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Cdk4 Recombinant Rabbit Monoclonal Antibody [SD205-1]

Usd: 385

Specification

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Catalog# ET1612-23

Cdk4 Recombinant Rabbit Monoclonal Antibody [SD205-1]

Application

  • WB

  • IF-Cell

  • IF-Tissue

  • IHC-P

  • FC

Reactivity

  • Human

  • Mouse

  • Rat

BSA and Azide free
Conjugation

  • unconjugated

This product has been cited in peer reviewed publications, see list HERE

Overview

Product Name

Cdk4 Recombinant Rabbit Monoclonal Antibody [SD205-1]

Antibody Type

Recombinant Rabbit monoclonal Antibody

Immunogen

Recombinant protein within mouse Cdk4 aa 180-303.

Species Reactivity

Human, Mouse, Rat

Validated Applications

WB, IF-Cell, IF-Tissue, IHC-P, FC

Molecular Weight

Predicted band size: 34 kDa

Positive Control

SK-OV-3 cell lysate, Jurkat cell lysate, HeLa cell lysate, MCF7 cell lysate, K-562 cell lysate, A549 cell lysate, NIH:OVCAR-3 cell lysate, NIH/3T3 cell lysate, C2C12 cell lysate, C6 cell lysate, PC-12 cell lysate, Mouse brain tissue lysate, Rat lung tissue lysate, human thyroid carcinoma tissue, mouse colon tissue, mouse lung tissue, rat colon tissue, rat lung tissue, NIH/3T3, HeLa.

Conjugation

unconjugated

Clone Number

SD205-1

RRID

AB_3070096

Product Features

Form

Liquid

Concentration

Storage Instructions

Shipped at 4℃. Store at +4℃ short term (1-2 weeks). It is recommended to aliquot into single-use upon delivery. Store at -20℃ long term.

Storage Buffer

1*TBS (pH7.4), 0.05% BSA, 40% Glycerol. Preservative: 0.05% Sodium Azide.

Isotype

IgG

Purification Method

Protein A affinity purified.

Application Dilution

  • WB

  • 1:2,000

  • IF-Cell

  • 1:50-1:200

  • IF-Tissue

  • 1:50-1:200

  • IHC-P

  • 1:200-1:1,000

  • FC

  • 1:1,000

Target

Function

The protein encoded by this gene is a member of the Ser/Thr protein kinase family. This protein is highly similar to the gene products of S. cerevisiae cdc28 and S. pombe cdc2. It is a catalytic subunit of the protein kinase complex that is important for cell cycle G1 phase progression. The activity of this kinase is restricted to the G1-S phase, which is controlled by the regulatory subunits D-type cyclins and CDK inhibitor p16INK4a. This kinase was shown to be responsible for the phosphorylation of retinoblastoma gene product (Rb).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 G1/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 G1 phase. Hypophosphorylates RB1 in early G1 phase. Cyclin D-CDK4 complexes are major integrators of various mitogenic 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.

Background References

1. Machado-Neto, JA. et al. 2014. ANKHD1, a novel component of the Hippo signaling pathway, promotes YAP1 activation and cell cycle progression in prostate cancer cells. Exp. Cell Res.. 324: 137-45.

2. Baranwal, S. et al. 2011. Molecular characterization of the tumor-suppressive function of nischarin in breast cancer. Journal of the National Cancer Institute. 103: 1513-28.

Sequence Similarity

Belongs to the protein kinase superfamily. CMGC Ser/Thr protein kinase family. CDC2/CDKX subfamily.

Post-translational Modification

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.

Subcellular Location

Cytoplasm, Nucleus, Membrane.

