Recombinant 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
Related conjugates and formulations
Overview
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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.
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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.
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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
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
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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...
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Purity
Protein A purified -
Clonality
Monoclonal -
Clone number
EPR17525 -
Isotype
IgG -
Research areas
Associated products
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Alternative Versions
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KO cell lines
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KO cell lysates
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KO cell pellets
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Recombinant Protein
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 |
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ICC/IF |
1/250.
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IP |
1/50.
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WB |
1/2000. Detects a band of approximately 34 kDa (predicted molecular weight: 34 kDa).
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Notes |
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ICC/IF
1/250. |
IP
1/50. |
WB
1/2000. Detects a band of approximately 34 kDa (predicted molecular weight: 34 kDa). |
Target
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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
modificationsPhosphorylation 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
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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
see all -
Alternative names
- Cdk 4 antibody
- cdk4 antibody
- CDK4 protein antibody
see all
Images
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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 kDaLanes 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.
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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. -
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 kDaLanes 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.
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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 secondsBlocking/Dilution buffer: 5% NFDM/TBST.
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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. -
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 minuteBlocking/Dilution buffer: 5% NFDM/TBST.
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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 secondsBlocking/Dilution buffer: 5% NFDM/TBST.
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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 secondsBlocking/Dilution buffer: 5% NFDM/TBST.
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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 kDaab199728 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.
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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
Protocols
Datasheets and documents
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Datasheet download
Certificate of Compliance
References (49)
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