Astana Biomed Group, an authorized Abcam distributor in Central Asia

Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [EPR20547] to 6X His tag® - ChIP Grade
  • Suitable for: WB, ICC/IF, IP, Flow Cyt, ChIP, IHC-P
  • Reacts with: Recombinant fragment

Overview

  • Product name

    Anti-6X His tag® antibody [EPR20547] - ChIP Grade
    See all 6X His tag® primary antibodies

  • Description

    Rabbit monoclonal [EPR20547] to 6X His tag® - ChIP Grade

  • Host species

    Rabbit

  • Tested applications

    Suitable for: WB, ICC/IF, IP, Flow Cyt, ChIP, IHC-Pmore details

  • Species reactivity

    Reacts with: Recombinant fragment

  • Immunogen

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

  • Positive control

    • WB: His-tagged mouse Cathepsin D and CDKN2A/p16INK4a proteins; HEK-293T transfected with His-tagged Staphylococcus aureus cas9, human Brachyury, human PRDM9 and human GAPDH cell lysates. IHC-P: HEK-293T cells transfected with His-tagged S.aureus cas9. ICC/IF: HEK-293T cells transfected with GFP-Myc-His. Flow Cyt: HEK-293T cells transfected with GFP-Myc-His. IP: HEK-293T cells transfected with His-tagged S.aureus cas9 cell lysate. ChIP: Chromatin from MCF7 cells transfected with 6X His-tagged GATA3.

  • General notes

    This product is a recombinant monoclonal antibody, which offers several advantages including:

    • - High batch-to-batch consistency and reproducibility
    • - Improved sensitivity and specificity
    • - Long-term security of supply
    • - Animal-free production
    For more information see here.

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.

    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 the problem with our range of recombinant monoclonal antibodies and knockout edited cell lines for gold-standard validation.

    One factor contributing to the crisis is the use of antibodies that are not suitable. This can lead to misleading results and the use of incorrect data informing project assumptions and direction. To help address this challenge, we have introduced an application and species grid on our primary antibody datasheets to make it easy to simplify identification of the right antibody for your needs.

    Learn more here.

Properties

  • Form

    Liquid

  • Storage instructions

    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term. Avoid freeze / thaw cycle.

  • Storage buffer

    pH: 7.2
    Preservative: 0.01% Sodium azide
    Constituents: 0.05% BSA, 40% Glycerol, PBS
  • Concentration information loading...

  • Purity

    Protein A purified

  • Clonality

    Monoclonal

  • Clone number

    EPR20547

  • Isotype

    IgG

  • Research areas

Images

  • Immunoprecipitation - Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204)
    Immunoprecipitation - Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204)

    His-tagged Staphylococcus aureus cas9 was immunoprecipitated from 0.35 mg of HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) transfected with a His-tagged Staphylococcus aureus cas9 (J7RUA5; aa1-1053; 125 kDa) construct, whole cell lysate with ab213204 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab213204 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10,000 dilution

    Lane 1: HEK-293T transfected with His-tagged Staphylococcus aureus cas9 construct, whole cell lysate 10 μg (Input). 

    Lane 2: ab213204 IP in HEK-293T transfected with His-tagged Staphylococcus aureus cas9 construct, whole cell lysate. 

    Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab213204 in HEK-293T transfected with His-tagged Staphylococcus aureus cas9 construct, whole cell lysate.

    Blocking/Dilution buffer: 5% NFDM/TBST.

    Exposure time: 1 second.

  • Immunocytochemistry/ Immunofluorescence - Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204)
    Immunocytochemistry/ Immunofluorescence - Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204)

    Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) cells transfected with GFP-Myc-His vector expression construct labeling 6X His tag® with ab213204 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (white). Confocal image showing positive staining on HEK-293T cells transfected with GFP-Myc-His vector expression construct.

    The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) (ab195889) (red) at 1/200 dilution.

