Anti-KMT6 / EZH2 antibody - ChIP Grade (ab3748)

Overview

  • Product nameAnti-KMT6 / EZH2 antibody - ChIP Grade
    See all KMT6 / EZH2 primary antibodies
  • Description
    Rabbit polyclonal to KMT6 / EZH2 - ChIP Grade
  • Specificityab3748 antibody recognises EZH2 in cells transfected with HA-EZH2 in IP.
  • Tested applicationsSuitable for: IHC-FoFr, WB, IP, ChIP, IHC-P, ICC/IF, RIPmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Cow, Human, Xenopus laevis
    Predicted to work with: Zebrafish
  • Immunogen

    Synthetic peptide conjugated to KLH derived from within residues 50 - 150 of Human EZH2.

    (Peptide available as ab13744.)

  • Positive control
    • WB: Hek293 whole cell lysate; EZH2 recombinant ICC: mouse embryonic stem cells IP: U20S cells transfected with 4ug of Myc-EZH2
  • General notes

    The Drosophila protein Enhancer of Zeste is a member of the Polycomb group, which maintains homeotic gene repression and is thought to control gene expression by regulating chromatin. EZH2 is thought to perform a similar role.

Properties

Applications

Our Abpromise guarantee covers the use of ab3748 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
IHC-FoFr Use at an assay dependent concentration.
WB Use a concentration of 0.4 - 1 µg/ml. Can be blocked with KMT6 / EZH2 peptide (ab13744).
IP Use at an assay dependent concentration.
ChIP Use at an assay dependent concentration.
IHC-P Use at an assay dependent concentration.
ICC/IF 1/100.
RIP Use at an assay dependent concentration. PubMed: 19571010

Target

  • FunctionPolycomb 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 specificityExpressed in many tissues. Overexpressed in numerous tumor types including carcinomas of the breast, colon, larynx, lymphoma and testis.
  • Sequence similaritiesBelongs to the histone-lysine methyltransferase family. EZ subfamily.
    Contains 1 SET domain.
  • Developmental stageExpression 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 localizationNucleus.
  • Information by UniProt
  • Database links
  • 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

Anti-KMT6 / EZH2 antibody - ChIP Grade images

  • All lanes : Anti-KMT6 / EZH2 antibody - ChIP Grade (ab3748) at 1/1000 dilution

    Lane 1 : HEK293 cell lysate
    Lane 2 : Mouse hippocampus cell lysate
    Lane 3 : Rat primary hippocampal neurons cell lysate

    Lysates/proteins at 30 µg per lane.

    Secondary
    HRP-conjugated Mouse anti-rabbit IgG at 1/5000 dilution
    developed using the ECL technique

    Performed under reducing conditions.

    Observed band size : 85 kDa (why is the actual band size different from the predicted?)
    Additional bands at : 100 kDa (possible non-specific binding),20 kDa (possible non-specific binding).

    Exposure time : 20 seconds

    This image is courtesy of an anonymous Abreview

    See Abreview

  • ab3748 staining KMT6/EZH2 in the HeLa cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehye permeabilized with 0.2% Triton X-100 in PBS and blocked with 2% BSA for 45 minutes at RT. Samples were incubated with primary antibody (1/2550 in PBS + 2% BSA) for 14 hours at 4°C. An  Alexa Fluor® 488-conjugated Goat anti-rabbit IgG polyclonal was used as the secondary antibody (1:10000).

    See Abreview

  • All lanes : Anti-KMT6 / EZH2 antibody - ChIP Grade (ab3748) at 1/1000 dilution

    Lane 1 : Human glioma cell LN229
    Lane 2 : Human glioma cell LN229 transfected with a Ezh2-siRNA

    Lysates/proteins at 20 µg per lane.

    developed using the ECL technique

    Performed under reducing conditions.

    Additional bands at : 90 kDa. We are unsure as to the identity of these extra bands.

    Exposure time : 1 minute

    Image courtesy of an anonymous Abreview.

    Western blot analysis of Human glioma cell lysate (20μg/lane) labelling KMT6/EMT2 with ab3748 at 1/1000 for 16 hours at 4ºC. A goat anti-rabbit HRP(1/10000) was used as the secondary antibody.

    Blocking was with 5% milk for 1 hour at 25ºC.

    See Abreview

  • ChIP analysis using ab3748 binding KMT6/EZH2 in rat Hippocampal primary Neurons. Cells were cross-linked for 10 minutes with 1% formaldehyde. Samples were incubated with primary antibody (1:100) for 16 hours at 4°C. Protein binding was detected using real-time PCR.

    Positive control: HoxA1, a PolyComb repressed gene in Hippocampus.

    Negative Control:b-act, an active gene, and normal Rabbit IgG.

    See Abreview

  • IP with rabbit polyclonal to EZH2 (ab3748).

    U20S cells were transfected with 4ug of Myc-EZH2. WB reprobe using 0.4ug/ml in TBS milk 2%, BSA 0.5%.

    NB. ab3748 did not detect endogenous EZH2.

  • ab3748 staining KMT6 / EZH2 in murine hippocampus tissue by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections).
    Tissue was fixed in paraformaldehyde and a heat mediated antigen retrieval step was performed using 10mM sodium citrate. Samples were then permeabilized using 0.3% Triton, blocked with 3% BSA for 30 minutes at 22°C and then incubated with ab3748 at a 1/3000 dilution for 16 hours at 4°C. The secondary used was a biotin conjugated horse polyclonal, used at a 1/2000 dilution. ABC method.

