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  1. Link

    products/primary-antibodies/cdk4-antibody-epr4513-32-7-ab108357.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 [EPR4513-32-7] (ab108357)

  • Datasheet
  • SDS
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Western blot - Anti-Cdk4 antibody [EPR4513-32-7] (ab108357)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cdk4 antibody [EPR4513-32-7] (ab108357)
  • Western blot - Anti-Cdk4 antibody [EPR4513-32-7] (ab108357)
  • Immunocytochemistry/ Immunofluorescence - Anti-Cdk4 antibody [EPR4513-32-7] (ab108357)
  • Flow Cytometry (Intracellular) - Anti-Cdk4 antibody [EPR4513-32-7] (ab108357)
  • Western blot - Anti-Cdk4 antibody [EPR4513-32-7] (ab108357)
  • Immunocytochemistry/ Immunofluorescence - Anti-Cdk4 antibody [EPR4513-32-7] (ab108357)
  • Flow Cytometry (Intracellular) - Anti-Cdk4 antibody [EPR4513-32-7] (ab108357)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cdk4 antibody [EPR4513-32-7] (ab108357)
  • Western blot - Anti-Cdk4 antibody [EPR4513-32-7] (ab108357)
  • OI-RD Scanning - Anti-Cdk4 antibody [EPR4513-32-7] (ab108357)
  • Anti-Cdk4 antibody [EPR4513-32-7] (ab108357)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [EPR4513-32-7] to Cdk4
  • Suitable for: WB, IHC-P, Flow Cyt (Intra), ICC/IF
  • Knockout validated
  • Reacts with: Human

Conjugates logo Related conjugates and formulations

Alexa Fluor® 488 Alexa Fluor® 555 Alexa Fluor® 647 Carrier Free HRP

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Knockout
Product image
Human CDK4 knockout HeLa cell line (ab255378)

View more associated products

Overview

  • Product name

    Anti-Cdk4 antibody [EPR4513-32-7]
    See all Cdk4 primary antibodies
  • Description

    Rabbit monoclonal [EPR4513-32-7] to Cdk4
  • Host species

    Rabbit
  • Tested applications

    Suitable for: WB, IHC-P, Flow Cyt (Intra), ICC/IFmore details
  • Species reactivity

    Reacts with: Human
  • Immunogen

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

  • Positive control

    • WB: HAP1, HeLa, MCF7, K562, and Ramos cell lysates. IHC-P: Human urothelial carcinoma and cervix carcinoma tissues. ICC/IF: Wild type HAP1 cells; MCF7 cells. Flow Cyt (intra): MCF-7 cells.
  • 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.

    We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.

    Mouse, Rat: We have preliminary internal testing data to indicate this antibody may not react with these species. Please contact us for more information.

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. Stable for 12 months at -20°C.
  • Dissociation constant (KD)

    KD = 1.86 x 10 -11 M
    Learn more about KD
  • Storage buffer

    pH: 7.20
    Preservative: 0.01% Sodium azide
    Constituents: 40% Glycerol, 0.05% BSA, 59% PBS
  • Concentration information loading...
  • Purity

    Protein A purified
  • Clonality

    Monoclonal
  • Clone number

    EPR4513-32-7
  • 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
    • Isotype/Loading Controls
    • Loading Controls
    • Cdk4

Associated products

  • Alternative Versions

    • Alexa Fluor® 488 Anti-Cdk4 antibody [EPR4513-32-7] (ab193966)
    • Alexa Fluor® 647 Anti-Cdk4 antibody [EPR4513-32-7] (ab193967)
    • HRP Anti-Cdk4 antibody [EPR4513-32-7] - Loading Control (ab193968)
    • Anti-Cdk4 antibody [EPR4513-32-7] - BSA and Azide free (ab213216)
    • Alexa Fluor® 555 Anti-Cdk4 antibody [EPR4513-32-7] (ab215318)
  • Compatible Secondaries

    • Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077)
    • Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (ab150081)
    • Goat Anti-Rabbit IgG H&L (HRP) (ab205718)
    • Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773)
    • Goat Anti-Rabbit IgG H&L (HRP) (ab97051)
  • Isotype control

    • Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730)
  • KO cell lines

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

    • Human CDK4 knockout HeLa cell lysate (ab263780)
  • Positive Controls

    • K-562 nuclear extract lysate (ab14851)
    • HeLa whole cell lysate (ab150035)
    • MCF7 whole cell lysate (ab29537)
    • MCF7 membrane extract lysate (ab29539)
    • HeLa whole cell lysate (ab29545)
    • HeLa membrane extract lysate (ab29547)
    • Ramos whole cell lysate (ab3955)
    • K-562 whole cell lysate (ab7911)
  • Recombinant Protein

    • Recombinant Human Cdk4 protein (ab126909)

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab108357 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
WB (1)
1/1000 - 1/10000. Detects a band of approximately 34 kDa (predicted molecular weight: 34 kDa).
IHC-P
1/100 - 1/250. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

See IHC antigen retrieval protocols.

