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Anti-Myc tag antibody [9E10] (ab32)

Reviews (29)Q&A (17)References (403)
Western blot - Anti-Myc tag antibody [9E10] (ab32)
  • Immunocytochemistry/ Immunofluorescence - Anti-Myc tag antibody [9E10] (ab32)
  • Western blot - Anti-Myc tag antibody [9E10] (ab32)
  • Immunocytochemistry/ Immunofluorescence - Anti-Myc tag antibody [9E10] (ab32)
  • Immunocytochemistry/ Immunofluorescence - Anti-Myc tag antibody [9E10] (ab32)
  • Western blot - Anti-Myc tag antibody [9E10] (ab32)

Key features and details

  • Mouse monoclonal [9E10] to Myc tag
  • Suitable for: ICC/IF, Flow Cyt, WB, IP, ELISA, IHC-Fr, Purification
  • Reacts with: Species independent
  • Isotype: IgG1

Conjugates logo Related conjugates and formulations

Alexa Fluor® 488 Alexa Fluor® 594 Alexa Fluor® 647 APC Biotin Biotin DyLight® 488 DyLight® 550 DyLight® 650 FITC HRP PE SureLight® APC

Overview

  • Product name

    Anti-Myc tag antibody [9E10]
    See all Myc tag primary antibodies

  • Description

    Mouse monoclonal [9E10] to Myc tag

  • Host species

    Mouse

  • Tested applications

    Suitable for: ICC/IF, Flow Cyt, WB, IP, ELISA, IHC-Fr, Purificationmore details

  • Species reactivity

    Reacts with: Species independent

  • Immunogen

    Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.

  • Positive control

    • Myc tagged proteins and myc tag expressing cells.

  • General notes

    If you require this antibody in a particular buffer formulation or a particular conjugate for your experiments, please contact orders@abcam.com or you can find further information here.

    The Life Science industry has been in the grips of a reproducibility crisis for a number of years. Abcam is leading the way in addressing this with our range of recombinant monoclonal antibodies and knockout edited cell lines for gold-standard validation. Please check that this product meets your needs before purchasing.

    If you have any questions, special requirements or concerns, please send us an inquiry and/or contact our Support team ahead of purchase. Recommended alternatives for this product can be found below, along with publications, customer reviews and Q&As

Properties

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab32 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
ICC/IF (6)
Use a concentration of 5 µg/ml.
Flow Cyt
Use 1µg for 106 cells.

ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.
WB (13)
1/500 - 1/1000.
IP (3)
Use at 6 µg/mg of lysate.
ELISA
Use at an assay dependent concentration.
IHC-Fr
1/1000.

See Abreviews.
Purification
Use at an assay dependent concentration.
Notes
ICC/IF
Use a concentration of 5 µg/ml.
Flow Cyt
Use 1µg for 106 cells.

ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.
WB
1/500 - 1/1000.
IP
Use at 6 µg/mg of lysate.
ELISA
Use at an assay dependent concentration.
IHC-Fr
1/1000.

See Abreviews.
Purification
Use at an assay dependent concentration.

Target

  • Relevance

    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.

  • Cellular localization

    Nuclear

  • Alternative names

    • c-myc tag antibody
    • Myc Epitope Tag antibody

Images

  • Western blot - Anti-Myc tag antibody [9E10] (ab32)
    Western blot - Anti-Myc tag antibody [9E10] (ab32)
    All lanes : Anti-Myc tag antibody [9E10] (ab32) at 1/200 dilution

    Lane 1 : Wild-type HEK-293T cell lysate
    Lane 2 : MYC CRISPR-Cas9 edited HEK-293T cell lysate
    Lane 3 : Jurkat cell lysate
    Lane 4 : SH-SY5Y cell lysate

    Lysates/proteins at 20 µg per lane.

    Performed under reducing conditions.


