重组Anti-Cyclin D2抗体[EPR19659] - BSA and Azide free (ab240377)
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [EPR19659] to Cyclin D2 - BSA and Azide free
- Suitable for: WB, Flow Cyt (Intra), ICC/IF, IP
- Knockout validated
- Reacts with: Human
Related conjugates and formulations
概述
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产品名称
Anti-Cyclin D2抗体[EPR19659] - BSA and Azide free
参阅全部 Cyclin D2 一抗 -
描述
兔单克隆抗体[EPR19659] to Cyclin D2 - BSA and Azide free -
宿主
Rabbit -
经测试应用
适用于: WB, Flow Cyt (Intra), ICC/IF, IPmore details -
种属反应性
与反应: Human -
免疫原
Recombinant full length protein. This information is proprietary to Abcam and/or its suppliers.
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阳性对照
- WB: Wild type HEK293T, HAP1 and HeLa whole cell lysate, 293T, U-2 OS, Hman heart tissue lysate, Human thyroid tissue lysate. Flow Cyt (intra): U-2 OS cells ICC/IF: Caco-2 and U-2 OS cells. IP: U-2 OS cell lysate.
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常规说明
ab240377 is the carrier-free version of ab207604.
Our carrier-free antibodies are typically supplied in a PBS-only formulation, purified and free of BSA, sodium azide and glycerol. The carrier-free buffer and high concentration allow for increased conjugation efficiency.
This conjugation-ready format is designed for use with fluorochromes, metal isotopes, oligonucleotides, and enzymes, which makes them ideal for antibody labelling, functional and cell-based assays, flow-based assays (e.g. mass cytometry) and Multiplex Imaging applications.
Use our conjugation kits for antibody conjugates that are ready-to-use in as little as 20 minutes with <1 minute hands-on-time and 100% antibody recovery: available for fluorescent dyes, HRP, biotin and gold.
This product is compatible with the Maxpar® Antibody Labeling Kit from Fluidigm, without the need for antibody preparation. Maxpar® is a trademark of Fluidigm Canada Inc.
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.
性能
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形式
Liquid -
存放说明
Shipped at 4°C. Store at +4°C. Do Not Freeze. -
存储溶液
pH: 7.2
Constituent: PBS -
无载体
是 -
Concentration information loading...
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纯度
Protein A purified -
克隆
单克隆 -
克隆编号
EPR19659 -
同种型
IgG -
研究领域
相关产品
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Alternative Versions
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Compatible Secondaries
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Conjugation kits
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Isotype control
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KO cell lines
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KO cell lysates
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Recombinant Protein
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Related Products
应用
The Abpromise guarantee
Abpromise™承诺保证使用ab240377于以下的经测试应用
“应用说明”部分 下显示的仅为推荐的起始稀释度;实际最佳的稀释度/浓度应由使用者检定。
应用 | Ab评论 | 说明 |
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WB |
Use at an assay dependent concentration. Detects a band of approximately 33 kDa (predicted molecular weight: 33 kDa).
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Flow Cyt (Intra) |
Use at an assay dependent concentration.
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ICC/IF |
Use at an assay dependent concentration.
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IP |
Use at an assay dependent concentration.
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说明 |
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WB
Use at an assay dependent concentration. Detects a band of approximately 33 kDa (predicted molecular weight: 33 kDa). |
Flow Cyt (Intra)
Use at an assay dependent concentration. |
ICC/IF
Use at an assay dependent concentration. |
IP
Use at an assay dependent concentration. |
靶标
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功能
Regulatory component of the cyclin D2-CDK4 (DC) complex that phosphorylates and inhibits members of the retinoblastoma (RB) protein family including RB1 and regulates the cell-cycle during G(1)/S transition. Phosphorylation of RB1 allows dissociation of the transcription factor E2F from the RB/E2F complex 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 substrate for SMAD3, phosphorylating SMAD3 in a cell-cycle-dependent manner and repressing its transcriptional activity. Component of the ternary complex, cyclin D2/CDK4/CDKN1B, required for nuclear translocation and activity of the cyclin D-CDK4 complex. -
序列相似性
Belongs to the cyclin family. Cyclin D subfamily.
