Validated using a knockout cell line
Recombinant
RabMAb

Recombinant Anti-Cdk4 antibody [EPR17525] (ab199728)

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

  • 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

Applications

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).

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

    Lane 1 : Wild-type Hap1 wildtype 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.

    Predicted band size: 34 kDa



    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 cells. Loss of signal was observed when knockout sample 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] (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.

  • 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)
    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

References

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

ab199728 has been referenced in 21 publications.

  • Wang K  et al. IL1RN mediates the suppressive effect of methionine deprivation on glioma proliferation. Cancer Lett 454:146-157 (2019). PubMed: 30978442
  • Zheng W  et al. The Role of miRNAs in Zearalenone-Promotion of TM3 Cell Proliferation. Int J Environ Res Public Health 16:N/A (2019). PubMed: 31035709
  • Sun L  et al. Swainsonine represses glioma cell proliferation, migration and invasion by reduction of miR-92a expression. BMC Cancer 19:247 (2019). PubMed: 30890138
  • Du J  et al. Astragaloside IV protects cardiomyocytes from hypoxia-induced injury by down-regulation of lncRNA GAS5. Biomed Pharmacother 116:109028 (2019). PubMed: 31136949
  • Cao A & Li X Bilobalide protects H9c2 cell from oxygen-glucose-deprivation-caused damage through upregulation of miR-27a. Artif Cells Nanomed Biotechnol 47:2980-2988 (2019). PubMed: 31322008
View all Publications for this product

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