UNIPROT

SwissProt: P11802 Human

SwissProt: P30285 Mouse

SwissProt: P35426 Rat

Synonyms

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

Expand

Images

  • Western blot analysis of Cdk4 on different lysates with Rabbit anti-Cdk4 antibody (<a href="/products/ET1612-23" style="font-weight: bold;text-decoration: underline;">ET1612-23</a>) at 1/2,000 dilution.<br /><br />Lane 1: SK-OV-3 cell lysate (20 µg/Lane)<br />Lane 2: Jurkat cell lysate (20 µg/Lane)<br />Lane 3: HeLa cell lysate (20 µg/Lane)<br />Lane 4: MCF7 cell lysate (20 µg/Lane)<br />Lane 5: K-562 cell lysate (20 µg/Lane)<br />Lane 6: A549 cell lysate (20 µg/Lane)<br />Lane 7: NIH:OVCAR-3 cell lysate (20 µg/Lane)<br />Lane 8: NIH/3T3 cell lysate (20 µg/Lane)<br />Lane 9: C2C12 cell lysate (20 µg/Lane)<br />Lane 10: C6 cell lysate (20 µg/Lane)<br />Lane 11: PC-12 cell lysate (20 µg/Lane)<br />Lane 12: Mouse brain tissue lysate (40 µg/Lane)<br />Lane 13: Rat lung tissue lysate (40 µg/Lane)<br /><br />Predicted band size: 34 kDa<br />Observed band size: 30 kDa<br /><br />Exposure time: 24 seconds; ECL: K1801;<br />4-20% SDS-PAGE gel.<br /><br />Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (<a href="/products/ET1612-23" style="font-weight: bold;text-decoration: underline;">ET1612-23</a>) at 1/2,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (<a href="/products/HA1001" style="font-weight: bold;text-decoration: underline;">HA1001</a>) at 1/50,000 dilution was used for 1 hour at room temperature.

    Western blot analysis of Cdk4 on different lysates with Rabbit anti-Cdk4 antibody (ET1612-23) at 1/2,000 dilution.

    Lane 1: SK-OV-3 cell lysate (20 µg/Lane)
    Lane 2: Jurkat cell lysate (20 µg/Lane)
    Lane 3: HeLa cell lysate (20 µg/Lane)
    Lane 4: MCF7 cell lysate (20 µg/Lane)
    Lane 5: K-562 cell lysate (20 µg/Lane)
    Lane 6: A549 cell lysate (20 µg/Lane)
    Lane 7: NIH:OVCAR-3 cell lysate (20 µg/Lane)
    Lane 8: NIH/3T3 cell lysate (20 µg/Lane)
    Lane 9: C2C12 cell lysate (20 µg/Lane)
    Lane 10: C6 cell lysate (20 µg/Lane)
    Lane 11: PC-12 cell lysate (20 µg/Lane)
    Lane 12: Mouse brain tissue lysate (40 µg/Lane)
    Lane 13: Rat lung tissue lysate (40 µg/Lane)

    Predicted band size: 34 kDa
    Observed band size: 30 kDa

    Exposure time: 24 seconds; ECL: K1801;
    4-20% SDS-PAGE gel.

    Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1612-23) at 1/2,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

  • ICC staining of Cdk4 in AGS cells (green). Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% Blocker BSA for 15 minutes at room temperature. Cells were probed with the primary antibody (<a href="/products/ET1612-23" style="font-weight: bold;text-decoration: underline;">ET1612-23</a>, 1/50) for 1 hour at room temperature, washed with PBS. Alexa Fluor&reg;488 Goat anti-Rabbit IgG was used as the secondary antibody at 1/1,000 dilution. The nuclear counter stain is DAPI (blue).

    ICC staining of Cdk4 in AGS cells (green). Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% Blocker BSA for 15 minutes at room temperature. Cells were probed with the primary antibody (ET1612-23, 1/50) for 1 hour at room temperature, washed with PBS. Alexa Fluor®488 Goat anti-Rabbit IgG was used as the secondary antibody at 1/1,000 dilution. The nuclear counter stain is DAPI (blue).

  • ICC staining of Cdk4 in NIH/3T3 cells (green). Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% Blocker BSA for 15 minutes at room temperature. Cells were probed with the primary antibody (<a href="/products/ET1612-23" style="font-weight: bold;text-decoration: underline;">ET1612-23</a>, 1/50) for 1 hour at room temperature, washed with PBS. Alexa Fluor&reg;488 Goat anti-Rabbit IgG was used as the secondary antibody at 1/1,000 dilution. The nuclear counter stain is DAPI (blue).

    ICC staining of Cdk4 in NIH/3T3 cells (green). Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% Blocker BSA for 15 minutes at room temperature. Cells were probed with the primary antibody (ET1612-23, 1/50) for 1 hour at room temperature, washed with PBS. Alexa Fluor®488 Goat anti-Rabbit IgG was used as the secondary antibody at 1/1,000 dilution. The nuclear counter stain is DAPI (blue).

  • Immunohistochemical analysis of paraffin-embedded human thyroid carcinoma tissue with Rabbit anti-Cdk4 antibody (<a href="/products/ET1612-23" style="font-weight: bold;text-decoration: underline;">ET1612-23</a>) at 1/1,000 dilution.<br /><br />The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH<sub>2</sub>O and PBS, and then probed with the primary antibody (<a href="/products/ET1612-23" style="font-weight: bold;text-decoration: underline;">ET1612-23</a>) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

    Immunohistochemical analysis of paraffin-embedded human thyroid carcinoma tissue with Rabbit anti-Cdk4 antibody (ET1612-23) at 1/1,000 dilution.

