Validated using a knockout cell line
Recombinant
RabMAb

Recombinant Anti-HP1 gamma/CBX3 antibody [EPR19802] - BSA and Azide free (ab223535)

Overview

  • Product name

    Anti-HP1 gamma/CBX3 antibody [EPR19802] - BSA and Azide free
    See all HP1 gamma/CBX3 primary antibodies
  • Description

    Rabbit monoclonal [EPR19802] to HP1 gamma/CBX3 - BSA and Azide free
  • Host species

    Rabbit
  • Tested applications

    Suitable for: ICC/IF, IP, Flow Cyt, WB, IHC-Pmore details
  • Species reactivity

    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide aa 100 to the C-terminus. The exact sequence is proprietary.
    Database link: P23198

  • Positive control

    • WB: Human brain lysate; HeLa, HepG2, PC-3, C2C12, 4T1, A549, LNCaP, C6 and NIH/3T3 whole cell lysates; Mouse and rat brain lysates. IHC-P: Human testis and bladder cancer tissues; Mouse liver tissue; Rat kidney tissue. ICC/IF: HeLa and NIH/3T3 cells. Flow Cyt: HeLa cells. IP: HeLa whole cell lysate.
  • General notes

    ab223535 is the carrier-free version of ab217999 This format is designed for use in antibody labeling, including fluorochromes, metal isotopes, oligonucleotides, enzymes.

     

    Our carrier-free formats are supplied in a buffer free of BSA, sodium azide and glycerol for higher conjugation efficiency.

    Use our conjugation kits  for antibody conjugates that are ready-to-use in as little as 20 minutes with <1 minute hands-on-time and 100% antibody recovery: available for fluorescent dyes, HRP, biotin and gold.

    Ab223535 is compatible with the Maxpar® Antibody Labeling Kit from Fluidigm.

    Maxpar® is a trademark of Fluidigm Canada Inc.

    This product was previously labelled as HP1 gamma

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

    This product is a recombinant rabbit monoclonal antibody.

Properties

Applications

Our Abpromise guarantee covers the use of ab223535 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
ICC/IF Use at an assay dependent concentration.
IP Use at an assay dependent concentration.
Flow Cyt Use at an assay dependent concentration.

ab199376 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

WB Use at an assay dependent concentration. Detects a band of approximately 21 kDa (predicted molecular weight: 21 kDa).
IHC-P Use at an assay dependent concentration. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Target

  • Function

    Seems to be involved in transcriptional silencing in heterochromatin-like complexes. Recognizes and binds histone H3 tails methylated at 'Lys-9', leading to epigenetic repression. May contribute to the association of the heterochromatin with the inner nuclear membrane through its interaction with lamin B receptor (LBR). Involved in the formation of functional kinetochore through interaction with MIS12 complex proteins.
  • Sequence similarities

    Contains 2 chromo domains.
  • Post-translational
    modifications

    Phosphorylated by PIM1. Phosphorylated during interphase and possibly hyper-phosphorylated during mitosis.
  • Cellular localization

    Nucleus. Associates with euchromatin and is largely excluded from constitutive heterochromatin. May be associated with microtubules and mitotic poles during mitosis.
  • Information by UniProt
  • Database links

  • Alternative names

    • CBX 3 antibody
    • CBX3 antibody
    • CBX3_HUMAN antibody
    • Chromobox homolog 3 (HP1 gamma homolog, Drosophila) antibody
    • Chromobox homolog 3 antibody
    • Chromobox protein homolog 3 antibody
    • GAMMA antibody
    • HECH antibody
    • Heterochromatin like protein 1 antibody
    • Heterochromatin protein 1 homolog gamma antibody
    • Heterochromatin protein HP1 gamma antibody
    • HP1 gamma antibody
    • HP1 gamma homolog antibody
    • HP1Hs gamma antibody
    • Modifier 2 protein antibody
    see all

Images

  • This WB data was generated using the same anti-HP1 gamma/CBX3 antibody clone [EPR19802] in a different buffer format (cat# ab217999).

    Lane 1: Wild type HAP1 whole cell lysate (20 µg)
    Lane 2: CBX3 knockout HAP1 whole cell lysate (20 µg)
    Lane 3: HepG2 whole cell lysate (20 µg)

    Lanes 1 - 3: Merged signal (red and green). Green - ab217999 observed at 25 kDa. Red - loading control, ab9484, observed at 37 kDa.

    ab217999 was shown to recognize CBX3 when CBX3 knockout samples were used, along with additional cross-reactive bands. Wild-type and CBX3 knockout samples were subjected to SDS-PAGE. Ab217999 and ab9484 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 2000 dilution and 1/10000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

  • Immunohistochemical analysis of paraffin-embedded human testis tissue labeling HP1 gamma/CBX3 with ab217999 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear staining on human testis is observed [PMID: 19786570]. 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.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab217999).

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

  • Immunohistochemical analysis of paraffin-embedded mouse liver tissue labeling HP1 gamma/CBX3 with ab217999 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear staining on hepatocytes of mouse liver 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.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab217999).

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

  • Immunohistochemical analysis of paraffin-embedded human bladder cancer tissue labeling HP1 gamma/CBX3 with ab217999 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear staining on tumor cells of human bladder cancer 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.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab217999).

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

  • Immunohistochemical analysis of paraffin-embedded rat kidney tissue labeling HP1 gamma/CBX3 with ab217999 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear staining on rat kidney 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.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab217999).

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

  • Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling HP1 gamma/CBX3 with ab217999 at 1/1000 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear staining on HeLa cell line.

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

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

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab217999).

  • Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NIH/3T3 (Mouse embryonic fibroblast cell line) cells labeling HP1 gamma/CBX3 with ab217999 at 1/1000 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear staining on NIH/3T3 cell line.

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

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

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab217999).

  • Flow cytometric analysis of 4% paraformaldehyde-fixed HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling HP1 gamma/CBX3 with ab217999 at 1/400 dilution (red) compared with a rabbit monoclonal IgG isotype control (ab172730; black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody; blue). Goat anti rabbit IgG (Alexa Fluor® 488) at 1/2000 dilution was used as the secondary antibody.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab217999).

  • HP1 gamma/CBX3 was immunoprecipitated from 0.35 mg of HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate with ab217999 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab217999 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, 10 µg (Input).

    Lane 2: ab217999 IP in HeLa whole cell lysate.

    Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab217999 in HeLa whole cell lysate.

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

    Exposure time: 1 second.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab217999).

References

ab223535 has not yet been referenced specifically in any publications.

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