Validated using a knockout cell lineRecombinantRabMAb

Recombinant Anti-Cyclin D2 antibody [EPR19659] (ab207604)

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Western blot - Anti-Cyclin D2 antibody [EPR19659] (ab207604)
  • Immunocytochemistry/ Immunofluorescence - Anti-Cyclin D2 antibody [EPR19659] (ab207604)
  • Flow Cytometry - Anti-Cyclin D2 antibody [EPR19659] (ab207604)
  • Western blot - Anti-Cyclin D2 antibody [EPR19659] (ab207604)
  • Western blot - Anti-Cyclin D2 antibody [EPR19659] (ab207604)
  • Western blot - Anti-Cyclin D2 antibody [EPR19659] (ab207604)
  • Western blot - Anti-Cyclin D2 antibody [EPR19659] (ab207604)
  • Immunocytochemistry/ Immunofluorescence - Anti-Cyclin D2 antibody [EPR19659] (ab207604)
  • Immunoprecipitation - Anti-Cyclin D2 antibody [EPR19659] (ab207604)
  • Anti-Cyclin D2 antibody [EPR19659] (ab207604)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [EPR19659] to Cyclin D2
  • Suitable for: WB, ICC/IF, IP, Flow Cyt
  • Knockout validated
  • Reacts with: Human, Recombinant fragment

Overview

  • Product name

    Anti-Cyclin D2 antibody [EPR19659]
    See all Cyclin D2 primary antibodies

  • Description

    Rabbit monoclonal [EPR19659] to Cyclin D2

  • Host species

    Rabbit

  • Tested Applications & Species

    Application Species
    Flow Cyt
    Human
    ICC/IF
    Human
    IP
    Human
    WB
    Human
    Recombinant fragment
    See all applications and species data

  • Immunogen

    Recombinant full length protein. This information is proprietary to Abcam and/or its suppliers.

  • Positive control

    • WB: Full length human Cyclin D2 recombinant protein; HAP1, HEK-293, Caco-2 and U-2 OS whole cell lysates; human fetal brain, fetal heart and fetal kidney lysates. ICC/IF: Caco-2 and U-2 OS cells. IP: U-2 OS whole cell lysate. Flow Cyt: U-2 OS 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.

    The Life Science industry has been in the grips of a reproducibility crisis for a number of years. Abcam is leading the way in addressing the problem with our range of recombinant monoclonal antibodies and knockout edited cell lines for gold-standard validation.

    One factor contributing to the crisis is the use of antibodies that are not suitable. This can lead to misleading results and the use of incorrect data informing project assumptions and direction. To help address this challenge, we have introduced an application and species grid on our primary antibody datasheets to make it easy to simplify identification of the right antibody for your needs.

    Learn more here.

Properties

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab207604 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Guaranteed

Tested applications are guaranteed to work and covered by our Abpromise guarantee.

Predicted

Predicted to work for this combination of applications and species but not guaranteed.

Incompatible

Does not work for this combination of applications and species.

Application Species
Flow Cyt
Human
ICC/IF
Human
IP
Human
WB
Human
Recombinant fragment
Application Abreviews Notes
WB
1/1000. Detects a band of approximately 33 kDa (predicted molecular weight: 33 kDa).
ICC/IF (1)
1/100.
IP
1/30.
Flow Cyt
1/450.
Notes
WB
1/1000. Detects a band of approximately 33 kDa (predicted molecular weight: 33 kDa).
ICC/IF
1/100.
IP
1/30.
Flow Cyt
1/450.

Target

  • Function

    Regulatory component of the cyclin D2-CDK4 (DC) complex that phosphorylates and inhibits members of the retinoblastoma (RB) protein family including RB1 and regulates the cell-cycle during G(1)/S transition. Phosphorylation of RB1 allows dissociation of the transcription factor E2F from the RB/E2F complex and the subsequent transcription of E2F target genes which are responsible for the progression through the G(1) phase. Hypophosphorylates RB1 in early G(1) phase. Cyclin D-CDK4 complexes are major integrators of various mitogenenic and antimitogenic signals. Also substrate for SMAD3, phosphorylating SMAD3 in a cell-cycle-dependent manner and repressing its transcriptional activity. Component of the ternary complex, cyclin D2/CDK4/CDKN1B, required for nuclear translocation and activity of the cyclin D-CDK4 complex.

  • Sequence similarities

    Belongs to the cyclin family. Cyclin D subfamily.
    Contains 1 cyclin N-terminal domain.