Flow Cyt (Intra)
1/10 - 1/100.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

ICC/IF
1/500.
Notes
WB
1/1000 - 1/10000. Detects a band of approximately 34 kDa (predicted molecular weight: 34 kDa).
IHC-P
1/100 - 1/250. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

See IHC antigen retrieval protocols.

Flow Cyt (Intra)
1/10 - 1/100.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

ICC/IF
1/500.

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
    • Omim: 123829 Human
    • SwissProt: P11802 Human
    • Unigene: 95577 Human
    • 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 [EPR4513-32-7] (ab108357)
      Western blot - Anti-Cdk4 antibody [EPR4513-32-7] (ab108357)
      All lanes : Anti-Cdk4 antibody [EPR4513-32-7] (ab108357) at 1/1000 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 - ab108357 observed at 34 kDa. Red - Anti-Vinculin antibody [VIN-54] observed at 124 kDa.

       ab108357 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. ab108357 and Anti-Vinculin antibody [VIN-54] overnight at 4°C at a 1 in 1000 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.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cdk4 antibody [EPR4513-32-7] (ab108357)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cdk4 antibody [EPR4513-32-7] (ab108357)

      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human cervix carcinoma tissue labeling Cdk4 with purified ab108357 at 1/100. Heat mediated antigen retrieval was performed using Tris/EDTA buffer (pH 9). ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L), was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

    • Western blot - Anti-Cdk4 antibody [EPR4513-32-7] (ab108357)
      Western blot - Anti-Cdk4 antibody [EPR4513-32-7] (ab108357)
      Lanes 1-2 : Anti-Cdk4 antibody [EPR4513-32-7] (ab108357) at 1/1000 dilution
      Lanes 3-4 : Anti-beta Actin antibody [mAbcam 8226] - Loading Control (ab8226) at 1/1000 dilution


      Lanes 1 & 3 & 5 : Wild-type HAP1 cell lysate
      Lanes 2 & 4 & 6 : CDK4 knockout HAP1 cell lysate

      Lysates/proteins at 20 µg per lane.

      Predicted band size: 34 kDa



      Lanes 5 and 6: Merged signal (red and green). Green - ab108357 observed at 34kDa. Red - loading control to beta actin observed at 40kDa.

      ab108357 was shown to specifically react with CDK4 in wild-type HAP1 cells. No band was observed when CDK4 knockout samples were examined. Wild-type and CDK4 knockout samples were subjected to SDS-PAGE. ab108357 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 1/10,000 dilution for 1 hour at room temperature before imaging.

    • Immunocytochemistry/ Immunofluorescence - Anti-Cdk4 antibody [EPR4513-32-7] (ab108357)
      Immunocytochemistry/ Immunofluorescence - Anti-Cdk4 antibody [EPR4513-32-7] (ab108357)

      Immunocytochemical analysis of MCF7 (human breast adenocarcinoma cell line) cells, labeling Cdk4 with purified ab108357 at 1/100. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/500), was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. ab7291, a mouse anti-tubulin (1/1000) and ab150120, an Alexa Fluor® 594-conjugated goat anti-mouse IgG (1/500) were also used.

      Control 1: primary antibody (1/100) and secondary antibody, ab150120, an Alexa Fluor® 594-conjugated goat anti-mouse IgG (1/500).

      Control 2: ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/500).

    • Flow Cytometry (Intracellular) - Anti-Cdk4 antibody [EPR4513-32-7] (ab108357)
      Flow Cytometry (Intracellular) - Anti-Cdk4 antibody [EPR4513-32-7] (ab108357)

      ab108357 staining CDK4in the human cell line MCF7 (human breast adenocarcinoma cell line) by intracellular flow cytometry. Cells were fixed with 4% paraformaldehyde, permeabilized with 90% methanol and the sample was incubated with the primary antibody at a dilution of 1/20. A goat anti rabbit IgG (Alexa Fluor® 488) at a dilution of 1/2000 was used as the secondary antibody. 

      Isoytype control: Rabbit monoclonal IgG (Black) 

      Unlabeled control: Cell without incubation with primary antibody and secondary antibody (Blue) 

       

       

    • Western blot - Anti-Cdk4 antibody [EPR4513-32-7] (ab108357)
      Western blot - Anti-Cdk4 antibody [EPR4513-32-7] (ab108357)
      All lanes : Anti-Cdk4 antibody [EPR4513-32-7] (ab108357) at 1/10000 dilution (purified)

      Lane 1 : HeLa (human epithelial cell line from cervix adenocarcinoma) cell lysate
      Lane 2 : K562 (human chronic myelogenous leukemia cell line from bone marrow) cell lysate
      Lane 3 : Ramos (human Burkitt's lymphoma cell line) cell lysate

      Lysates/proteins at 20 µg per lane.

      Secondary
      All lanes : Peroxidase-conjugated goat anti-rabbit IgG, (H+L) at 1/1000 dilution

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



      Blocking and dilution buffer: 5% NFDM/TBST.

