Validated using a knockout cell lineRecombinantRabMAb

Recombinant Anti-Cdk4 antibody [EPR17525] - BSA and Azide free (ab236019)

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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 [EPR17525] to Cdk4 - BSA and Azide free
  • Suitable for: IP, ICC/IF, WB
  • Knockout validated
  • Reacts with: Mouse, Rat, Human

Overview

  • Product name

    Anti-Cdk4 antibody [EPR17525] - BSA and Azide free
    See all Cdk4 primary antibodies

  • Description

    Rabbit monoclonal [EPR17525] to Cdk4 - BSA and Azide free

  • Host species

    Rabbit

  • Tested applications

    Suitable for: IP, ICC/IF, WBmore details

  • Species reactivity

    Reacts with: Mouse, Rat, Human

  • Immunogen

    Recombinant fragment within Human Cdk4 aa 200 to the C-terminus. The exact sequence is proprietary.
    Database link: P30285

  • Positive control

    • WB: HAP1 and HeLa cell lysates. IP: NIH/3T3 cells. ICC/IF: NIH/3T3 and HeLa cells.

  • General notes

    Ab236019 is the carrier-free version of ab199728. 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.

    ab236019 is compatible with the Maxpar® Antibody Labeling Kit from Fluidigm.

    Maxpar® is a trademark of Fluidigm Canada Inc.

    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.

    Reproducibility is key to advancing scientific discovery and accelerating scientists’ next breakthrough.

    Abcam is leading the way with our range of recombinant antibodies, knockout-validated antibodies and knockout cell lines, all of which support improved reproducibility.

    We are also planning to innovate the way in which we present recommended applications and species on our product datasheets, so that only applications & species that have been tested in our own labs, our suppliers or by selected trusted collaborators are covered by our Abpromise™ guarantee.

    In preparation for this, we have started to update the applications & species that this product is Abpromise guaranteed for.

    We are also updating the applications & species that this product has been “predicted to work with,” however this information is not covered by our Abpromise guarantee.

    Applications & species from publications and Abreviews that have not been tested in our own labs or in those of our suppliers are not covered by the Abpromise guarantee.

    Please check that this product meets your needs before purchasing. If you have any questions, special requirements or concerns, please send us an inquiry and/or contact our Support team ahead of purchase. Recommended alternatives for this product can be found below, as well as customer reviews and Q&As.

Properties

Applications

Our Abpromise guarantee covers the use of ab236019 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
IP Use at an assay dependent concentration.
ICC/IF Use at an assay dependent concentration.
WB Use at an assay dependent concentration. Detects a band of approximately 34 kDa (predicted molecular weight: 34 kDa).

Target

  • Function

    Ser/Thr-kinase component of cyclin D-CDK4 (DC) complexes that phosphorylate and inhibit members of the retinoblastoma (RB) protein family including RB1 and regulate the cell-cycle during G(1)/S transition. Phosphorylation of RB1 allows dissociation of the transcription factor E2F from the RB/E2F complexes 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 phosphorylates SMAD3 in a cell-cycle-dependent manner and represses its transcriptional activity. Component of the ternary complex, cyclin D/CDK4/CDKN1B, required for nuclear translocation and activity of the cyclin D-CDK4 complex.

  • Involvement in disease

    Defects in CDK4 are a cause of susceptibility to cutaneous malignant melanoma type 3 (CMM3) [MIM:609048]. Malignant melanoma is a malignant neoplasm of melanocytes, arising de novo or from a pre-existing benign nevus, which occurs most often in the skin but also may involve other sites.

  • Sequence similarities

    Belongs to the protein kinase superfamily. CMGC Ser/Thr protein kinase family. CDC2/CDKX subfamily.
    Contains 1 protein kinase domain.

  • Post-translational
    modifications

    Phosphorylation at Thr-172 is required for enzymatic activity. Phosphorylated, in vitro, at this site by CCNH-CDK7, but, in vivo, appears to be phosphorylated by a proline-directed kinase. In the cyclin D-CDK4-CDKN1B complex, this phosphorylation and consequent CDK4 enzyme activity, is dependent on the tyrosine phosphorylation state of CDKN1B. Thus, in proliferating cells, CDK4 within the complex is phosphorylated on Thr-172 in the T-loop. In resting cells, phosphorylation on Thr-172 is prevented by the non-tyrosine-phosphorylated form of CDKN1B.

  • Cellular localization

    Cytoplasm. Nucleus. Membrane. Cytoplasmic when non-complexed. Forms a cyclin D-CDK4 complex in the cytoplasm as cells progress through G(1) phase. The complex accumulates on the nuclear membrane and enters the nucleus on transition from G(1) to S phase. Also present in nucleoli and heterochromatin lumps. Colocalizes with RB1 after release into the nucleus.
  • Information by UniProt

  • Database links

    see all

  • Alternative names

    • Cdk 4 antibody
    • cdk4 antibody
    • CDK4 protein antibody
    • CDK4_HUMAN antibody
    • Cell division kinase 4 antibody
    • Cell division protein kinase 4 antibody
    • CMM 3 antibody
    • CMM3 antibody
    • Crk3 antibody
    • Cyclin dependent kinase 4 antibody
    • Cyclin-dependent kinase 4 antibody
    • Melanoma cutaneous malignant 3 antibody
    • MGC14458 antibody
    • p34 cdk4 antibody
    • PSK J3 antibody
    • PSK-J3 antibody
    see all

Images

  • Western blot - Anti-Cdk4 antibody [EPR17525] - BSA and Azide free (ab236019)
    Western blot - Anti-Cdk4 antibody [EPR17525] - BSA and Azide free (ab236019)
    All lanes : Anti-Cdk4 antibody [EPR17525] (ab199728) at 1/2000 dilution

    Lane 1 : Wild-type HAP1 cell lysate
    Lane 2 : CDK4 knockout HAP1 cell lysate
    Lane 3 : Wild-type HeLa cell lysate
    Lane 4 : CDK4 knockout HeLa cell lysate

    Lysates/proteins at 20 µg per lane.

