重组Anti-Cyclin E1抗体[EP435E] - BSA and Azide free (ab208696)
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [EP435E] to Cyclin E1 - BSA and Azide free
- Suitable for: Flow Cyt (Intra), IHC-P, WB, IP, ICC/IF
- Knockout validated
- Reacts with: Human
Related conjugates and formulations
概述
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产品名称
Anti-Cyclin E1抗体[EP435E] - BSA and Azide free
参阅全部 Cyclin E1 一抗 -
描述
兔单克隆抗体[EP435E] to Cyclin E1 - BSA and Azide free -
宿主
Rabbit -
特异性
This antibody recognises Cyclin E1. It is predicted to detect the splice isoform 2 based on sequence analysis.
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经测试应用
适用于: Flow Cyt (Intra), IHC-P, WB, IP, ICC/IFmore details -
种属反应性
与反应: Human -
免疫原
Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.
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阳性对照
- WB: HAP1 and HeLa cell lysates, Human testis and placenta tissue lysates IP: HeLa cell lysate Flow Cyt (intra): HeLa and MCF7 Cells ICC/IF: HeLa cells IHC-P: Human placenta, Human colon carcinoma, wild type HAP-1.
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常规说明
ab208696 is the carrier-free version of ab33911.
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.
Mouse, Rat: We have preliminary internal testing data to indicate this antibody may not react with these species. Please contact us for more information.
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
EP435E -
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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Positive Controls
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Recombinant Protein
应用
The Abpromise guarantee
Abpromise™承诺保证使用ab208696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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Flow Cyt (Intra) |
Use at an assay dependent concentration.
ab199376 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody. |
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IHC-P |
Use at an assay dependent concentration. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
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WB |
Use at an assay dependent concentration. Detects a band of approximately 50 kDa (predicted molecular weight: 47 kDa).
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IP |
Use at an assay dependent concentration.
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ICC/IF | (1) |
Use at an assay dependent concentration.
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说明 |
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Flow Cyt (Intra)
Use at an assay dependent concentration. ab199376 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody. |
IHC-P
Use at an assay dependent concentration. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol. |
WB
Use at an assay dependent concentration. Detects a band of approximately 50 kDa (predicted molecular weight: 47 kDa). |
IP
Use at an assay dependent concentration. |
ICC/IF
Use at an assay dependent concentration. |
靶标
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功能
Essential for the control of the cell cycle at the G1/S (start) transition. -
组织特异性
Highly expressed in testis and placenta. Low levels in bronchial epithelial cells. -
序列相似性
Belongs to the cyclin family. Cyclin E subfamily. -
翻译后修饰
Phosphorylation of Thr-395 by GSK3 and of Ser-399 by CDK2 accelerates degradation via the ubiquitin proteasome pathway. Phosphorylated upon DNA damage, probably by ATM or ATR. -
细胞定位
Nucleus. - Information by UniProt
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数据库链接
- Entrez Gene: 898 Human
- Omim: 123837 Human
- SwissProt: P24864 Human
- Unigene: 244723 Human
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别名
- CCNE antibody
- Ccne1 antibody
- CCNE1_HUMAN antibody
see all
图片
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All lanes : Anti-Cyclin E1 antibody [EP435E] (ab33911) at 1/1000 dilution
Lane 1 : Wild-type MCF7 cell lysate
Lane 2 : CCNE1 knockout MCF7 cell lysate
Lane 3 : HT-29 cell lysate
Lane 4 : PC-3 cell lysate
Lysates/proteins at 20 µg per lane.
Developed using the ECL technique.
Performed under reducing conditions.
Predicted band size: 47 kDa
Observed band size: 47 kDaThis data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab33911).
Western blot: Anti-CCNE1 antibody [EP435E] (ab33911) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab33911 was shown to bind specifically to CCNE1. A band was observed at 47 kDa in wild-type MCF7 cell lysates with no signal observed at this size in CCNE1 knockout cell line ab286303 (knockout cell lysate AB300211). To generate this image, wild-type and CCNE1 knockout MCF7 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 800CW and Goat anti-Mouse IgG H&L 680RD 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 (ab33911).
Immunohistochemical analysis of paraffin-embedded (A) Wild-type HAP1 (human chronic myelogenous leukemia near-haploid cell) cell pellets (B)CCNE1 KO HAP1 cell pellets tissue labeling Cyclin E1 with ab33911 at 1/2000 (0.12 μg/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Nuclear staining on (A) Wild-type HAP1 cell pellets, no staining on (B) CCNE1 KO HAP1 cell pellets.The section was incubated with ab33911 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.
Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins
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This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab33911).
Immunohistochemical analysis of paraffin-embedded Human colon carcinoma tissue labeling Cyclin E1 with ab33911 at 1/2000 (0.12 μg/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Nuclear staining on the human colon carcinoma. The section was incubated with ab33911 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.
Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins
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This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab33911).
Immunohistochemical analysis of paraffin-embedded Human placenta tissue labeling Cyclin E1 with ab33911 at 1/2000 (0.12 μg/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Nuclear staining on the human placenta. The section was incubated with ab33911 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.
Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins
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All lanes : Anti-Cyclin E1 antibody [EP435E] (ab33911) at 1/1000 dilution
Lane 1 : Wild-type HEK-293T cell lysate
Lane 2 : CCNE1 knockout HAP1 cell lysate
Lane 3 : Jurkat cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 47 kDa
Observed band size: 47 kDaFalse colour image of Western blot: Anti-Cyclin E1 antibody [EP435E] staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab33911 was shown to bind specifically to Cyclin E1. A band was observed at 47 kDa in wild-type HAP1 cell lysates with no signal observed at this size in CCNE1 knockout cell line. To generate this image, wild-type and CCNE1 knockout HAP1 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.
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All lanes : Anti-Cyclin E1 antibody [EP435E] (ab33911) at 1/1000 dilution
Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : CCNE1 (Cyclin E1) knockout HAP1 whole cell lysate
Lysates/proteins at 40 µg per lane.
Predicted band size: 47 kDaLanes 1 - 2: Merged signal (red and green). Green - ab33911 observed at 47 kDa. Red - loading control, ab9484, observed at 37 kDa.
ab33911 was shown to recognize CCNE1 in wild-type HAP1 cells as signal was lost at the expected MW in CCNE1 (Cyclin E1) knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and CCNE1 (Cyclin E1) knockout samples were subjected to SDS-PAGE. Ab33911 and ab9484 (Mouse anti-GAPDH 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.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab33911) (unpurified).
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Immunocytochemistry/Immunofluorescence analysis of HeLa (human cervix adenocarcinoma) cells labeling Cyclin E1 (green) with purified ab33911 at 1/500. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. Nuclei were counterstained with DAPI (blue).
Secondary Only Control: PBS was used instead of the primary antibody as the negative control.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab33911).
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All lanes : Anti-Cyclin E1 antibody [EP435E] (ab33911) at 1/1000 dilution (Purified)
Lane 1 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates
Lane 2 : Human testis lysates
Lane 3 : Human placenta lysates
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: 47 kDa
Observed band size: 50 kDa why is the actual band size different from the predicted?Cyclin E1 is highly expressed in testis and placenta which is described in PMID: 9840943.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab33911).
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Purified ab33911 at 1/30 dilution (2ug) immunoprecipitating Cyclin E1 in HeLa whole cell lysate.
Lane 1 (input): HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate (10µg)
Lane 2 (+): ab33911 + HeLa whole cell lysate.
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab33911 in HeLa whole cell lysate.
VeriBlot for IP Detection Reagent (HRP) (ab131366) (1/1000) was used for Western blotting.
Blocking Buffer and concentration: 5% NFDM/TBST.
Diluting buffer and concentration: 5% NFDM/TBST.
Observed band size: 50 kDa
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab33911). -
Intracellular Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling Cyclin E1 with Purified ab33911 at 1/30 dilution (10 µg/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. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue). This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab33911).
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Immunocytochemistry/Immunofluorescence analysis of HeLa cells labeling Cyclin E1 with ab33911 at 1/500 dilution. Cells were fixed in paraformaldehyde and permeabilized with 0.5% Triton X-100 in PBS. Staining with ab33911 at 1/500 was carried out for 1 hour at 22°C in PBS buffer. ab150081, a Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed secondary antibody, was used at 1/200 dilution. DAPI was used to counterstain.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab33911) (unpurified).
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ab33911 staining Cyclin E1 in HeLa cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde, permeabilized with 0.2% Triton X-100 and blocked with 2% BSA for 45 minutes at room temperature. Samples were incubated with primary antibody (1/300 in PBS + 2% BSA) for 14 hours at 4°C. An Alexa Fluor® 488-conjugated goat anti-rabbit IgG polyclonal (1/500) was used as the secondary antibody.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab33911) (unpurified).
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Overlay histogram showing MCF7 cells stained with ab33911 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab33911, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1µg/1x106 cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. This antibody gave a positive signal in MCF7 cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab33911) (unpurified).
实验方案
数据表及文件
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Datasheet download
Certificate of Compliance
文献 (4)
ab208696 被引用在 4 文献中.
- Fu YP et al. The 19q12 bladder cancer GWAS signal: association with cyclin E function and aggressive disease. Cancer Res 74:5808-18 (2014). PubMed: 25320178
- Brodowska K et al. Effects of metformin on retinoblastoma growth in vitro and in vivo. Int J Oncol 45:2311-24 (2014). PubMed: 25215935
- Zhang X et al. MicroRNA-7 arrests cell cycle in G1 phase by directly targeting CCNE1 in human hepatocellular carcinoma cells. Biochem Biophys Res Commun 443:1078-84 (2014). WB ; Human . PubMed: 24370822
- Dai G et al. Maternal hepatic growth response to pregnancy in the mouse. Exp Biol Med (Maywood) 236:1322-32 (2011). PubMed: 21969712