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204)

    Immunohistochemical analysis of agarose-embedded HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) transfected with a His-tagged Staphylococcus aureus cas9 (J7RUA5; aa1-1053; 125kDa) construct labeling 6X His tag® with ab213204 at 1/16000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. 

    Left image: Positive staining on HEK-293T transfected with a His-tagged Staphylococcus aureus cas9 (J7RUA5; aa1-1053; 125kDa) construct. Right image: No staining on HEK-293T transfected with an empty expression vector. 

    Counter stained with Hematoxylin.

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

    Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204)

    Immunohistochemical analysis of paraffin-embedded human liver tissue labeling 6X His tag® with ab213204 at 1/16000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

    Negative control: No staining on human liver.

    Counter stained with Hematoxylin.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204)

    Immunohistochemical analysis of paraffin-embedded rat stomach tissue labeling 6X His tag® with ab213204 at 1/16000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

    Negative control: No staining on rat stomach.

    Counter stained with Hematoxylin.

     

  • ChIP - Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204)
    ChIP - Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204)

    Chromatin was prepared from MCF7 (human breast adenocarcinoma cell line) cells transfected with 6X His-tagged GATA3 according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 minutes. The ChIP was performed with 25µg of chromatin, 2µg of ab213204 (blue), and 20µl of A/G sepharose beads slurry (10µl of sepharose A beads + 10µl of sepharose G beads). 2μg of rabbit normal IgG was added to the beads control (yellow).  The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).

    ChIP was performed according to the literature (PMID:22951069).

  • Flow Cytometry - Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204)
    Flow Cytometry - Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204)

    Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol permeabilized HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) transfected with GFP-Myc-His vector labeling 6X His tag® with ab213204 (right panel) at 1/5000 dilution compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (left panel). Goat Anti-Rabbit IgG H&L (Alexa Fluor® 647) (ab150079) at 1/2000 dilution was used as the secondary antibody.

    Gate is set between transfected and untransfected HEK-293T cells.

  • Western blot - Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204)
    Western blot - Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204)
    All lanes : Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204) at 1/20000 dilution

    Lane 1 : His-tagged mouse Cathepsin D recombinant protein (aa65-310; 27kDa)
    Lane 2 : His-tagged mouse CDKN2A/p16INK4a recombinant protein (aa1-168;18kDa)

    Lysates/proteins at 0.02 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

    Developed using the ECL technique.

    Observed band size: 18,27 kDa why is the actual band size different from the predicted?


    Exposure time: 1 second


    Blocking/Dilution buffer: 5% NFDM/TBST.

  • Western blot - Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204)
    Western blot - Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204)
    All lanes : Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204) at 1/5000 dilution

    Lane 1 : HEK-293T (human epithelial cell from embryonic kidney transformed with large T antigen) transfected with an empty expression vector, whole cell lysate
    Lane 2 : HEK-293T (human epithelial cell from embryonic kidney transformed with large T antigen) transfected with a His-tagged Staphylococcus aureus cas9 (J7RUA5; aa1-1053;125kDa) construct, whole cell lysate
    Lane 3 : HEK-293T (human epithelial cell from embryonic kidney transformed with large T antigen) transfected with a His-tagged human Brachyury (O15178; aa1-435;50kDa) construct, whole cell lysate
    Lane 4 : HEK-293T (human epithelial cell from embryonic kidney transformed with large T antigen) transfected with a His-tagged human PRDM9 (Q9NQV7; aa1-894;100kDa) construct, whole cell lysate
    Lane 5 : HEK-293T (human epithelial cell from embryonic kidney transformed with large T antigen) transfected with a His-tagged human GAPDH (P04406; aa1- 335;36kDa) construct, whole cell lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

    Developed using the ECL technique.

    Observed band size: 100,125,36,50 kDa why is the actual band size different from the predicted?



    Blocking/Dilution buffer: 5% NFDM/TBST.

    Exposure time: Lane 1-3: 1 second; Lane 4-5: 10 seconds.

  • Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204)
    Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204)

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