人CDH1 (E Cadherin) knockout A-431 cell line (ab273747)
概述
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产品名称
人CDH1 (E Cadherin) knockout A-431 cell line -
Parental Cell Line
A431 -
Organism
Human -
Mutation description
Knockout achieved by CRISPR/Cas9; X = 1 bp insertion after Phe38 (allele 1), 4 bp deletion after Thr37 (allele 2), and 1 bp deletion after Thr37 (allele 3) of the WT protein Frameshift = 95% -
Passage number
<20 -
Knockout validation
Next Generation Sequencing (NGS), Western Blot (WB) -
经测试应用
适用于: WBmore details -
Biosafety level
1 -
常规说明
Recommended control: Human wild-type A-431 cell line (ab263975). Please note a wild-type cell line is not automatically included with a knockout cell line order, if required please add recommended wild-type cell line at no additional cost using the code WILDTYPE-TMTK1.
Cryopreservation cell medium: Cell Freezing Medium-DMSO Serum free media, contains 8.7% DMSO in MEM supplemented with methyl cellulose.
Culture medium: DMEM (High Glucose) + 10% FBS
Initial handling guidelines: Upon arrival, the vial should be stored in liquid nitrogen vapor phase and not at -80°C. Storage at -80°C may result in loss of viability.
1. Thaw the vial in 37°C water bath for approximately 1-2 minutes.
2. Transfer the cell suspension (0.8 mL) to a 15 mL/50 mL conical sterile polypropylene centrifuge tube containing 8.4 mL pre-warmed culture medium, wash vial with an additional 0.8 mL culture medium (total volume 10 mL) to collect remaining cells, and centrifuge at 201 x g (rcf) for 5 minutes at room temperature. 10 mL represents minimum recommended dilution. 20 mL represents maximum recommended dilution.
3. Resuspend the cell pellet in 5 mL pre-warmed culture medium and count using a haemocytometer or alternative cell counting method. Based on cell count, seed cells in an appropriate cell culture flask at a density of 2x104 cells/cm2. Seeding density is given as a guide only and should be scaled to align with individual lab schedules.
4. Incubate the culture at 37°C incubator with 5% CO2. Cultures should be monitored daily.Subculture guidelines:
- All seeding densities should be based on cell counts gained by established methods.
- A guide seeding density of 2x104 cells/cm2 is recommended.
- A partial media change 24 hours prior to subculture may be helpful to encourage growth, if required.
- Cells should be passaged when they have achieved 80-90% confluence.
This product is subject to limited use licenses from The Broad Institute and ERS Genomics Limited, and is developed with patented technology. For full details of the limited use licenses and relevant patents please refer to our limited use license and patent pages.
We will provide viable cells that proliferate on revival.
性能
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Number of cells
1 x 106 cells/vial, 1 mL -
Adherent /Suspension
Adherent -
Tissue
Skin -
Cell type
epithelial -
Disease
Epidermoid Carcinoma -
Gender
Female -
Mycoplasma free
Yes -
存放说明
Shipped on Dry Ice. Store in liquid nitrogen. -
存储溶液
Constituents: 8.7% Dimethylsulfoxide, 2% Cellulose, methyl ether -
研究领域
靶标
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功能
Cadherins are calcium-dependent cell adhesion proteins. They preferentially interact with themselves in a homophilic manner in connecting cells; cadherins may thus contribute to the sorting of heterogeneous cell types. CDH1 is involved in mechanisms regulating cell-cell adhesions, mobility and proliferation of epithelial cells. Has a potent invasive suppressor role. It is a ligand for integrin alpha-E/beta-7.
E-Cad/CTF2 promotes non-amyloidogenic degradation of Abeta precursors. Has a strong inhibitory effect on APP C99 and C83 production. -
组织特异性
Non-neural epithelial tissues. -
疾病相关
Defects in CDH1 are the cause of hereditary diffuse gastric cancer (HDGC) [MIM:137215]. An autosomal dominant cancer predisposition syndrome with increased susceptibility to diffuse gastric cancer. Diffuse gastric cancer is a malignant disease characterized by poorly differentiated infiltrating lesions resulting in thickening of the stomach. Malignant tumors start in the stomach, can spread to the esophagus or the small intestine, and can extend through the stomach wall to nearby lymph nodes and organs. It also can metastasize to other parts of the body. Note=Heterozygous germline mutations CDH1 are responsible for familial cases of diffuse gastric cancer. Somatic mutations in the has also been found in patients with sporadic diffuse gastric cancer and lobular breast cancer.
Defects in CDH1 are a cause of susceptibility to endometrial cancer (ENDMC) [MIM:608089].