    • Immunocytochemistry/ Immunofluorescence - Anti-Cdk4 antibody [EPR4513-32-7] (ab108357)
      Immunocytochemistry/ Immunofluorescence - Anti-Cdk4 antibody [EPR4513-32-7] (ab108357)

      ab108357 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 ab108357 at 1/500 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.

    • Flow Cytometry (Intracellular) - Anti-Cdk4 antibody [EPR4513-32-7] (ab108357)
      Flow Cytometry (Intracellular) - Anti-Cdk4 antibody [EPR4513-32-7] (ab108357)

      Intracellular Flow Cytometry analysis of MCF7 (human breast adenocarcinoma cell line) cells labelling Cdk4 with purified ab108357 at 1/40 (red). Cells were fixed with 2% paraformaldehyde. A FITC-conjugated goat anti-rabbit IgG (1/150) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabelled control, cells without incubation with primary and secondary antibodies. 

       

       

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cdk4 antibody [EPR4513-32-7] (ab108357)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cdk4 antibody [EPR4513-32-7] (ab108357)

      Immunohistochemical analysis of formalin/PFA-fixed paraffin-embedded human urothelial carcinoma tissue labelling Cdk4 with unpurified ab108357 at a dilution of 1/100.

      Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

    • Western blot - Anti-Cdk4 antibody [EPR4513-32-7] (ab108357)
      Western blot - Anti-Cdk4 antibody [EPR4513-32-7] (ab108357)This image is courtesy of an Abreview submitted by Sonia Rocha
      All lanes : Anti-Cdk4 antibody [EPR4513-32-7] (ab108357) at 1/1000 dilution (unpurified)

      Lane 1 : Human osteosarcoma whole cell lysate - control, non-targeting siRNA
      Lane 2 : Human osteosarcoma whole cell lysate - siRNA for CDK4

      Lysates/proteins at 20 µg per lane.

      Secondary
      All lanes : HRP-conjugated goat anti-rabbit IgG polyclonal at 1/2000 dilution

      Developed using the ECL technique.

      Performed under reducing conditions.

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


      Exposure time: 5 seconds

      See Abreview

    • OI-RD Scanning - Anti-Cdk4 antibody [EPR4513-32-7] (ab108357)
      OI-RD Scanning - Anti-Cdk4 antibody [EPR4513-32-7] (ab108357)
      Equilibrium disassociation constant (KD)
      Learn more about KD

      Click here to learn more about KD
    • Anti-Cdk4 antibody [EPR4513-32-7] (ab108357)
      Anti-Cdk4 antibody [EPR4513-32-7] (ab108357)

    Protocols

    • Flow cytometry protocols
    • Immunohistochemistry protocols
    • Immunocytochemistry & immunofluorescence protocols
    • Western blot protocols

    Click here to view the general protocols

    Datasheets and documents

    • SDS download

    • Datasheet download

      Download

    References (114)

    Publishing research using ab108357? Please let us know so that we can cite the reference in this datasheet.

    ab108357 has been referenced in 114 publications.

    • Xu W  et al. m6A RNA methylation-mediated NDUFA4 promotes cell proliferation and metabolism in gastric cancer. Cell Death Dis 13:715 (2022). alpha smooth muscle Actin ; Human . PubMed: 35977935
    • Ren M  et al. Simultaneously targeting SOAT1 and CPT1A ameliorates hepatocellular carcinoma by disrupting lipid homeostasis. Cell Death Discov 7:125 (2021). PubMed: 34052835
    • Pack LR  et al. Clinical CDK4/6 inhibitors induce selective and immediate dissociation of p21 from cyclin D-CDK4 to inhibit CDK2. Nat Commun 12:3356 (2021). PubMed: 34099663
    • Deng H  et al. YAP Promotes Cell Proliferation and Epithelium-Derived Cytokine Expression via NF-?B Pathway in Nasal Polyps. J Asthma Allergy 14:839-850 (2021). PubMed: 34276219
    • Chen S  et al. CLK1/SRSF5 pathway induces aberrant exon skipping of METTL14 and Cyclin L2 and promotes growth and metastasis of pancreatic cancer. J Hematol Oncol 14:60 (2021). PubMed: 33849617
    View all Publications for this product

    Customer reviews and Q&As

    Show All Reviews Q&A
    Submit a review Submit a question

    Western blot abreview for Anti-Cdk4 antibody [EPR4513] - Loading Control

    Excellent
    Abreviews
    Abreviews
    abreview image
    Application
    Western blot
    Loading amount
    20 µg
    Gel Running Conditions
    Reduced Denaturing (10%)
    Sample
    Human Cell lysate - whole cell (osteosarcoma cells)
    Specification
    osteosarcoma cells
    Treatment
    lane 1 control, lane 2 siRNA for CDK4, 48h
    Blocking step
    Milk as blocking agent for 10 minute(s) · Concentration: 10% · Temperature: 21°C
    Read More
    The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

    DR. Sonia Rocha

    Verified customer

    Submitted Sep 26 2014

    Please note: All products are "FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES"
    For licensing inquiries, please contact partnerships@abcam.com

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