Name: Anti-Cdk4 antibody [EPR17525]
Brand: RabMAb
SKU: ab199728

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

Run BLAST with Run BLAST with
Positive control
- WB: 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

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.

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

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

Protein A purified
Clonality

Monoclonal
Clone number

EPR17525
Research areas

Alternative Versions
- Anti-Cdk4 antibody [EPR17525] (Alexa Fluor® 647) (ab225420)
- Anti-Cdk4 antibody [EPR17525] - BSA and Azide free (ab236019)

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).

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

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

Lane 1: Wild-type HAP1 whole cell lysate (20 µg)
Lane 2: Cdk4 knockout HAP1 whole cell lysate (20 µg)
Lane 3: HeLa whole cell lysate (20 µg)

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)

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)

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)

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)

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)

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)

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)

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)

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

Western blot protocols
Immunoprecipitation protocols
Immunocytochemistry & immunofluorescence protocols

Datasheet

This product has been referenced in:

Zheng WL et al. ROS-Mediated Cell Cycle Arrest and Apoptosis Induced by Zearalenone in Mouse Sertoli Cells via ER Stress and the ATP/AMPK Pathway. Toxins (Basel) 10:N/A (2018). Read more (PubMed: 29301253) »
Xu L et al. Morphological adaptation of sheep's rumen epithelium to high-grain diet entails alteration in the expression of genes involved in cell cycle regulation, cell proliferation and apoptosis. J Anim Sci Biotechnol 9:32 (2018). Read more (PubMed: 29686866) »

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Anti-Cdk4 antibody [EPR17525] (ab199728)

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