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    kmt6--ezh2-mutated-y646n-antibody-epr24904-99-ab300491.pdf

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Epigenetics and Nuclear Signaling Chromatin Modifying Enzymes Methylation
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RecombinantRabMAb

Recombinant Anti-KMT6 / EZH2 (mutated Y646N) antibody [EPR24904-99] (ab300491)

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-KMT6 / EZH2 (mutated Y646N) antibody [EPR24904-99] (AB300491)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-KMT6 / EZH2 (mutated Y646N) antibody [EPR24904-99] (AB300491)
  • Western blot - Anti-KMT6 / EZH2 (mutated Y646N) antibody [EPR24904-99] (AB300491)
  • Immunocytochemistry/ Immunofluorescence - Anti-KMT6 / EZH2 (mutated Y646N) antibody [EPR24904-99] (ab300491)
  • Flow Cytometry (Intracellular) - Anti-KMT6 / EZH2 (mutated Y646N) antibody [EPR24904-99] (AB300491)
  • Immunoprecipitation - Anti-KMT6 / EZH2 (mutated Y646N) antibody [EPR24904-99] (AB300491)
  • Dot Blot - Anti-KMT6 / EZH2 (mutated Y646N) antibody [EPR24904-99] (AB300491)
  • Anti-KMT6 / EZH2 (mutated Y646N) antibody [EPR24904-99] (AB300491)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [EPR24904-99] to KMT6 / EZH2 (mutated Y646N)
  • Suitable for: Flow Cyt (Intra), IP, WB, Dot blot, IHC-P, ICC/IF
  • Reacts with: Human

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Overview

  • Product name

    Anti-KMT6 / EZH2 (mutated Y646N) antibody [EPR24904-99]
    See all KMT6 / EZH2 primary antibodies
  • Description

    Rabbit monoclonal [EPR24904-99] to KMT6 / EZH2 (mutated Y646N)
  • Host species

    Rabbit
  • Tested applications

    Suitable for: Flow Cyt (Intra), IP, WB, Dot blot, IHC-P, ICC/IFmore details
  • Species reactivity

    Reacts with: Human
    Does not react with: Mouse, Rat
  • Immunogen

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

  • Positive control

    • WB: HEK-293T cells transfected with a human KMT6 / EZH2 mutated Y646N expression vector containing a myc-his tag, whole cell lysate. Dot Blot: Human KMT6 / EZH2 mutated Y646N peptide. IHC-P: Transfected HEK-293T cells. ICC/IF: Transfected HEK-293T cells. Flow Cyt (Intra): Transfected HEK-293T cells. IP: Transfected HEK-293T cells.
  • 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.

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: 7.20
    Preservative: 0.01% Sodium azide
    Constituents: 59% PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
  • Concentration information loading...
  • Purity

    Protein A purified
  • Clonality

    Monoclonal
  • Clone number

    EPR24904-99
  • Isotype

    IgG
  • Research areas

    • Epigenetics and Nuclear Signaling
    • Chromatin Modifying Enzymes
    • Methylation
    • Epigenetics and Nuclear Signaling
    • Chromatin Remodeling
    • Polycomb Silencing
    • PRC2
    • Epigenetics and Nuclear Signaling
    • Chromatin Remodeling
    • Polycomb Silencing
    • PRC3
    • Epigenetics and Nuclear Signaling
    • Chromatin Modifying Enzymes
    • Methylation
    • Lysine methylation
    • Cancer
    • Tumor biomarkers
    • Enzymes
    • Other

Associated products

  • Alternative Versions

    • Anti-KMT6 / EZH2 (mutated Y646N) antibody [EPR24904-99] (BSA and Azide free) (ab300492)
  • Compatible Secondaries

    • VeriBlot for IP Detection Reagent (HRP) (ab131366)
    • Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (ab150081)
    • Goat Anti-Rabbit IgG H&L (HRP) (ab97051)

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab300491 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
Flow Cyt (Intra)
1/500.
IP
1/30.
WB
1/1000. Predicted molecular weight: 85 kDa.
Dot blot
1/1000.
IHC-P
1/200. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
ICC/IF
1/50.
Notes
Flow Cyt (Intra)
1/500.
IP
1/30.
WB
1/1000. Predicted molecular weight: 85 kDa.
Dot blot
1/1000.
IHC-P
1/200. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
ICC/IF
1/50.

