Validated using a knockout cell line
Recombinant
RabMAb

Recombinant Anti-KDM4A / JHDM3A / JMJD2A antibody [EPR18466] (ab191433)

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Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [EPR18466] to KDM4A / JHDM3A / JMJD2A
  • Suitable for: WB, IHC-P, IP
  • Knockout validated
  • Reacts with: Mouse, Rat, Human

Overview

  • Product name

    Anti-KDM4A / JHDM3A / JMJD2A antibody [EPR18466]
    See all KDM4A / JHDM3A / JMJD2A primary antibodies

  • Description

    Rabbit monoclonal [EPR18466] to KDM4A / JHDM3A / JMJD2A

  • Host species

    Rabbit

  • Specificity

    IHC application is recommended for human and rat.

  • Tested applications

    Suitable for: WB, IHC-P, IPmore details

  • Species reactivity

    Reacts with: Mouse, Rat, Human

  • Immunogen

    Synthetic peptide within Human KDM4A/ JHDM3A/ JMJD2A aa 550-650. The exact sequence is proprietary.
    Database link: O75164

  • Positive control

    • WB: LNCaP, U-2 OS, HEK-293, PC-12 and NIH/3T3 whole cell lysates; human fetal kidney lysate. IHC-P: Human colon, rat colon and rat skeletal muscle tissues. IP: HeLa whole cell lysate.

  • 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

Applications

Our Abpromise guarantee covers the use of ab191433 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
WB 1/5000. Detects a band of approximately 150 kDa (predicted molecular weight: 120 kDa).
IHC-P 1/250. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
IP 1/80.

Target

  • Function

    Histone demethylase that specifically demethylates 'Lys-9' and 'Lys-36' residues of histone H3, thereby playing a central role in histone code. Does not demethylate histone H3 'Lys-4', H3 'Lys-27' nor H4 'Lys-20'. Demethylates trimethylated H3 'Lys-9' and H3 'Lys-36' residue, while it has no activity on mono- and dimethylated residues. Demethylation of Lys residue generates formaldehyde and succinate. Participates in transcriptional repression of ASCL2 and E2F-responsive promoters via the recruitment of histone deacetylases and NCOR1, respectively.
    Isoform 2: Crucial for muscle differentiation, promotes transcriptional activation of the Myog gene by directing the removal of repressive chromatin marks at its promoter. Lacks the N-terminal demethylase domain.

  • Tissue specificity

    Ubiquitous.

  • Sequence similarities

    Belongs to the JHDM3 histone demethylase family.
    Contains 1 C2HC pre-PHD-type zinc finger.
    Contains 1 JmjC domain.
    Contains 1 JmjN domain.
    Contains 2 PHD-type zinc fingers.
    Contains 2 Tudor domains.

  • Domain

    The 2 Tudor domains recognize and bind methylated histone H3 'Lys-4' residue (H3K4me). Double Tudor domain has an interdigitated structure and the unusual fold is required for its ability to bind methylated histone tails. Trimethylated H3 'Lys-4' (H3K4me3) is bound in a cage of 3 aromatic residues, 2 of which are from the Tudor domain 2, while the binding specificity is determined by side-chain interactions involving residues from the Tudor domain 1. The Tudor domains are also able to bind trimethylated histone H3 'Lys-9' (H3K9me3), di- and trimethylated H4 'Lys-20' (H4K20me2 and H4K20me3). Has high affinity for H4K20me2, blocking recruitment of proteins such as TP53BP1.

  • Post-translational
    modifications

    Ubiquitinated by RNF8 and RNF168 following DNA damage, leading to its degradation. Degradation promotes accessibility of H4K20me2 mark for DNA repair protein TP53BP1, which is then recruited.

  • Cellular localization

    Nucleus.
  • Information by UniProt

  • Database links

    see all

  • Alternative names

    • JHDM3A antibody
    • JmjC domain containing histone demethylation protein 3A antibody
    • JmjC domain-containing histone demethylation protein 3A antibody
    • JMJD2 antibody
    • JMJD2A antibody
    • jumonji C domain containing histone demethylase 3A antibody
    • Jumonji domain containing 2 antibody
    • Jumonji domain containing 2A antibody
    • Jumonji domain containing protein 2A antibody
    • Jumonji domain-containing protein 2A antibody
    • KDM4A antibody
    • KDM4A_HUMAN antibody
    • KIAA0677 antibody
    • Lysine (K) specific demethylase 4A antibody
    • Lysine-specific demethylase 4A antibody
    • TDRD14A antibody
    • Tudor domain containing 14A antibody
    see all

Images

  • Western blot - Anti-KDM4A / JHDM3A / JMJD2A antibody [EPR18466] (ab191433)
    Western blot - Anti-KDM4A / JHDM3A / JMJD2A antibody [EPR18466] (ab191433)