    False colour image of Western blot: Anti-Myc tag antibody [9E10] staining at 1/200 dilution, shown in green; Rabbit Anti-GAPDH antibody [EPR16891] (ab181602) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab32 was shown to bind specifically to Myc tag. A band was observed at 57 kDa in wild-type HEK-293T cell lysates with no signal observed at this size in MYC CRISPR-Cas9 edited cell line ab256500 (CRISPR-Cas9 edited cell lysate ab263850). The band observed in the CRISPR-Cas9 edited lysate lane below 57 kDa is likely to represent a truncated form of Myc tag. This has not been investigated further and the functional properties of the gene product have not been determined. To generate this image, wild-type and MYC CRISPR-Cas9 edited HEK-293T cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Mouse IgG H&L (IRDye® 800CW) preabsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preabsorbed (ab216777) at 1/20000 dilution.

  • Immunocytochemistry/ Immunofluorescence - Anti-Myc tag antibody [9E10] (ab32)
    Immunocytochemistry/ Immunofluorescence - Anti-Myc tag antibody [9E10] (ab32)Image from Harris CJ et al., PLoS Genet. 2016;12(5):e1005998. Fig 5.; doi: 10.1371/journal.pgen.1005998. Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/

    (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] (ab32)
    Western blot - Anti-Myc tag antibody [9E10] (ab32)
    Anti-Myc tag antibody [9E10] (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.

    Performed under reducing conditions.

    Observed band size: 45 kDa why is the actual band size different from the predicted?


    Exposure time: 1 minute


    Lysate from E. coli recombinantly expressing 11 commonly used tags including myc tag.

  • Immunocytochemistry/ Immunofluorescence - Anti-Myc tag antibody [9E10] (ab32)
    Immunocytochemistry/ Immunofluorescence - Anti-Myc tag antibody [9E10] (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.

    See Abreview

  • Immunocytochemistry/ Immunofluorescence - Anti-Myc tag antibody [9E10] (ab32)
    Immunocytochemistry/ Immunofluorescence - Anti-Myc tag antibody [9E10] (ab32)Image from Molla-Herman A et al., PLoS One. 2008;3(11):e3728. Fig 8(B).; doi: 10.1371/journal.pone.0003728. Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/

    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] (ab32)
    Western blot - Anti-Myc tag antibody [9E10] (ab32)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).

References (403)

Publishing research using ab32? Please let us know so that we can cite the reference in this datasheet.

ab32 has been referenced in 403 publications.

  • Xu WN  et al. Sesn2 Serves as a Regulator between Mitochondrial Unfolded Protein Response and Mitophagy in Intervertebral Disc Degeneration. Int J Biol Sci 19:571-592 (2023). PubMed: 36632468
  • Feng T  et al. OsMADS14 and NF-YB1 cooperate in the direct activation of OsAGPL2 and Waxy during starch synthesis in rice endosperm. New Phytol 234:77-92 (2022). PubMed: 35067957
  • Sengupta S  et al. GABA transmission from mAL interneurons regulates aggression in Drosophila males. Proc Natl Acad Sci U S A 119:N/A (2022). PubMed: 35082150
  • Cohen A  et al. TOR complex 2 contributes to regulation of gene expression via inhibiting Gcn5 recruitment to subtelomeric and DNA replication stress genes. PLoS Genet 18:e1010061 (2022). PubMed: 35157728
  • Li X  et al. Regulation of P53 signaling in breast cancer by the E3 ubiquitin ligase RNF187. Cell Death Dis 13:149 (2022). PubMed: 35165289
View all Publications for this product

Customer reviews and Q&As

Show All Reviews Q&A
Submit a question

1-10 of 17 Q&A

Question

The data sheet of ab32 states it's good for IP but NOT suitable for immunoprecipitation of native c-myc protein.
Could you please explain above statement? How can the antibody be good for CHIP that requires it to react with the native C-myc form and not be good for IP of the native form?

Read More
Answer


The antibody was originally designed for pull down (IP) and ChIP of c-myc-tagged proteins. And that is how it has been used by other customers who submitted IP and ChIP reviews.

The information that "This antibody is NOT suitable for immunoprecipitation of native c-myc protein." is information from the originating lab. I believe the antibody was not tested this way in ChIP by the originating lab, which is why we did not add the same statement for the ChIP application.

It was later shown that the antibody also detects the human protein.

Read More

Question

what is the affinity of the antibody?