Target

  • Function

    Polycomb group (PcG) protein. Catalytic subunit of the PRC2/EED-EZH2 complex, which methylates 'Lys-9' and 'Lys-27' of histone H3, leading to transcriptional repression of the affected target gene. Able to mono-, di- and trimethylate 'Lys-27' of histone H3 to form H3K27me1, H3K27me2 and H3K27me3, respectively. Compared to EZH2-containing complexes, it is more abundant in embryonic stem cells and plays a major role in forming H3K27me3, which is required for embryonic stem cell identity and proper differentiation. The PRC2/EED-EZH2 complex may also serve as a recruiting platform for DNA methyltransferases, thereby linking two epigenetic repression systems. Genes repressed by the PRC2/EED-EZH2 complex include HOXC8, HOXA9, MYT1, CDKN2A and retinoic acid target genes.
  • Tissue specificity

    Expressed in many tissues. Overexpressed in numerous tumor types including carcinomas of the breast, colon, larynx, lymphoma and testis.
  • Sequence similarities

    Belongs to the histone-lysine methyltransferase family. EZ subfamily.
    Contains 1 SET domain.
  • Developmental stage

    Expression decreases during senescence of embryonic fibroblasts (HEFs). Expression peaks at the G1/S phase boundary.
  • Post-translational
    modifications

    Phosphorylated by AKT1. Phosphorylation by AKT1 reduces methyltransferase activity.
  • Cellular localization

    Nucleus.
  • Target information above from: UniProt accession Q15910 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt
  • Database links

    • Entrez Gene: 2146 Human
    • Omim: 601573 Human
    • SwissProt: Q15910 Human
    • Unigene: 444082 Human
    • Alternative names

      • Enhancer of zeste 2 antibody
      • enhancer of zeste 2 polycomb repressive complex 2 subunit antibody
      • Enhancer of zeste homolog 2 (Drosophila) antibody
      • Enhancer of zeste homolog 2 antibody
      • Enhancer of zeste, Drosophila, homolog 2 antibody
      • ENX 1 antibody
      • Enx 1h antibody
      • ENX-1 antibody
      • ENX1 antibody
      • Enx1h antibody
      • EZH 2 antibody
      • EZH1 antibody
      • EZH2 antibody
      • EZH2_HUMAN antibody
      • EZH2b antibody
      • Histone-lysine N-methyltransferase EZH2 antibody
      • KMT 6 antibody
      • KMT6 antibody
      • KMT6A antibody
      • Lysine N-methyltransferase 6 antibody
      • MGC9169 antibody
      • WVS antibody
      • WVS2 antibody
      see all

    Images

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-KMT6 / EZH2 (mutated Y646N) antibody [EPR24904-99] (AB300491)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-KMT6 / EZH2 (mutated Y646N) antibody [EPR24904-99] (AB300491)

      Immunohistochemical analysis of paraffin-embedded sections labeling KMT6 / EZH2 (mutated Y646N) with ab300491 at 1/200 dilution (2.345 µg/ml) followed by a ready to use Leica DS9800 (BOND™ Polymer Refine Detection) kit.
      Panel A: HEK-293T cells transfected with a human EZH2 Y646N expression vector containing a myc-His tag.
      Panel B: HEK-293T cells transfected with a human EZH2 WT expression vector containing a myc-His tag.
      Panel C: HEK-293T transfected with an empty vector.
      Positive staining on HEK-293T cells transfected with a human EZH2 Y646N expression vector (Panel A), no staining on HEK-293T cells transfected with a human EZH2 WT expression vector (Panel B), no staining on HEK-293T transfected with an empty vector (Panel C). The section was incubated with ab300491 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin. Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (BOND™ Polymer Refine Detection). Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-KMT6 / EZH2 (mutated Y646N) antibody [EPR24904-99] (AB300491)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-KMT6 / EZH2 (mutated Y646N) antibody [EPR24904-99] (AB300491)

      Immunohistochemical analysis of paraffin-embedded human testis tissue labeling KMT6 / EZH2 (mutated Y646N) with ab300491 at 1/200 dilution (2.345 µg/ml) followed by a ready to use Leica DS9800 (BOND™ Polymer Refine Detection) kit.
      Negative control: No staining on human testis.
      The section was incubated with ab300491 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin. Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (BOND™ Polymer Refine Detection). Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

    • Western blot - Anti-KMT6 / EZH2 (mutated Y646N) antibody [EPR24904-99] (AB300491)
      Western blot - Anti-KMT6 / EZH2 (mutated Y646N) antibody [EPR24904-99] (AB300491)
      All lanes : Anti-KMT6 / EZH2 (mutated Y646N) antibody [EPR24904-99] (ab300491) at 1/1000 dilution

      Lane 1 : HEK-293T cells transfected with a human KMT6 / EZH2 mutated Y646N expression vector containing a myc-his tag, whole cell lysate
      Lane 2 : HEK-293T cells transfected with a human wild type KMT6 / EZH2 expression vector containing a myc-his tag, whole cell lysate

      Lysates/proteins at 10 µg per lane.

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

      Predicted band size: 85 kDa
      Observed band size: 98 kDa why is the actual band size different from the predicted?