    Lane 1: Wild-type HAP1 cell lysate (20 µg)
    Lane 2: KDM4A / JHDM3A / JMJD2A knockout HAP1 cell lysate (20 µg)
    Lane 3: U2OS cell lysate (20 µg)
    Lane 4: PC12 cell lysate (20 µg)
    Lanes 1 - 4: Merged signal (red and green). Green - ab191433 observed at 110 kDa. Red - loading control, ab8245, observed at 37 kDa.
    ab191433 was shown to specifically react with KDM4A / JHDM3A / JMJD2A when KDM4A / JHDM3A / JMJD2A knockout samples were used. Wild-type and KDM4A / JHDM3A / JMJD2A knockout samples were subjected to SDS-PAGE. ab191433 and ab8245 (loading control to GAPDH) were diluted 1/500 and 1/10000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-KDM4A / JHDM3A / JMJD2A antibody [EPR18466] (ab191433)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-KDM4A / JHDM3A / JMJD2A antibody [EPR18466] (ab191433)

    Immunohistochemical analysis of paraffin-embedded human colon tissue labeling KDM4A / JHDM3A / JMJD2A with ab191433 at 1/250 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

    Nuclear staining in epithelial cells of human colon is observed.

    Counter stained with Hematoxylin.

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

    Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

  • Immunoprecipitation - Anti-KDM4A / JHDM3A / JMJD2A antibody [EPR18466] (ab191433)
    Immunoprecipitation - Anti-KDM4A / JHDM3A / JMJD2A antibody [EPR18466] (ab191433)

    KDM4A / JHDM3A / JMJD2A was immunoprecipitated from 1mg of HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate with ab191433 at 1/80 dilution.

    Western blot was performed from the immunoprecipitate using ab191433 at 1/1000 dilution.

    VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

    Lane 1: HeLa whole cell lysate 10µg (Input).

    Lane 2: ab191433 IP in HeLa whole cell lysate.

    Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab191433 in HeLa whole cell lysate.

    Blocking and dilution buffer and concentration: 5% NFDM/TBST.

    Exposure time: 3 seconds.

  • Western blot - Anti-KDM4A / JHDM3A / JMJD2A antibody [EPR18466] (ab191433)
    Western blot - Anti-KDM4A / JHDM3A / JMJD2A antibody [EPR18466] (ab191433)
    Lanes 1-3 : Anti-KDM4A / JHDM3A / JMJD2A antibody [EPR18466] (ab191433) at 1/5000 dilution
    Lane 4 : Anti-MTHFD2 antibody [7] (ab191443) at 1/5000 dilution

    Lane 1 : Human fetal kidney at 10 µg
    Lane 2 : HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate at 20 µg
    Lane 3 : LNCaP (Human prostate cancer cell line) whole cell lysate at 20 µg
    Lane 4 : U-2 OS (Human bone osteosarcoma epithelial cell line) whole call lysate at 20 µg

    Secondary
    Lane 1 : Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG at 1/100000 dilution
    Lanes 2-3 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution
    Lane 4 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/5000 dilution

    Developed using the ECL technique.

    Predicted band size: 120 kDa
    Observed band size: 150 kDa
    why is the actual band size different from the predicted?



    Exposure time: Lane 1: 1 minute; Lane 2: 3 minutes; Lane 3 and 4: 1 minute

    Blocking/Dilution buffer: 5% NFDM/TBST

     

     

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-KDM4A / JHDM3A / JMJD2A antibody [EPR18466] (ab191433)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-KDM4A / JHDM3A / JMJD2A antibody [EPR18466] (ab191433)

    Immunohistochemical analysis of paraffin-embedded rat colon tissue labeling KDM4A / JHDM3A / JMJD2A with ab191433 at 1/250 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

    Nuclear staining in epithelial cells of rat colon is observed.

    Counter stained with Hematoxylin.

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

    Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

  • Western blot - Anti-KDM4A / JHDM3A / JMJD2A antibody [EPR18466] (ab191433)
    Western blot - Anti-KDM4A / JHDM3A / JMJD2A antibody [EPR18466] (ab191433)
    All lanes : Anti-KDM4A / JHDM3A / JMJD2A antibody [EPR18466] (ab191433) at 1/5000 dilution

    Lane 1 : PC-12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysate
    Lane 2 : NIH/3T3 (Mouse embryonic fibroblast cell line) 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

    Developed using the ECL technique.

    Predicted band size: 120 kDa
    Observed band size: 150 kDa why is the actual band size different from the predicted?


    Exposure time: 30 seconds


    Blocking/Dilution buffer: 5% NFDM/TBST.

References

Publishing research using ab191433? Please let us know so that we can cite the reference in this datasheet.

ab191433 has not yet been referenced specifically in any publications.

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