概述

  • 产品名称

    Anti-Myc tag抗体[9E10] - ChIP Grade
    参阅全部 Myc tag 一抗
  • 描述

    小鼠单克隆抗体[9E10] to Myc tag - ChIP Grade
  • 宿主

    Mouse
  • 特异性

    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 (PubMed ID: 26826706 DOI: 10.1111/his.12939) shows the IHC staining generated by the 9E10 clone does not correlate with c-myc mRNA expression.

  • 经测试应用

    适用于: ICC/IF, ICC, ChIP, IHC (Methanol fixed), Flow Cyt, WB, IP, ELISA, IHC-P, IHC-Fr, Purificationmore details
  • 免疫原

    Synthetic peptide corresponding to Human Myc tag aa 400-500 (C terminal) conjugated to keyhole limpet haemocyanin.

  • 表位

    aa 410-419 of human Myc.
  • 阳性对照

    • Myc tagged proteins and myc tag expressing cells.
  • 常规说明

    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.

性能

应用

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.

应用 Ab评论 说明
ICC/IF Use a concentration of 5 µg/ml.
ICC 1/2000.

See Abreviews.

ChIP Use at an assay dependent concentration.
IHC (Methanol fixed) 1/200. PubMed: 17329357
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-P 1/250 - 1/500.
IHC-Fr 1/1000. See Abreviews.
Purification Use at an assay dependent concentration.

靶标

  • 相关性

    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.
  • 细胞定位

    Nuclear
  • 别名

    • c-myc tag antibody
    • Myc Epitope Tag antibody

图片

  • (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.

     

  • 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.

    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.

  • 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

  • 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.

  • 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).

  • 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.

文献

This product has been referenced in:

  • Feng S  et al. Tripartite motif-containing 14 (TRIM14) promotes epithelial-mesenchymal transition via ZEB2 in glioblastoma cells. J Exp Clin Cancer Res 38:57 (2019). Read more (PubMed: 30728039) »
  • Crerar H  et al. Regulation of NGF Signaling by an Axonal Untranslated mRNA. Neuron 102:553-563.e8 (2019). Read more (PubMed: 30853298) »
See all 211 Publications for this product

客户评价及客户问答

1-10 of 17 Q&A

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.

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Question
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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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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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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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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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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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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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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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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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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