Overview

  • Product name
    Anti-Rb antibody [EPR17512]
    See all Rb primary antibodies
  • Description
    Rabbit monoclonal [EPR17512] to Rb
  • Host species
    Rabbit
  • Tested applications
    Suitable for: Flow Cyt, WB, IP, ICC/IF, IHC-Pmore details
  • Species reactivity
    Reacts with: Mouse, Human, African green monkey
  • Immunogen

    Recombinant fragment within Mouse Rb aa 700 to the C-terminus. The exact sequence is proprietary.
    Database link: P13405

  • Positive control
    • WB: Jurkat, Hek294, K562, WEHI-3, COS-1, MCF7 and F9 whole cell lysates; Mouse brain and lung lysates; Human fetal brain lysate. IHC-P: Human lung, Human breast cancer, Mouse lung and Mouse cerebral cortex tissues. ICC/IF: MCF7 cells. IP: MCF7 whole cell lysate.
  • General notes

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

    We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.

    This product is a recombinant rabbit monoclonal antibody.

Properties

Applications

Our Abpromise guarantee covers the use of ab181616 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 Use at an assay dependent concentration.
WB 1/2000. Detects a band of approximately 105 kDa (predicted molecular weight: 105 kDa).

For Lysate preparation protocol, please refer to the protocol book in the protocol section and/or here (downloadable copy).

IP 1/80.
ICC/IF 1/500.
IHC-P 1/1000. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Target

  • Function
    Key regulator of entry into cell division that acts as a tumor suppressor. Promotes G0-G1 transition when phosphorylated by CDK3/cyclin-C. Acts as a transcription repressor of E2F1 target genes. The underphosphorylated, active form of RB1 interacts with E2F1 and represses its transcription activity, leading to cell cycle arrest. Directly involved in heterochromatin formation by maintaining overall chromatin structure and, in particular, that of constitutive heterochromatin by stabilizing histone methylation. Recruits and targets histone methyltransferases SUV39H1, KMT5B and KMT5C, leading to epigenetic transcriptional repression. Controls histone H4 'Lys-20' trimethylation. Inhibits the intrinsic kinase activity of TAF1. Mediates transcriptional repression by SMARCA4/BRG1 by recruiting a histone deacetylase (HDAC) complex to the c-FOS promoter. In resting neurons, transcription of the c-FOS promoter is inhibited by BRG1-dependent recruitment of a phospho-RB1-HDAC1 repressor complex. Upon calcium influx, RB1 is dephosphorylated by calcineurin, which leads to release of the repressor complex (By similarity). In case of viral infections, interactions with SV40 large T antigen, HPV E7 protein or adenovirus E1A protein induce the disassembly of RB1-E2F1 complex thereby disrupting RB1's activity.
  • Tissue specificity
    Expressed in the retina.
  • Involvement in disease
    Childhood cancer retinoblastoma
    Bladder cancer
    Osteogenic sarcoma
  • Sequence similarities
    Belongs to the retinoblastoma protein (RB) family.
  • Domain
    The Pocket domain binds to the threonine-phosphorylated domain C, thereby preventing interaction with heterodimeric E2F/DP transcription factor complexes.
  • Post-translational
    modifications
    Phosphorylated by CDK6 and CDK4, and subsequently by CDK2 at Ser-567 in G1, thereby releasing E2F1 which is then able to activate cell growth. Dephosphorylated at the late M phase. SV40 large T antigen, HPV E7 and adenovirus E1A bind to the underphosphorylated, active form of pRb. Phosphorylation at Thr-821 and Thr-826 promotes interaction between the C-terminal domain C and the Pocket domain, and thereby inhibits interactions with heterodimeric E2F/DP transcription factor complexes. Dephosphorylated at Ser-795 by calcineruin upon calcium stimulation. CDK3/cyclin-C-mediated phosphorylation at Ser-807 and Ser-811 is required for G0-G1 transition. Phosphorylated by CDK1 and CDK2 upon TGFB1-mediated apoptosis.
    N-terminus is methylated by METTL11A/NTM1 (By similarity). Monomethylation at Lys-810 by SMYD2 enhances phosphorylation at Ser-807 and Ser-811, and promotes cell cycle progression. Monomethylation at Lys-860 by SMYD2 promotes interaction with L3MBTL1.
    Acetylation at Lys-873 and Lys-874 regulates subcellular localization, at least during keratinocytes differentiation.
  • Cellular localization
    Nucleus.
  • Information by UniProt
  • Database links
  • Alternative names
    • Exon 17 tumor GOS561 substitution mutation causes premature stop antibody
    • GOS563 exon 17 substitution mutation causes premature stop antibody
    • OSRC antibody
    • Osteosarcoma antibody
    • p105-Rb antibody
    • P105RB antibody
    • PP105 antibody
    • pp110 antibody
    • PPP1R130 antibody
    • pRb antibody
    • Prepro retinoblastoma associated protein antibody
    • Protein phosphatase 1 regulatory subunit 130 antibody
    • Rb antibody
    • RB transcriptional corepressor 1 antibody
    • RB_HUMAN antibody
    • RB1 antibody
    • RB1 gene antibody
    • Retinoblastoma 1 antibody
    • Retinoblastoma suspectibility protein antibody
    • Retinoblastoma-associated protein antibody
    see all

Images

  • Lane 1: Wild-type HAP1 whole cell lysate (20 µg)
    Lane 2: Rb knockout HAP1 whole cell lysate (20 µg)
    Lane 3: HeLa whole cell lysate (20 µg)
    Lane 4: HEK293 whole cell lysate (20 µg)

    Lanes 1 - 4: Merged signal (red and green). Green - ab181616 observed at 120 kDa. Red - loading control, ab8245, observed at 37 kDa.

    ab181616 was shown to recognize Rb in wild-type HAP1 cells as signal was lost at the expected MW in Rb knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and RB1 knockout samples were subjected to SDS-PAGE. Ab181616 and ab8245 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/2000 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 1/10000 dilution for 1 hour at room temperature before imaging.

