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  1. Link

    cdk4-antibody-epr17525-ab199728.pdf

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Cell Biology Cell Cycle Kinases/Phosphatases Cdks
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Validated using a knockout cell lineRecombinantRabMAb

Recombinant Anti-Cdk4 antibody [EPR17525] (ab199728)

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  • Certificate of Compliance
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Western blot - Anti-Cdk4 antibody [EPR17525] (ab199728)
  • Immunocytochemistry/ Immunofluorescence - Anti-Cdk4 antibody [EPR17525] (ab199728)
  • Western blot - Anti-Cdk4 antibody [EPR17525] (ab199728)
  • Western blot - Anti-Cdk4 antibody [EPR17525] (ab199728)
  • Immunocytochemistry/ Immunofluorescence - Anti-Cdk4 antibody [EPR17525] (ab199728)
  • Western blot - Anti-Cdk4 antibody [EPR17525] (ab199728)
  • Western blot - Anti-Cdk4 antibody [EPR17525] (ab199728)
  • Western blot - Anti-Cdk4 antibody [EPR17525] (ab199728)
  • Western blot - Anti-Cdk4 antibody [EPR17525] (ab199728)
  • Immunoprecipitation - Anti-Cdk4 antibody [EPR17525] (ab199728)
  • Anti-Cdk4 antibody [EPR17525] (ab199728)

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
  • Suitable for: ICC/IF, IP, WB
  • Knockout validated
  • Reacts with: Mouse, Rat, Human

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Overview

  • Product name

    Anti-Cdk4 antibody [EPR17525]
    See all Cdk4 primary antibodies
  • Description

    Rabbit monoclonal [EPR17525] to Cdk4
  • Host species

    Rabbit
  • Tested applications

    Suitable for: ICC/IF, IP, 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, HeLa, C6, PC-12, C2C12, NIH/3T3 whole cell lysate; mouse brain and heart cell lysate. ICC: HeLa and NIH3T3 cells. IP: NIH/3T3 whole cell lysate.
  • 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.

Properties

  • Form

    Liquid
  • Storage instructions

    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term. Avoid freeze / thaw cycle.
  • Storage buffer

    pH: 7.2
    Preservative: 0.01% Sodium azide
    Constituents: PBS, 40% Glycerol, 0.05% BSA
  • Concentration information loading...
  • Purity

    Protein A purified
  • Clonality

    Monoclonal
  • Clone number

    EPR17525
  • Isotype

    IgG
  • Research areas

    • Cell Biology
    • Cell Cycle
    • Kinases/Phosphatases
    • Cdks
    • Epigenetics and Nuclear Signaling
    • Cell cycle
    • Kinases/Phosphatases
    • Cdks
    • Cancer
    • Cell cycle
    • Kinases/phosphatases
    • Cdks

Associated products

  • Alternative Versions

    • Alexa Fluor® 647 Anti-Cdk4 antibody [EPR17525] (ab225420)
    • Anti-Cdk4 antibody [EPR17525] - BSA and Azide free (ab236019)
  • KO cell lines

    • Human CDK4 knockout HeLa cell line (ab255378)
  • KO cell lysates

    • Human CDK4 knockout HeLa cell lysate (ab263780)
  • KO cell pellets

    • Human CDK4 knockout HeLa cell pellet (ab278889)
  • Recombinant Protein

    • Recombinant Human Cdk4 protein (ab126909)

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab199728 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
ICC/IF
1/250.
IP
1/50.
WB
1/2000. Detects a band of approximately 34 kDa (predicted molecular weight: 34 kDa).
Notes
ICC/IF
1/250.
IP
1/50.
WB
1/2000. 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.
  • Target information above from: UniProt accession P11802 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt
  • Database links

    • Entrez Gene: 1019 Human
    • Entrez Gene: 12567 Mouse
    • Entrez Gene: 94201 Rat
    • Omim: 123829 Human
    • SwissProt: P11802 Human
    • SwissProt: P30285 Mouse
    • SwissProt: P35426 Rat
    • Unigene: 95577 Human
    • Unigene: 6839 Mouse
    • Unigene: 6115 Rat
    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] (ab199728)
    Western blot - Anti-Cdk4 antibody [EPR17525] (ab199728)
    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



    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.

  • Immunocytochemistry/ Immunofluorescence - Anti-Cdk4 antibody [EPR17525] (ab199728)
    Immunocytochemistry/ Immunofluorescence - Anti-Cdk4 antibody [EPR17525] (ab199728)

    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.

  • Western blot - Anti-Cdk4 antibody [EPR17525] (ab199728)
    Western blot - Anti-Cdk4 antibody [EPR17525] (ab199728)
    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 : 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.

