Validated using a knockout cell line
Recombinant
RabMAb

Recombinant Anti-Cdk4 antibody [EPR2513Y] - BSA and Azide free (ab236042)

Overview

  • Product name

    Anti-Cdk4 antibody [EPR2513Y] - BSA and Azide free
    See all Cdk4 primary antibodies
  • Description

    Rabbit monoclonal [EPR2513Y] to Cdk4 - BSA and Azide free
  • Host species

    Rabbit
  • Tested applications

    Suitable for: IP, ICC/IF, Flow Cyt, WBmore details
  • Species reactivity

    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide corresponding to Human Cdk4 aa 1-100 (N terminal).

  • Positive control

    • WB: WT HAP1 whole cell lysate.
  • General notes

    Ab236042 is the carrier-free version of ab68266. 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.

    ab236042 is compatible with the Maxpar® Antibody Labeling Kit from Fluidigm.

    Maxpar® is a trademark of Fluidigm Canada Inc.

    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 ab236042 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.
Flow Cyt Use at an assay dependent concentration.

ab172730 - 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 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

  • 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

  • Lanes 1, 3 and 5: Wild-type HAP1 cell lysate (20 µg)
    Lanes 2, 4 and 6: CDK4 knockout HAP1 cell lysate (20 µg)
    Lanes 1 and 2: Green signal from target - ab68266 observed at 34 kDa 
    Lanes 3 and 4: Red signal from loading control - ab8226 observed at 42 kDa
    Lanes 5 and 6: Merged (red and green) signal 
    Unpurified ab68266 was shown to specifically react with CDK4 when CDK4 knockout samples were used. Wild-type and CDK4 knockout samples were subjected to SDS-PAGE. ab68266 and ab8226 (loading control to beta actin) were both diluted at 1/1000 and incubated overnight at 4°C. 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 1/10000 dilution for 1 h 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 (ab68266).

  • ab68266 (purified) at 1:30 dilution (2µg) immunoprecipitating Cdk4 in K562 whole cell lysate.
    Lane 1 (input): K562 (Human chronic myelogenous leukemia lymphoblast) whole cell lysate 10µg
    Lane 2 (+): ab68266 & K562 whole cell lysate
    Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab68266 in K562 whole cell lysate
    For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1:1000 dilution.
    Blocking and diluting buffer: 5% NFDM/TBST.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab68266).

  • Flow Cytometry analysis of A549 (Human lung carcinoma epithelial cell) cells labeling Cdk4 with purified ab68266 at 1:50 dilution (1 µg/ml) (red). Cells were fixed with 4% Paraformaldehyde. A Goat anti rabbit IgG (Alexa Fluor® 488) secondary antibody was used at 1:2000 dilution. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab68266).

  • Immunocytochemistry/ Immunofluorescence analysis of MCF7 (Human breast adenocarcinoma epithelial cell) cells labeling Cdk4 with Purified ab68266 at 1:100 (5.4 µg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with None. Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1:1000 (2 ?g/ml) dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab68266).

  • Unpurified ab68266 staining Cdk4 in wild-type HAP1 cells (top panel) and Cdk4 knockout HAP1 cells (bottom panel). The cells were fixed with 4% formaldehyde (10min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab68266 at 1/250 dilution and ab195889 at 1/250 dilution (shown in pseudo colour red) overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab68266).

References

ab236042 has not yet been referenced specifically in any publications.

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