    The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1612-23) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

  • Immunohistochemical analysis of paraffin-embedded mouse colon tissue with Rabbit anti-Cdk4 antibody (<a href="/products/ET1612-23" style="font-weight: bold;text-decoration: underline;">ET1612-23</a>) at 1/200 dilution.<br /><br />The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH<sub>2</sub>O and PBS, and then probed with the primary antibody (<a href="/products/ET1612-23" style="font-weight: bold;text-decoration: underline;">ET1612-23</a>) at 1/200 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

    Immunohistochemical analysis of paraffin-embedded mouse colon tissue with Rabbit anti-Cdk4 antibody (ET1612-23) at 1/200 dilution.

    The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1612-23) at 1/200 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

  • Immunohistochemical analysis of paraffin-embedded mouse lung tissue with Rabbit anti-Cdk4 antibody (<a href="/products/ET1612-23" style="font-weight: bold;text-decoration: underline;">ET1612-23</a>) at 1/1,000 dilution.<br /><br />The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH<sub>2</sub>O and PBS, and then probed with the primary antibody (<a href="/products/ET1612-23" style="font-weight: bold;text-decoration: underline;">ET1612-23</a>) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

    Immunohistochemical analysis of paraffin-embedded mouse lung tissue with Rabbit anti-Cdk4 antibody (ET1612-23) at 1/1,000 dilution.

    The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1612-23) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

  • Immunohistochemical analysis of paraffin-embedded rat colon tissue with Rabbit anti-Cdk4 antibody (<a href="/products/ET1612-23" style="font-weight: bold;text-decoration: underline;">ET1612-23</a>) at 1/200 dilution.<br /><br />The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH<sub>2</sub>O and PBS, and then probed with the primary antibody (<a href="/products/ET1612-23" style="font-weight: bold;text-decoration: underline;">ET1612-23</a>) at 1/200 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

    Immunohistochemical analysis of paraffin-embedded rat colon tissue with Rabbit anti-Cdk4 antibody (ET1612-23) at 1/200 dilution.

    The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1612-23) at 1/200 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

  • Immunohistochemical analysis of paraffin-embedded rat lung tissue with Rabbit anti-Cdk4 antibody (<a href="/products/ET1612-23" style="font-weight: bold;text-decoration: underline;">ET1612-23</a>) at 1/200 dilution.<br /><br />The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH<sub>2</sub>O and PBS, and then probed with the primary antibody (<a href="/products/ET1612-23" style="font-weight: bold;text-decoration: underline;">ET1612-23</a>) at 1/200 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

    Immunohistochemical analysis of paraffin-embedded rat lung tissue with Rabbit anti-Cdk4 antibody (ET1612-23) at 1/200 dilution.

    The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1612-23) at 1/200 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

  • Immunocytochemistry analysis of NIH/3T3 cells labeling Cdk4 with Rabbit anti-Cdk4 antibody (<a href="/products/ET1612-23" style="font-weight: bold;text-decoration: underline;">ET1612-23</a>) at 1/100 dilution.<br /><br />Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Cdk4 antibody (<a href="/products/ET1612-23" style="font-weight: bold;text-decoration: underline;">ET1612-23</a>) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor&trade; 488, <a href="/products/HA1121" style="font-weight: bold;text-decoration: underline;">HA1121</a>) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.<br /><br />Beta tubulin (<a href="/products/M1305-2" style="font-weight: bold;text-decoration: underline;">M1305-2</a>, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor&trade; 594, <a href="/products/HA1126" style="font-weight: bold;text-decoration: underline;">HA1126</a>) was used as the secondary antibody at 1/1,000 dilution.

    Immunocytochemistry analysis of NIH/3T3 cells labeling Cdk4 with Rabbit anti-Cdk4 antibody (ET1612-23) at 1/100 dilution.

    Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Cdk4 antibody (ET1612-23) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

    Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.

  • Flow cytometric analysis of HeLa cells labeling Cdk4.<br /><br />Cells were fixed and permeabilized. Then stained with the primary antibody (<a href="/products/ET1612-23" style="font-weight: bold;text-decoration: underline;">ET1612-23</a>, 1μg/mL) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor&trade; 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (<a href="/products/HA1121" style="font-weight: bold;text-decoration: underline;">HA1121</a>) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).

    Flow cytometric analysis of HeLa cells labeling Cdk4.

    Cells were fixed and permeabilized. Then stained with the primary antibody (ET1612-23, 1μg/mL) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).

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Citation

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