      Exposure time: 6 seconds


      Blocking / Diluting buffer: 5% NFDM/TBST.

      This blot was developed using a high sensitivity ECL substrate.

      Loading controls
      All Lanes:  ab191250, Recombinant Anti-KMT6 / EZH2 antibody and ab213204, Recombinant Anti-6X His tag® antibody

    • Immunocytochemistry/ Immunofluorescence - Anti-KMT6 / EZH2 (mutated Y646N) antibody [EPR24904-99] (ab300491)
      Immunocytochemistry/ Immunofluorescence - Anti-KMT6 / EZH2 (mutated Y646N) antibody [EPR24904-99] (ab300491)

      Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HEK293T (human embryonic kidney epithelial cells) labelling KMT6 / EZH2 (mutated Y646N) with ab300491 at 1/50 dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/2000 dilution (green). Confocal image showing nuclear staining in HEK-293T cells transfected with a human EZH2 Y646N expression vector containing a myc tag. An anti Myc-Tag Mouse monoclonal antibody (Alexa Fluor® 647) at 1/100 dilution was used as a counterstain (red). The nuclear counterstain is DAPI (blue).

    • Flow Cytometry (Intracellular) - Anti-KMT6 / EZH2 (mutated Y646N) antibody [EPR24904-99] (AB300491)
      Flow Cytometry (Intracellular) - Anti-KMT6 / EZH2 (mutated Y646N) antibody [EPR24904-99] (AB300491)

      Flow Cytometry (Intracellular) analysis of HEK-293T (human embryonic kidney) cells transfected with a human wild type KMT6 / EZH2 expression vector containing a myc-his tag (Left) / HEK-293T transfected with a human KMT6 / EZH2 mutated Y646N expression vector containing a myc-his tag (Right). Cells were fixed with 4% paraformaldehyde and permeabilized with 90% Methanol. ab300491 used at a 1/500 dilution. A Goat Anti-Rabbit IgG (Alexa Fluor® 488, (ab150081)) was used as the secondary antibody at a 1/2000 dilution.

    • Immunoprecipitation - Anti-KMT6 / EZH2 (mutated Y646N) antibody [EPR24904-99] (AB300491)
      Immunoprecipitation - Anti-KMT6 / EZH2 (mutated Y646N) antibody [EPR24904-99] (AB300491)

      KMT6 / EZH2 (mutated Y646N) was immunoprecipitated from 10 μg HEK-293T cells transfected with a human KMT6 / EZH2 mutated Y646N expression vector containing a myc-his tag, whole cell lysate with ab300491 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab300491 at 1/1000 dilution. VeriBlot for IP secondary antibody (HRP) (ab131366), was used as secondary antibody at 1/5000 dilution.

      Lane 1: HEK-293T cells transfected with a human KMT6 / EZH2 mutated Y646N expression vector containing a myc-his tag, whole cell lysate 10 μg (Input).

      Lane 2: HEK-293T cells transfected with a human KMT6 / EZH2 mutated Y646N expression vector containing a myc-his tag, whole cell lysate. 

      Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab300491 in HEK-293T cells transfected with a human KMT6 / EZH2 mutated Y646N expression vector containing a myc-his tag, whole cell lysate.

      Blocking and dilution buffer: 5% NFDM/TBST.
      Exposure time: 3 minutes.

      Observed MW(KDa): 98

      This blot was developed using a high sensitivity ECL substrate.

    • Dot Blot - Anti-KMT6 / EZH2 (mutated Y646N) antibody [EPR24904-99] (AB300491)
      Dot Blot - Anti-KMT6 / EZH2 (mutated Y646N) antibody [EPR24904-99] (AB300491)

      Dot Blot - Anti KMT6 / EZH2 (mutated Y646N)

      Concentration of ab300491: 1/1000 dilution (0.469 μg/ml)

      Secondary ab: Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051), 1/100000 dilution

      Blocking/diluting buffer: 5% NFDM/TBST

      Lane 1: Human KMT6 / EZH2 mutated Y646N peptide

      Lane 2: Human wild type KMT6 / EZH2 peptide

      Exposure time: 3 minutes

    • Anti-KMT6 / EZH2 (mutated Y646N) antibody [EPR24904-99] (AB300491)
      Anti-KMT6 / EZH2 (mutated Y646N) antibody [EPR24904-99] (AB300491)

    Protocols

    To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.

    Click here to view the general protocols

    Datasheets and documents

    • SDS download

    • Datasheet download

      Download

    Certificate of Compliance

    To download a Certificate of Compliance, please enter your Lot number below:

    References (0)

    Publishing research using ab300491? Please let us know so that we can cite the reference in this datasheet.

    ab300491 has not yet been referenced specifically in any publications.

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