctions. J Neurochem140:307-319 (2017).
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Cheung RS et al. Ubiquitination-Linked Phosphorylation of the FANCI S/TQ Cluster Contributes to Activation of the Fanconi Anemia I/D2 Complex. Cell Rep19:2432-2440 (2017).
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