Read More
Answer

Vielen Dank für Ihren Anruf.

In dem folgenden Artikel wurde die Affinität des Antikörpers zu c-Myc tags getestet. Mit ELISA wurde eine Kd von 5.6 "107 für die Sequenz EQKLISEEDLN gemessen.
http://peds.oxfordjournals.org/content/14/10/803.full

Ich hoffe, dies hilft Ihnen weiter. Bitte zögern Sie nicht, sich wieder bei uns zu melden, falls Sie weitere Fragen haben.

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Question

A potential customer would like to detect human Oct4, Sox-2, C-myc in
HFF and HEK293T using these 3 antibodies in western blotting:
AB18976 Abcam Oct4 antibody, 100 ug
AB97959 Abcam SOX2 antibody, 100ug
AB32 Abcam c-Myc antibody [9E10], 100 µg
Please advise if it is feasible?

Read More
Answer

Thank you for contacting us.

Please advice customer to consult publication whole selecting the antibodies.

HEK293T cells do not express c-Myc and Oct4. They express SOX2.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1289416/

Human embryonic fibroblasts have basal level of Oct4 and Sox2 proteins. You may need to induce the expression of these proteins.

http://sklrm.shsmu.edu.cn/manage/edit/UploadFile/200982102734474.pdf

c-myc is also OK in HEF cells; http://www.pnas.org/content/106/31/12759.full

I hope this information is helpful to you.

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Question

Hello, I want to purchase you anti-c-Myc antibody, but before I have couple questions: 1) I want to use it in Western blot against the natural endogenic c-Myc, not Myc-tagged protein, as I saw most people did. can I still use ab32 for this purpose? 2) about ab dilution, what would be preferable dilution if the amounts of c-myc I want to detect are very low? should I dilute it more than usual? what about cases of over-expression? thank you

Read More
Answer

Thank you for your enquiry. Ab32 recognizes recombinant proteins incorporating a c-Myc epitope tag and it also detects human c-Myc protein. The optimal dilution range to use in your samples does indeed depend on the abundance of protein in them; it is hard to tell what will work for your samples, I therefore recommend trying a range of dilutions in a preliminary experiment; I would recommend trying 1:100, 1:500, 1:1000, 1:2000 and optimise following the results of those tests.

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Question

I am looking for a myc tag antibody to detect transfected myc tag protein in rat neuronal cells. It appears to me that both ab 32 as well as ab 9106 may be suitable for immunofluorescence. Do you know whether any of these antibodies would be preferrable over the other one to use for immunofluorescent staining on 4% paraformaldehyde fixed cells? Alternatively if there is another myc tag antibody that would be recommendable for this application please let me know. Thank you.

Read More
Answer

I'm afraid I have received very little information from the source of ab9106. I was informed just a few minutes ago " a customer of mine had sent me the anti-Myc image, but only provided me with the working dilution. I have been unable to track them down at their university". I am therefore unable to find out what type of fixative was used. For ab32, the information that it works in IF dates from 2001, where a customer told us it worked well for him. At the time we did not request further details and therefore unfortunately again we do not know if 4%PFA was the fixative successfully used. If you are able to try different fixatives on your cells I would therefore recommend to try both 4% PFA and acetone as fixatives and see which one works well for you. If you cannot have your cells fixed in anything other than 4% PFA I would recommend not to purchase these products as I'm not sure they will work with 4% PFA. I would be happy to enquire if ab9132 will work on 4% PFA fixed cells, however the detection method was chromogenic, not fluorescent, therefore the antibody may or may not work in IF, we don't know at this stage. I'm sorry I cannot help you more on this occasion,

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Question

Was this antibody purified from ascites or TC supernatant? What is the purification process?

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Answer

Thank you for your enquiry. This monoclonal antibody was purified from tissue culture supernatant using a standard protein A, binding in ammonium sulphate Ph 9. The sample is eluted in a commercially bought Neutral Elution Buffer and exchanged into PBS using a G25 column. I hope this information helps. Please do not hesitate to contact us if you need anything further.