  • Cellular localization

    Nucleus. Cytoplasm. Membrane. Cyclin D-CDK4 complexes accumulate at the nuclear membrane and are then translocated into the nucleus through interaction with KIP/CIP family members.
  • Information by UniProt

  • Database links

    • Alternative names

      • CCND 2 antibody
      • ccnd2 antibody
      • CCND2_HUMAN antibody
      • CyclinD2 antibody
      • G1/S specific cyclin D2 antibody
      • G1/S-specific cyclin-D2 antibody
      • KIAK0002 antibody
      • MGC102758 antibody
      • MPPH3 antibody
      see all

    Images

    • Western blot - Anti-Cyclin D2 antibody [EPR19659] (ab207604)
      Western blot - Anti-Cyclin D2 antibody [EPR19659] (ab207604)
      All lanes : Anti-Cyclin D2 antibody [EPR19659] (ab207604) at 1/1000 dilution

      Lane 1 : Wild-type HEK293T cell lysate
      Lane 2 : CCND2 knockout HEK293T cell lysate

      Lysates/proteins at 20 µg per lane.

      Predicted band size: 33 kDa
      Observed band size: 36 kDa why is the actual band size different from the predicted?



      Lanes 1-2: Merged signal (red and green). Green - ab207604 observed at 36 kDa. Red - loading control ab8245 observed at 36 kDa. 

       ab207604 Anti-Cyclin D2 antibody [EPR19659] was shown to specifically react with Cyclin D2 in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab267318 (knockout cell lysate ab257875) was used.  Wild-type and Cyclin D2 knockout samples were subjected to SDS-PAGE.  ab207604 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated at room temperature for 2. 5 hours at 1 in 1000 dilution and 1 in 20000 dilution respectively.   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 in 20000 dilution for 1 hour at room temperature before imaging.

    • Immunocytochemistry/ Immunofluorescence - Anti-Cyclin D2 antibody [EPR19659] (ab207604)
      Immunocytochemistry/ Immunofluorescence - Anti-Cyclin D2 antibody [EPR19659] (ab207604)

      Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized Caco-2 (Human colorectal adenocarcinoma cell line) cells labeling Cyclin D2 with ab207604 at 1/100 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear and weak cytoplasmic staining on Caco-2 cells. The nuclear counter stain is DAPI (blue).

      Tubulin is detected with Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) (ab150120) secondary antibody at 1/1000 dilution (red).

      The negative controls are as follows:
      -ve control 1: ab207604 at 1/100 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) (ab150120) secondary antibody at 1/1000 dilution.
      -ve control 2: Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution.

    • Flow Cytometry - Anti-Cyclin D2 antibody [EPR19659] (ab207604)
      Flow Cytometry - Anti-Cyclin D2 antibody [EPR19659] (ab207604)

      Flow Cytometry analysis of U-2 OS (Human bone osteosarcoma epithelial cell) cells labeling Cyclin D2 with purified ab207604 at 1/450 dilution (1.00 µg/mL) (Red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) secondary antibody was used at 1/2000 dilution. Isotype control - Rabbit monoclonal IgG (ab172730) (black). Unlabeled control - Unlabelled cells (blue).

    • Western blot - Anti-Cyclin D2 antibody [EPR19659] (ab207604)
      Western blot - Anti-Cyclin D2 antibody [EPR19659] (ab207604)

      Lane 1: Wild type HAP1 whole cell lysate (20 µg)
      Lane 2: CCND2 (Cyclin D2) knockout HAP1 whole cell lysate (20 µg)
      Lane 3: Hek293 whole cell lysate (20 µg)
      Lane 4: HeLa whole cell lysate (20 µg)

      Lanes 1 - 4: Merged signal (red and green). Green - ab207604 observed at 34 kDa. Red - loading control, ab18058, observed at 130 kDa.

      ab207604 was shown to recognize CCND2 (Cyclin D2) in wild type cells as signal was lost at the expected MW in CCND2 (Cyclin D2) knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and CCND2 (Cyclin D2) knockout samples were subjected to SDS-PAGE. Ab207604 and ab18058 (Mouse anti Vinculin loading control) were incubated overnight at 4°C at a 1/1000 dilution and 1/20000 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.

    • Western blot - Anti-Cyclin D2 antibody [EPR19659] (ab207604)
      Western blot - Anti-Cyclin D2 antibody [EPR19659] (ab207604)
      All lanes : Anti-Cyclin D2 antibody [EPR19659] (ab207604) at 1/1000 dilution

      Lane 1 : Full length human Cyclin D2 recombinant protein
      Lane 2 : Full length human Cyclin D1 recombinant protein

      Lysates/proteins at 0.01 µg per lane.

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

      Predicted band size: 33 kDa
      Observed band size: 35 kDa why is the actual band size different from the predicted?


      Exposure time: 3 seconds


      Blocking/Dilution buffer: 5% NFDM/TBST.

      Full length human Cyclin D2 recombinant protein contains aa1-289 with a His-Tag. Full length human Cyclin D1 recombinant protein contains aa1-295 with a His-Tag. These two recombinant proteins were made in-house.