  • Immunohistochemical analysis of paraffin-embedded Human lung tissue labeling Rb with ab181616 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Nuclear staining on Human lung 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.

  • Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized MCF7 (Human breast adenocarcinoma cell line) cells labeling Rb with ab181616 at 1/500 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/500 dilution (green). Confocal image showing nuclear staining on MCF7 cells. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution (red).
    The negative controls are as follows:
    -ve control 1: ab181616 at 1/500 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
    -ve control 2: ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/500 dilution.

  • ab181616 staining Rb in the human cell line MCF-7(human breast carcinoma) by flow cytometry. Cells were fixed with 4% paraformaldehyde, permeabilized with 90% methanol and the sample was incubated with the primary antibody at a dilution of 1/140. A goat anti rabbit IgG (Alexa Fluor® 488) at a dilution of 1/2000 was used as the secondary antibody.

    Isoytype control: Rabbit monoclonal IgG (Black)

    Unlabelled control: Cell without incubation with primary antibody and secondary antibody (Blue)

  • All lanes : Anti-Rb antibody [EPR17512] (ab181616) at 1/1000 dilution

    Lane 1 : HepG2 (Human hepatocellular carcinoma epithelial cell) whole cell lysates prepared in RIPA lysis method with 5% NFDM/TBST
    Lane 2 : HepG2 (Human hepatocellular carcinoma epithelial cell) whole cell lysates prepared in 1%SDS Hot lysis method with 5% NFDM/TBST
    Lane 3 : K-562 (Human chronic myelogenous leukemia lymphoblast) whole cell lysates prepared in RIPA lysis method with 5% NFDM/TBST
    Lane 4 : K-562 (Human chronic myelogenous leukemia lymphoblast) whole cell lysates prepared in 1%SDS Hot lysis method with 5% NFDM/TBST

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/1000 dilution

    Predicted band size: 105 kDa



    Exposure time:

    Lane 1 and 2: 100 seconds
    Lane 3 and 4: 10 seconds


    We recommend to use 1%SDS Hot lysis method to get clear band.
    We are unsure how to define the extra bands.

  • Rb was immunoprecipitated from 1mg of MCF7 (Human breast adenocarcinoma cell line) whole cell lysate with ab181616 at 1/80 dilution. Western blot was performed from the immunoprecipitate using ab181616 at 1/1000 dilution. Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG, was used as secondary antibody at 1/1500 dilution.

    Lane 1: MCF7 whole cell lysate 10 µg (Input). Lane 2: ab181616 IP in MCF7 whole cell lysate. Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab181616 in MCF7 whole cell lysate.
    Blocking and dilution buffer and concentration: 5% NFDM/TBST.

  • All lanes : Anti-Rb antibody [EPR17512] (ab181616) at 1/20000 dilution

    Lane 1 : Jurkat (Human T cell leukemia cells from peripheral blood) whole cell lysate
    Lane 2 : K562 (Human chronic myelogenous leukemia cells from bone marrow) whole cell lysate
    Lane 3 : WEHI-3 (Mouse leukemia) whole cell lysate
    Lane 4 : COS-1 (African green monkey kidney fibroblast-like cell line) whole cell lysate
    Lane 5 : MCF7 (Human breast adenocarcinoma cell line) whole cell lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

    Predicted band size: 105 kDa
    Observed band size: 105 kDa


    Exposure time: 5 seconds


    Blocking/Dilution buffer: 5% NFDM/TBST.

    The lysates were all prepared using 1%SDS Hot lysis method.

  • All lanes : Anti-Rb antibody [EPR17512] (ab181616) at 1/10000 dilution

    Lane 1 : F9 (Mouse embyro testicular cancer cell line) whole cell lysate
    Lane 2 : Mouse brain lysate
    Lane 3 : Mouse lung lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

    Predicted band size: 105 kDa
    Observed band size: 105 kDa


    Exposure time: 1 minute


    Blocking/Dilution buffer: 5% NFDM/TBST.

    The lysates were all prepared using 1%SDS Hot lysis method.

  • Anti-Rb antibody [EPR17512] (ab181616) at 1/2000 dilution + Human fetal brain lysate at 10 µg

    Secondary
    Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

    Predicted band size: 105 kDa
    Observed band size: 105 kDa


    Exposure time: 1 minute


    Blocking/Dilution buffer: 5% NFDM/TBST.

    The lysates were all prepared using 1%SDS Hot lysis method.

  • Immunohistochemical analysis of paraffin-embedded Human breast cancer tissue labeling Rb with ab181616 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Nuclear staining on cancer cells of Human breast 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.

  • Immunohistochemical analysis of paraffin-embedded Mouse lung tissue labeling Rb with ab181616 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Nuclear staining on mouse lung 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.

  • Immunohistochemical analysis of paraffin-embedded Mouse cerebral cortex tissue labeling Rb with ab181616 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Nuclear staining on neuron of mouse cerebral cortex 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.

References

This product has been referenced in:
  • Peng Y  et al. Epigenetic knockdown of Notch1 inhibits hepatitis B virus X protein-induced hepatocarcinogenesis of L02/HBx cells. Oncol Rep 41:1151-1159 (2019). Read more (PubMed: 30431136) »
  • Huang H  et al. RIPK1 Inhibition Enhances Pirarubicin Cytotoxic Efficacy through AKT-P21-dependent Pathway in Hepatocellular Carcinoma. Int J Med Sci 15:1648-1657 (2018). Read more (PubMed: 30588188) »
See all 12 Publications for this product

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