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Question

We have purchased ab32 for use on formalin fixed wax embedded transgenic mouse heart tissue which contains a membrane protein with a myc epitope tag. The ab32 works fine on transfected cells but we aren't getting anywhere with the fixed tissue. We have been using citrate plus microwave antigen retrieval. Would enzymatic antigen retrieval be better with this Ab. Secondly we would like to use something as a positive control as it may be that levels of our protein are just too low to detect. Do you have any suggestions for something we could obtain commercially.

Read More
Answer

I'm sorry to hear you are experiencing problems with ab32 in IHC-P. The antibody was tested in this application by Jennifer Edwards who submitted a review on the 29 November 2004 of her experience of this product. Unfortunately she does not mention what antigen retrieval was used. You can contact her directly via the link on her review, hopefully she can tell you more protocol details. We can also look into details at your protocol to make some suggestions to improve your staining, indeed changing antigen retrieval method may help. I enclose the link to a protocol questionnaire where you can put your protocol easily together, we will gladly look at it. https://www.abcam.com/index.html?section=ihc&pageconfig=technical&intAbID=32&mode=questionaire We look forward to hearing from you to help you more,

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Question

ORDER NUMBER 111584 DESCRIPTION OF THE PROBLEM Non-specific band SAMPLE Control (rat primary muscle precursor cells - differentiating) Positive: same sample with adenovirus with c-myc tagged protein PRIMARY ANTIBODY 1:1,000 (1.2 ug/ml) based upon previous question to Abcam diluted in blocking buffer DETECTION METHOD ECL Plus (western lighning) POSITIVE AND NEGATIVE CONTROLS USED See sample ANTIBODY STORAGE CONDITIONS Aliquotted and stored at -20C SAMPLE PREPARATION Lysed in cell lysing buffer (cell lytic); boile sample in Laemli buffer AMOUNT OF PROTEIN LOADED 30 ug ELECTROPHORESIS/GEL CONDITIONS SDS-Page; 4-20% TRANSFER AND BLOCKING CONDITIONS Overnight at 30V (mini blot from Biorad); 5% NFDM in TBS-T SECONDARY ANTIBODY HRP conjugated anti-mouse from {a competitor} HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 2 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? No primary control ADDITIONAL NOTES Comment from customer: You can see that this non-specific signal poses a problem for us, not to mention that it looks like we are not over-expressing our protein, but that is a problem for another day (and of course, not your problem). Also, I included some of the information about the samples and our protocol.

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Answer

Thank you for your enquiry and I'm sorry to hear that your customer is experiencing difficulty with this antibody. For the tagged samples - was human c-myc used? We do have a Western blotting protocol available for this antibody (I have included it below), and would suggest that your customer incubate with the primary for 1 hr at RT and block overnight at 4C. Your customer may have already tried this, please let me know. If this does not help, I can certainly offer a free of charge replacement vial or refund.

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Question

I would like to use the myc tag ab 9E10 for immunoprecipitation. Can I use protein A to IP which I have already got or it is better to use Protein G which I have read has a higher affinity for IgG1.

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Answer

You are right, not all the classes or subclasses of immunoglobulin from all species bind tightly to Protein A. For those that do not insolubilized Protein G, Protein A/G or a second antibody is used to allow precipitation. As a general rule, for mouse IgG1 (this antibody is raised in mouse and the subclasses is IgG1) Protein G would be a better choice. However, you may also be able to use Protein A successfully (although the affinity is lower), so just have a go since you already have this material in your lab. We would like to draw your attention to the published reviews on-line, particularly the IP-related ones. One particular customer has reported that Protein G worked nicely. We hope this information will be useful for you.

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Question

with regards to the Myc tag antibody [9E10] (ab32) datasheet; would it be any good for western blotting proteins expressed in HEK293 or TSA201 cells? Would the cross reactivity mask any sort of signal?

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Answer

Thank you for your email. All the information that we currently have regarding ab32 is located on the online datasheet. This antibody gives variable results on native protein and is mainly used for tagged recombinant proteins. You may also want to look at ab56 - Mouse monoclonal [9E11] to c-Myc. If you have any more questions, please let us know.

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1-10 of 17 Q&A

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