Validated using a knockout cell lineRecombinantRabMAb

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

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

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

Conjugates logo Related conjugates and formulations

Alexa Fluor® 647 Unconjugated

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. This information is proprietary to Abcam and/or its suppliers.

  • 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.

    Our carrier-free antibodies are typically supplied in a PBS-only formulation, purified and free of BSA, sodium azide and glycerol. The carrier-free buffer and high concentration allow for increased conjugation efficiency.

    This conjugation-ready format is designed for use with fluorochromes, metal isotopes, oligonucleotides, and enzymes, which makes them ideal for antibody labelling, functional and cell-based assays, flow-based assays (e.g. mass cytometry) and Multiplex Imaging applications.

    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.

    This product is compatible with the Maxpar® Antibody Labeling Kit from Fluidigm, without the need for antibody preparation. 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.

Properties

Applications

The Abpromise guarantee

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).
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 HeLa cell lysate
    Lane 2 : 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- 2: Merged signal (red and green). Green - ab199728 observed at 34 kDa. Red - Anti-Vinculin antibody [VIN-54] observed at 124 kDa.

     ab199728 was shown to react with Cdk4 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab255378 (knockout cell lysate ab263780) was used. Wild-type HeLa and CDK4 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab199728 and Anti-Vinculin antibody [VIN-54] overnight at 4°C at a 1 in 2000 dilution and a 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.

  • 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).

  • 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).

  • 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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