Defects in CDH1 are a cause of susceptibility to ovarian cancer (OC) [MIM:167000]. Ovarian cancer common malignancy originating from ovarian tissue. Although many histologic types of ovarian neoplasms have been described, epithelial ovarian carcinoma is the most common form. Ovarian cancers are often asymptomatic and the recognized signs and symptoms, even of late-stage disease, are vague. Consequently, most patients are diagnosed with advanced disease. -
序列相似性
Contains 5 cadherin domains. -
翻译后修饰
During apoptosis or with calcium influx, cleaved by a membrane-bound metalloproteinase (ADAM10), PS1/gamma-secretase and caspase-3 to produce fragments of about 38 kDa (E-CAD/CTF1), 33 kDa (E-CAD/CTF2) and 29 kDa (E-CAD/CTF3), respectively. Processing by the metalloproteinase, induced by calcium influx, causes disruption of cell-cell adhesion and the subsequent release of beta-catenin into the cytoplasm. The residual membrane-tethered cleavage product is rapidly degraded via an intracellular proteolytic pathway. Cleavage by caspase-3 releases the cytoplasmic tail resulting in disintegration of the actin microfilament system. The gamma-secretase-mediated cleavage promotes disassembly of adherens junctions. -
细胞定位
Cell junction. Cell membrane. Endosome. Golgi apparatus > trans-Golgi network. Colocalizes with DLGAP5 at sites of cell-cell contact in intestinal epithelial cells. Anchored to actin microfilaments through association with alpha-, beta- and gamma-catenin. Sequential proteolysis induced by apoptosis or calcium influx, results in translocation from sites of cell-cell contact to the cytoplasm. Colocalizes with RAB11A endosomes during its transport from the Golgi apparatus to the plasma membrane. - Information by UniProt
应用
The Abpromise guarantee
Abpromise™承诺保证使用ab273747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 |
|---|---|---|
| WB |
Use at an assay dependent concentration. Predicted molecular weight: 97 kDa.
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| 说明 |
|---|
|
WB
Use at an assay dependent concentration. Predicted molecular weight: 97 kDa. |
图片
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Next Generation Sequencing - Human CDH1 (E Cadherin) knockout A-431 cell line (ab273747)1 bp insertion after Phe38 (allele 1), 4 bp deletion after Thr37 (allele 2), and 1 bp deletion after Thr37 (allele 3) of the WT protein
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Western blot - Human CDH1 (E Cadherin) knockout A-431 cell line (ab273747)All lanes : Anti-E Cadherin antibody [EP700Y] - Intercellular Junction Marker (ab40772) at 1/1000 dilution
Lane 1 : Wild-type A431 cell lysate
Lane 2 : CDH1 knockout A431 cell lysate
Lane 3 : Caco-2 cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 97 kDa
Observed band size: 110,130,40,55,80 kDa why is the actual band size different from the predicted?Western blot: Anti-CDH1 antibody [EP700Y] (ab40772) 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, ab40772 was shown to bind specifically to CDH1. A band was observed at 130, 110, 80, 55, 40 kDa in wild-type A431 cell lysates with no signal observed at this size in CDH1 knockout cell line ab273747 (knockout cell lysate ab273781). To generate this image, wild-type and CDH1 knockout A431 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.
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Western blot - Human CDH1 (E Cadherin) knockout A-431 cell line (ab273747)All lanes : Anti-E Cadherin antibody [4A2] (ab231303) at 1 µg/ml
Lane 1 : Wild-type A431 cell lysate
Lane 2 : CDH1 knockout A431 cell lysate
Lane 3 : Caco-2 cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 97 kDa
Observed band size: 110,130,55 kDa why is the actual band size different from the predicted?Western blot: Anti-CDH1 antibody [4A2] (ab231303) staining at 1 ug/ml, shown in green; Rabbit Anti-GAPDH antibody [EPR16891] (ab181602) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab231303 was shown to bind specifically to CDH1. A band was observed at 130, 110, 55 kDa in wild-type A431 cell lysates with no signal observed at this size in CDH1 knockout cell line ab273747 (knockout cell lysate ab273781). To generate this image, wild-type and CDH1 knockout A431 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5 % 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-Mouse IgG H&L 800CW and Goat anti-Rabbit IgG H&L 680RD at 1/20000 dilution.
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Western blot - Human CDH1 (E Cadherin) knockout A-431 cell line (ab273747)All lanes : Anti-E Cadherin antibody [EP700Y] - Intercellular Junction Marker (ab40772) at 1/10000 dilution
Lane 1 : Wild-type Raji cell lysate
Lane 2 : CDH1 knockout Raji cell lysate
Lane 3 : MCF7 cell lysate
Lane 4 : MDA-MB-231 cell lysate
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution
Performed under reducing conditions.
Predicted band size: 97 kDa
Observed band size: 105,130 kDa why is the actual band size different from the predicted?False colour image of Western blot: Anti-E Cadherin antibody [EP700Y] - Intercellular Junction Marker staining at 1/10000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab40772 was shown to bind specifically to E Cadherin. A band was observed at 105/130 kDa in wild-type Raji cell lysates with no signal observed at this size in CDH1 knockout cell line ab273747 (knockout cell lysate ab273781). To generate this image, wild-type and CDH1 knockout Raji 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.
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Western blot - Human CDH1 (E Cadherin) knockout A-431 cell line (ab273747)All lanes : Anti-E Cadherin antibody [SP64] (ab227639) at 1/100 dilution
Lane 1 : Wild-type A431 cell lysate
Lane 2 : CDH1 knockout A431 cell lysate
Lane 3 : MCF7 cell lysate
Lane 4 : MDA-MB-231 cell lysate
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution
Performed under reducing conditions.
Predicted band size: 97 kDa
Observed band size: 105,130 kDa why is the actual band size different from the predicted?False colour image of Western blot: Anti-E Cadherin antibody [SP64] staining at 1/100 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab227639 was shown to bind specifically to E Cadherin. A band was observed at 105/130 kDa in wild-type A431 cell lysates with no signal observed at this size in CDH1 knockout cell line ab273747 (knockout cell lysate ab273781).
To generate this image, wild-type and CDH1 knockout A431 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.
实验方案
数据表及文件
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SDS download
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Datasheet download
文献 (0)
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