  • Western blot - Anti-Cdk4 antibody [EPR17525] (ab199728)
    Western blot - Anti-Cdk4 antibody [EPR17525] (ab199728)
    Anti-Cdk4 antibody [EPR17525] (ab199728) at 1/2000 dilution + NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate at 10 µg

    Secondary
    Goat Anti-Rabbit IgG H&L Peroxidase conjugated at 1/1000 dilution

    Developed using the ECL technique.

    Predicted band size: 34 kDa


    Exposure time: 30 seconds


    Blocking/Dilution buffer: 5% NFDM/TBST.

  • Immunocytochemistry/ Immunofluorescence - Anti-Cdk4 antibody [EPR17525] (ab199728)
    Immunocytochemistry/ Immunofluorescence - Anti-Cdk4 antibody [EPR17525] (ab199728)

    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.

  • Western blot - Anti-Cdk4 antibody [EPR17525] (ab199728)
    Western blot - Anti-Cdk4 antibody [EPR17525] (ab199728)
    All lanes : Anti-Cdk4 antibody [EPR17525] (ab199728) at 1/10000 dilution

    Lane 1 : Mouse brain whole cell lysate
    Lane 2 : Mouse heart whole cell lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L Peroxidase conjugated at 1/1000 dilution

    Developed using the ECL technique.

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


    Exposure time: 1 minute


    Blocking/Dilution buffer: 5% NFDM/TBST.

  • Western blot - Anti-Cdk4 antibody [EPR17525] (ab199728)
    Western blot - Anti-Cdk4 antibody [EPR17525] (ab199728)
    Anti-Cdk4 antibody [EPR17525] (ab199728) at 1/20000 dilution + C2C12 (Mouse myoblast cell line) whole cell lysate at 10 µg

    Secondary
    Goat Anti-Rabbit IgG H&L, Peroxidase conjugated at 1/1000 dilution

    Developed using the ECL technique.

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


    Exposure time: 30 seconds


    Blocking/Dilution buffer: 5% NFDM/TBST.

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

    Lane 1 : C6 (Rat glial tumor cell line)
    Lane 2 : PC-12 (Rat adrenal gland pheochromocytoma cell line)

    Lysates/proteins at 10 µg per lane.

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

    Developed using the ECL technique.

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


    Exposure time: 15 seconds


    Blocking/Dilution buffer: 5% NFDM/TBST.

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

    Lane 1 : WT HAP1 cell lysate
    Lane 2 : CDK4 knockout HAP1 cell lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) at 1/10000 dilution

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



    ab199728 was shown to specifically react with CDK4 when CDK4 knockout samples were used. Wild-type and CDK4 knockout samples were subjected to SDS-PAGE. ab199728 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) and goat anti-mouse IgG (H + L) secondary antibodies at 1/10 000 dilution for 1 h at room temperature before imaging.

  • Immunoprecipitation - Anti-Cdk4 antibody [EPR17525] (ab199728)
    Immunoprecipitation - Anti-Cdk4 antibody [EPR17525] (ab199728)

    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

  • Anti-Cdk4 antibody [EPR17525] (ab199728)
    Anti-Cdk4 antibody [EPR17525] (ab199728)

Protocols

  • Western blot protocols
  • Immunoprecipitation protocols
  • Immunocytochemistry & immunofluorescence protocols

Click here to view the general protocols

Datasheets and documents

  • Datasheet download

    Download

Certificate of Compliance

To download a Certificate of Compliance, please enter your Lot number below:

References (49)

Publishing research using ab199728? Please let us know so that we can cite the reference in this datasheet.

ab199728 has been referenced in 49 publications.

  • Buitrago-Molina LE  et al. p53-Independent Induction of p21 Fails to Control Regeneration and Hepatocarcinogenesis in a Murine Liver Injury Model. Cell Mol Gastroenterol Hepatol 11:1387-1404 (2021). PubMed: 33484913
  • Sun L & Zhang X Report of a rare case of congenital mitral valve prolapse with chronic kidney disease--reconsidered genotype-phenotypic correlations. Mol Genet Genomic Med 9:e1558 (2021). PubMed: 33225636
  • Zhang C  et al. Ethanol Extracts of Solanum lyratum Thunb Regulate Ovarian Cancer Cell Proliferation, Apoptosis, and Epithelial-to-Mesenchymal Transition (EMT) via the ROS-Mediated p53 Pathway. J Immunol Res 2021:5569354 (2021). PubMed: 33869638
  • Yang L  et al. LINC00221 silencing prevents the progression of hepatocellular carcinoma through let-7a-5p-targeted inhibition of MMP11. Cancer Cell Int 21:202 (2021). PubMed: 33836753
  • Zou N  et al. Elevated HNF1A expression promotes radiation-resistance via driving PI3K/AKT signaling pathway in esophageal squamous cell carcinoma cells. J Cancer 12:5013-5024 (2021). PubMed: 34234870
View all Publications for this product

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