Anti-Cdk4 抗体 [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
製品の概要
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製品名
Anti-Cdk4 antibody [EPR17525]
Cdk4 一次抗体 製品一覧 -
製品の詳細
Rabbit monoclonal [EPR17525] to Cdk4 -
由来種
Rabbit -
アプリケーション
適用あり: ICC/IF, IP, WBmore details -
種交差性
交差種: Mouse, Rat, Human -
免疫原
Recombinant fragment. This information is proprietary to Abcam and/or its suppliers.
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ポジティブ・コントロール
- 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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特記事項
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.
製品の特性
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製品の状態
Liquid -
保存方法
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. -
バッファー
pH: 7.2
Preservative: 0.01% Sodium azide
Constituents: PBS, 40% Glycerol, 0.05% BSA -
Concentration information loading...
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精製度
Protein A purified -
ポリ/モノ
モノクローナル -
クローン名
EPR17525 -
アイソタイプ
IgG -
研究分野
関連製品
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Alternative Versions
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KO cell lines
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KO cell lysates
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Recombinant Protein
アプリケーション
The Abpromise guarantee
Abpromise保証は、 次のテスト済みアプリケーションにおけるab199728の使用に適用されます
アプリケーションノートには、推奨の開始希釈率がありますが、適切な希釈率につきましてはご検討ください。
アプリケーション | Abreviews | 特記事項 |
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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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特記事項 |
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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). |
ターゲット情報
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機能
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. -
関連疾患
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. -
配列類似性
Belongs to the protein kinase superfamily. CMGC Ser/Thr protein kinase family. CDC2/CDKX subfamily.
Contains 1 protein kinase domain. -
翻訳後修飾
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. -
細胞内局在
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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参照データベース
- 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 -
別名
- Cdk 4 antibody
- cdk4 antibody
- CDK4 protein antibody
see all
画像
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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 : Wild-type 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
プロトコール
データシートおよび資料
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SDS download
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Datasheet download
Certificate of Compliance
参考文献 (61)
ab199728 は 61 報の論文で使用されています。
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- Ren BX et al. The antibiotic drug trimethoprim suppresses tumour growth and metastasis via targeting Snail. Br J Pharmacol 179:2659-2677 (2022). PubMed: 34855201
- Cheng LZ et al. Structural Optimization and Improving Antitumor Potential of Moreollic Acid from Gamboge. Molecules 27:N/A (2022). PubMed: 35056797
- Fang Y et al. Age-related GSK3β overexpression drives podocyte senescence and glomerular aging. J Clin Invest 132:N/A (2022). PubMed: 35166234
- Xu F et al. SLC1A5 Prefers to Play as an Accomplice Rather Than an Opponent in Pancreatic Adenocarcinoma. Front Cell Dev Biol 10:800925 (2022). PubMed: 35419359