    • Western blot - Anti-Cyclin D2 antibody [EPR19659] (ab207604)
      Western blot - Anti-Cyclin D2 antibody [EPR19659] (ab207604)
      All lanes : Anti-Cyclin D2 antibody [EPR19659] (ab207604) at 1/1000 dilution

      Lane 1 : HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate
      Lane 2 : Caco-2 (Human colorectal adenocarcinoma cell line) whole cell lysate
      Lane 3 : U-2 OS (Human bone osteosarcoma epithelial 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

      Predicted band size: 33 kDa
      Observed band size: 33 kDa


      Exposure time: 3 minutes


      Blocking/Dilution buffer: 5% NFDM/TBST.

       

    • Western blot - Anti-Cyclin D2 antibody [EPR19659] (ab207604)
      Western blot - Anti-Cyclin D2 antibody [EPR19659] (ab207604)
      All lanes : Anti-Cyclin D2 antibody [EPR19659] (ab207604) at 1/1000 dilution

      Lane 1 : Human fetal brain lysate
      Lane 2 : Human fetal heart lysate
      Lane 3 : Human fetal kidney lysate

      Lysates/proteins at 10 µg per lane.

      Secondary
      All lanes : Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG at 1/10000 dilution

      Predicted band size: 33 kDa
      Observed band size: 33 kDa


      Exposure time: 3 minutes


      Blocking/Dilution buffer: 5% NFDM/TBST.

    • Immunocytochemistry/ Immunofluorescence - Anti-Cyclin D2 antibody [EPR19659] (ab207604)
      Immunocytochemistry/ Immunofluorescence - Anti-Cyclin D2 antibody [EPR19659] (ab207604)

      Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized U-2 OS (Human bone osteosarcoma epithelial cell line) cells labeling Cyclin D2 with ab207604 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear and weak cytoplasmic staining on U-2 OS cells. The nuclear counter stain is DAPI (blue).

      Tubulin is detected with Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) (ab150120) secondary antibody at 1/1000 dilution (red).

      The negative controls are as follows:
      -ve control 1: ab207604 at 1/100 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) (ab150120) secondary antibody at 1/1000 dilution.
      -ve control 2: Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary at 1/1000 dilution.

    • Immunoprecipitation - Anti-Cyclin D2 antibody [EPR19659] (ab207604)
      Immunoprecipitation - Anti-Cyclin D2 antibody [EPR19659] (ab207604)

      Cyclin D2 was immunoprecipitated from 0.35 mg of U-2 OS (Human bone osteosarcoma epithelial cell line) whole cell lysate with ab207604 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab207604 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

      Lane 1: U-2 OS whole cell lysate, 10µg (Input).

      Lane 2: ab207604 IP in U-2 OS whole cell lysate.

      Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab207604 in U-2 OS whole cell lysate.

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

      Exposure time: 3 minutes.

    • Anti-Cyclin D2 antibody [EPR19659] (ab207604)
      Anti-Cyclin D2 antibody [EPR19659] (ab207604)

    References (11)

    Publishing research using ab207604? Please let us know so that we can cite the reference in this datasheet.

    ab207604 has been referenced in 11 publications.

    • Jiang Z  et al. Long non-coding RNA LINC00673 silencing inhibits proliferation and drug resistance of prostate cancer cells via decreasing KLF4 promoter methylation. J Cell Mol Med 24:1878-1892 (2020). PubMed: 31881124
    • Yao Y  et al. miR-671-3p is downregulated in non-small cell lung cancer and inhibits cancer progression by directly targeting CCND2. Mol Med Rep 19:2407-2412 (2019). PubMed: 30664171
    • Zhou X  et al. UCA1 promotes cell viability, proliferation and migration potential through UCA1/miR-204/CCND2 pathway in primary cystitis glandularis cells. Biomed Pharmacother 114:108872 (2019). PubMed: 30999112
    • Caller LG  et al. Temporal Proteomic Analysis of BK Polyomavirus Infection Reveals Virus-Induced G2 Arrest and Highly Effective Evasion of Innate Immune Sensing. J Virol 93:N/A (2019). PubMed: 31142673
    • Jiang D  et al. Long noncoding RNA HCG22 suppresses proliferation and metastasis of bladder cancer cells by regulation of PTBP1. J Cell Physiol N/A:N/A (2019). PubMed: 31304601
    View all Publications for this product

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    Immunocytochemistry/ Immunofluorescence abreview for Anti-Cyclin D2 antibody [EPR19659]

     Poor
    Abreviews
    abreview image
    Application
    Immunocytochemistry/ Immunofluorescence
    Sample
    Human Cell (HeLa)
    Permeabilization
    Yes - 0.5% Triton X-100
    Specification
    HeLa
    Fixative
    Paraformaldehyde
    Read More

    Dr. Kirk Mcmanus

    Verified customer

    Submitted Jan 15 2019

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