    See Abreview

  • ab3748 staining KMT6 / EZH2 in human U-2 OS cells by Immunocytochemistry/ Immunofluorescence. Cells were fixed with paraformaldehyde, permeabilized with 0.1% Triton ×100 and blocking with 1% BSA was done for 1 hour at RT. Samples were incubated with primary antibody (1/100) for 2 hours. An undiluted Alexa Fluor®594-conjugated goat polyclonal to rabbit IgG was used undiluted as secondary antibody.

    See Abreview

  • Anti-KMT6 / EZH2 antibody - ChIP Grade (ab3748) at 1 µg/ml + EZH2 - Recombinant Protein at 0.1 µg

    Secondary
    Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

    Performed under reducing conditions.

    Observed band size : 40 kDa (why is the actual band size different from the predicted?)
    ab3748 detects a band at 40 kDa which corresponds to the predicted molecular weight of the Human EZH2 Recombinant Protein.
  • ChIP analysis of Human glioma cells using ab3748 to bind KMT6 / EZH2. Chromatin was obtained by cross-linking with 1% formaldehyde for 10 minutes and incubated with primary antibody (1/250) for 16 hours at 4°C. Protein binding was detected using semi-quantitive PCR.

    See Abreview

References for Anti-KMT6 / EZH2 antibody - ChIP Grade (ab3748)

This product has been referenced in:
  • McLaughlin N  et al. In situ histone landscape of nephrogenesis. Epigenetics 9:222-35 (2014). IHC-P ; Mouse . Read more (PubMed: 24169366) »
  • Rossetto CC  et al. Regulation of viral and cellular gene expression by Kaposi's sarcoma-associated herpesvirus polyadenylated nuclear RNA. J Virol 87:5540-53 (2013). Read more (PubMed: 23468496) »

See all 22 Publications for this product

Product Wall

Application Western blot
Sample Rat Cell lysate - whole cell (Epithelial Cells)
Gel Running Conditions Reduced Denaturing (10%)
Loading amount 75 µg
Specification Epithelial Cells
Blocking step Milk as blocking agent for 12 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C
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Submitted Nov 11 2015

Application ChIP
Sample Human Cell lysate - other (Epithelial cells)
Negative control IgG
Specification Epithelial cells
Detection step Real-time PCR
Type Cross-linking (X-ChIP)
Duration of cross-linking step: 10 minute(s) and 0 second(s)
Positive control Input DNA
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Submitted Jun 22 2015

Application Flow Cytometry
Sample Human Cell (Ovarian)
Permeabilization Yes - 90% Methanol
Gating Strategy Cells were selected from FSC-A vs SSC-A, and single cells were selected from FSW-W vs FSC-H and SSC-W vs SSC-H. The gated cells were then analyzed for green fluorescence (FITC-A channel) and plotted against FSC-W.
Specification Ovarian
Preparation Cell harvesting/tissue preparation method: Fixed in 4% formaldehyde, permeabolized in 90% methanol
Sample buffer: 3% goat serum albumin in PBS
Fixation Formaldehyde
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Submitted Apr 21 2015

Abcam has not validated the combination of species/application used in this Abreview.
Application Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample Sheep Tissue sections (1-2 month old testis)
Antigen retrieval step Heat mediated - Buffer/Enzyme Used: 0.01M citric acide/sodium citrate
Permeabilization No
Specification 1-2 month old testis
Blocking step BSA as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 25°C
Fixative 4%PFA
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Submitted Jan 14 2015

Application Immunoprecipitation
Immuno-precipitation step Protein A/G
Sample Human Cell lysate - whole cell (HeLa cells)
Specification HeLa cells
Total protein in input 30 µg
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Submitted Jan 07 2015

Application Immunocytochemistry/ Immunofluorescence
Blocking step BSA as blocking agent for 45 minute(s) · Concentration: 2% · Temperature: RT°C
Sample Human Cell (HeLa cells)
Specification HeLa cells
Permeabilization Yes - 0.2% Triton-X100
Fixative Paraformaldehyde
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Submitted Nov 24 2014

Application ChIP
Detection step Real-time PCR
Sample Rat Cell lysate - nuclear (Hippocampal primary Neurons)
Specification Hippocampal primary Neurons
Negative control b-act, an active gene, and normal Rabbit IgG
Type Cross-linking (X-ChIP)
Duration of cross-linking step: 10 minute(s) and 0 second(s)
Specification of the cross-linking agent: Formaldehide 1%
Positive control HoxA1, a PolyComb repressed gene in Hippocampus
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Submitted Oct 07 2014

Application Western blot
Loading amount 15 µg
Gel Running Conditions Non-reduced Non-Denaturing (Native)
Sample Mouse Tissue lysate - whole (Whole brain tissue lysate)
Specification Whole brain tissue lysate
Blocking step BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
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Submitted Sep 11 2014

Application Western blot
Loading amount 25 µg
Gel Running Conditions Reduced Denaturing (4-20% gradient Tris-Gly gel)
Sample Mouse Cell lysate - whole cell (MEFs)
Specification MEFs
Blocking step LI-COR­ Odyssey­ Blocking Buffer as blocking agent for 45 minute(s) · Concentration: 50% · Temperature: RT°C
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Submitted Sep 01 2014

Application Western blot
Loading amount 25 µg
Gel Running Conditions Reduced Denaturing (4-20% gradient Tris-Gly gel)
Sample Human Cell lysate - whole cell (Human primary fibroblasts)
Specification Human primary fibroblasts
Blocking step LI-COR­ Odyssey­ Blocking Buffer as blocking agent for 45 minute(s) · Concentration: 50% · Temperature: RT°C
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Submitted Aug 26 2014

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"