Contains 1 cyclin N-terminal domain. -
细胞定位
Nucleus. Cytoplasm. Membrane. Cyclin D-CDK4 complexes accumulate at the nuclear membrane and are then translocated into the nucleus through interaction with KIP/CIP family members. - Information by UniProt
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数据库链接
- Entrez Gene: 894 Human
- Omim: 123833 Human
- SwissProt: P30279 Human
- Unigene: 376071 Human
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别名
- CCND 2 antibody
- ccnd2 antibody
- CCND2_HUMAN antibody
see all
图片
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All lanes : Anti-Cyclin D2 antibody [EPR19659] (ab207604) at 1/1000 dilution
Lane 1 : 293T (Human embryonic kidney epithelial cell) whole cell lysate
Lane 2 : U-2 OS (Human bone osteosarcoma epithelial cell) whole cell lysate
Lane 3 : Human heart tissue lysate
Lane 4 : Human thyroid tissue lysate
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution
Predicted band size: 33 kDa
Observed band size: 31 kDa why is the actual band size different from the predicted?
Exposure time: 40 secondsBlocking and diluting buffer and concentration: 5% NFDM/TBST.ab181602 was used as a GAPDH loading control.We recommend using a higher sensitive ECL substrate to increase the band intensity.This data was developed using ab207604, the same antibody clone in a different buffer formulation. -
All lanes : Anti-Cyclin D2 antibody [EPR19659] (ab207604) at 1/1000 dilution
Lane 1 : Wild-type HEK-293T cell lysate
Lane 2 :Human CCND2 (Cyclin D2) knockout HEK-293T cell lysate (ab257875)
Lane 3 : HeLa cell lysate
Lane 4 : U-2 OS cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 33 kDa
Observed band size: 33 kDaThis data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab207604).
False colour image of Western blot: Anti-Cyclin D2 antibody [EPR19659] staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab207604 was shown to bind specifically to Cyclin D2.
A band was observed at 33 kDa in wild-type HEK-293T cell lysates with no signal observed at this size in Ccnd2 knockout cell line ab267318 (knockout cell lysate ab257875). To generate this image, wild-type and Ccnd2 knockout HEK-293T cell lysates were analysed.
First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged.
Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/20000 dilution.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab207604).
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Lane 1: Wild type HAP1 whole cell lysate (20 µg)
Lane 2: CCND2 (Cyclin D2) knockout HAP1 whole cell lysate (20 µg)
Lane 3: Hek293 whole cell lysate (20 µg)
Lane 4: HeLa whole cell lysate (20 µg)Lanes 1 - 4: Merged signal (red and green). Green - ab207604 observed at 34 kDa. Red - loading control, ab18058, observed at 130 kDa.
ab207604 was shown to recognize CCND2 (Cyclin D2) in wild type cells as signal was lost at the expected MW in CCND2 (Cyclin D2) knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and CCND2 (Cyclin D2) knockout samples were subjected to SDS-PAGE. Ab207604 and ab18058 (Mouse anti Vinculin loading control) were incubated overnight at 4°C at a 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.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab207604).
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Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized Caco-2 (Human colorectal adenocarcinoma cell line) cells labeling Cyclin D2 with ab207604 at 1/100 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear and weak cytoplasmic staining on Caco-2 cells. The nuclear counter stain is DAPI (blue).
Tubulin is detected with Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) (ab150120) secondary antibody at 1/1000 dilution (red).
The negative controls are as follows:
-ve control 1: ab207604 at 1/100 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) (ab150120) secondary antibody at 1/1000 dilution.
-ve control 2: Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution.This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab207604).
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Intracellular Flow Cytometry analysis of U-2 OS (Human bone osteosarcoma epithelial cell) cells labeling Cyclin D2 with purified ab207604 at 1/450 dilution (1.00µg/mL) (Red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) secondary antibody was used at 1/2000 dilution. Isotype control - Rabbit monoclonal IgG (ab172730) (black). Unlabeled control - Unlabelled cells (blue). This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab207604).
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Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized U-2 OS (Human bone osteosarcoma epithelial cell line) cells labeling Cyclin D2 with ab207604 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear and weak cytoplasmic staining on U-2 OS cells. The nuclear counter stain is DAPI (blue).
Tubulin is detected with Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) (ab150120) secondary antibody at 1/1000 dilution (red).
The negative controls are as follows:
-ve control 1: ab207604 at 1/100 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) (ab150120) secondary antibody at 1/1000 dilution.
-ve control 2: Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary at 1/1000 dilution.This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab207604).
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Cyclin D2 was immunoprecipitated from 0.35 mg of U-2 OS (Human bone osteosarcoma epithelial cell line) whole cell lysate with ab207604 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab207604 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.
Lane 1: U-2 OS whole cell lysate, 10µg (Input).
Lane 2: ab207604 IP in U-2 OS whole cell lysate.
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab207604 in U-2 OS whole cell lysate.
Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 3 minutes.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab207604).
实验方案
To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.
数据表及文件
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Datasheet download
Certificate of Compliance
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