    Performed under reducing conditions.

    Predicted band size: 34 kDa
    Observed band size: 34 kDa



    This data was developed using the same antibody clone in a different buffer formulation (ab199728).

    Lanes 1 - 4: Merged signal (red and green). Green - ab199728 observed at 34 kDa. Red - loading control, ab130007 observed at 125 kDa.  

     ab199728 was shown to react with Cdk4 in wild-type HeLa. Loss of signal was observed when knockout cell line ab255378 (knockout cell lysate ab263780) was used. Wild-type and Cdk4 knockout samples were subjected to SDS-PAGE. ab199728 and Anti-Vinculin antibody [VIN-54] (ab130007) were incubated overnight at 4°C at 1 in 2000 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.

     

     

  • Western blot - Anti-Cdk4 antibody [EPR17525] - BSA and Azide free (ab236019)
    Western blot - Anti-Cdk4 antibody [EPR17525] - BSA and Azide free (ab236019)
    All lanes : Anti-Cdk4 antibody [EPR17525] (ab199728) at 1/1000 dilution

    Lane 1 : Wild-type HAP1 whole cell lysate
    Lane 2 : Cdk4 knockout HAP1 whole cell lysate
    Lane 3 : Wild-type HeLa whole cell lysate

    Lysates/proteins at 20 µg per lane.

    Predicted band size: 34 kDa



    Lanes 1 - 3: Merged signal (red and green). Green - ab199728 observed at 34 kDa. Red - loading control, ab18058, observed at 130 kDa.

    ab199728 was shown to specifically recognize Cdk4 in wild-type HAP1 cells along with additional cross-reactive bands. No band was observed when Cdk4 knockout samples were examined. Wild-type and Cdk4 knockout samples were subjected to SDS-PAGE. Ab199728 and ab18058 (Mouse anti Vinculin loading control) were incubated overnight at 4°C at 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.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab199728).

  • Immunoprecipitation - Anti-Cdk4 antibody [EPR17525] - BSA and Azide free (ab236019)
    Immunoprecipitation - Anti-Cdk4 antibody [EPR17525] - BSA and Azide free (ab236019)

    Cdk4 was immunoprecipitated from 1mg NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate with ab199728 at 1/50 dilution. Western blot was performed from the immunoprecipitate using ab199728 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: NIH/3T3 whole cell lysate, 10 (Input).

    Lane 2: ab199728 IP in NIH/3T3 whole cell lysate.

    Lane 3: Rabbit IgG,monoclonal [EPR25A]- Isotype Control (ab172730) instead of ab199728 in NIH/3T3 whole cell lysate.

    Blocking and dilution buffer and concentration: 5% NFDM/TBST.
    Exposure time: 10 seconds

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab199728).

  • Immunocytochemistry/ Immunofluorescence - Anti-Cdk4 antibody [EPR17525] - BSA and Azide free (ab236019)
    Immunocytochemistry/ Immunofluorescence - Anti-Cdk4 antibody [EPR17525] - BSA and Azide free (ab236019)

    Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NIH/3T3 (Mouse embryonic fibroblast cell line) cells labeling Cdk4 with ab199728 at 1/250 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/500 dilution (green). Confocal image showing cytoplasmic and nuclear staining on NIH/3T3 cell line. The nuclear counter stain is DAPI (blue).

    Tubulin is detected with Anti-alpha Tubulin antibody- Loading Control (ab7291) at 1/1000 dilution and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed (ab150120) at 1/500 dilution (red).
    The negative controls are as follows:
    -ve control 1: ab199728 at 1/250 dilution followed by ab150120 at 1/500 dilution.
    -ve control 2: ab7291 at 1/1000 dilution followed by ab150077 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 (ab199728).

  • Immunocytochemistry/ Immunofluorescence - Anti-Cdk4 antibody [EPR17525] - BSA and Azide free (ab236019)
    Immunocytochemistry/ Immunofluorescence - Anti-Cdk4 antibody [EPR17525] - BSA and Azide free (ab236019)

    Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling Cdk4 with ab199728 at 1/250 dilution, followed by Goat Anti-Rabbit IgG H & L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/500 dilution (green). Confocal image showing cytoplasmic, nuclear and membrane staining on HeLa cell line. The nuclear counter stain is DAPI (blue).

    Tubulin is detected with Anti-alpha Tubulin antibody - Loading Control (ab7291) at 1/1000 dilution and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed (ab150120) at 1/500 dilution (red).
    The negative controls are as follows:
    -ve control 1: ab199728 at 1/250 dilution followed by ab150120 at 1/500 dilution.
    -ve control 2: ab7291 at 1/1000 dilution followed by ab150077 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 (ab199728).

  • Anti-Cdk4 antibody [EPR17525] - BSA and Azide free (ab236019)
    Anti-Cdk4 antibody [EPR17525] - BSA and Azide free (ab236019)

Protocols

To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.

Click here to view the general protocols

References (0)

Publishing research using ab236019? Please let us know so that we can cite the reference in this datasheet.

ab236019 has not yet been referenced specifically in any publications.

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