人CTNNB1 (beta Catenin II) knockout HCT116 cell line (ab273712)
概述
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产品名称
人CTNNB1 (beta Catenin II) knockout HCT116 cell line
参阅全部 beta Catenin 细胞裂解液 -
Parental Cell Line
HCT116 -
Organism
Human -
Mutation description
Knockout achieved by using CRISPR/Cas9, Homozygous: 14 bp deletion and 7 bp insertion in exon 3 -
Passage number
<20 -
Knockout validation
Sanger Sequencing, 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 HCT116 cell line (ab273730). 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: McCoY5a + 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, ERS Genomics Limited and Sigma-Aldrich Co. LLC, and is developed with patented technology. For full details of the licenses and 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
Colon -
Cell type
epithelial -
Disease
Carcinoma -
Gender
Male -
Antibiotic resistance
Puromycin 1.00µg/ml -
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
Key dowstream component of the canonical Wnt signaling pathway. In the absence of Wnt, forms a complex with AXIN1, AXIN2, APC, CSNK1A1 and GSK3B that promotes phosphorylation on N-terminal Ser and Thr residues and ubiquitination of CTNNB1 via BTRC and its subsequent degradation by the proteasome. In the presence of Wnt ligand, CTNNB1 is not ubiquitinated and accumulates in the nucleus, where it acts as a coactivator for transcription factors of the TCF/LEF family, leading to activate Wnt responsive genes.
Involved in the regulation of cell adhesion. The majority of beta-catenin is localized to the cell membrane and is part of E-cadherin/catenin adhesion complexes which are proposed to couple cadherins to the actin cytoskeleton. -
组织特异性
Expressed in several hair follicle cell types: basal and peripheral matrix cells, and cells of the outer and inner root sheaths. Expressed in colon. -
疾病相关
Defects in CTNNB1 are associated with colorectal cancer (CRC) [MIM:114500].
Note=Activating mutations in CTNNB1 have oncogenic activity resulting in tumor development. Somatic mutations are found in various tumor types, including colon cancers, ovarian and prostate carcinomas, hepatoblastoma (HB), hepatocellular carcinoma (HCC). HBs are malignant embryonal tumors mainly affecting young children in the first three years of life.
Defects in CTNNB1 are a cause of pilomatrixoma (PTR) [MIM:132600]; a common benign skin tumor.
Defects in CTNNB1 are a cause of medulloblastoma (MDB) [MIM:155255]. MDB is a malignant, invasive embryonal tumor of the cerebellum with a preferential manifestation in children.
Defects in CTNNB1 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.
Note=A chromosomal aberration involving CTNNB1 is found in salivary gland pleiomorphic adenomas, the most common benign epithelial tumors of the salivary gland. Translocation t(3;8)(p21;q12) with PLAG1. -
序列相似性
Belongs to the beta-catenin family.
Contains 12 ARM repeats. -
翻译后修饰
Phosphorylation by GSK3B requires prior phosphorylation of Ser-45 by another kinase. Phosphorylation proceeds then from Thr-41 to Ser-37 and Ser-33.
EGF stimulates tyrosine phosphorylation. Phosphorylation on Tyr-654 decreases CDH1 binding and enhances TBP binding.
Ubiquitinated by the SCF(BTRC) E3 ligase complex when phosphorylated by GSK3B, leading to its degradation. Ubiquitinated by a E3 ubiquitin ligase complex containing UBE2D1, SIAH1, CACYBP/SIP, SKP1, APC and TBL1X, leading to its subsequent proteasomal degradation. -
细胞定位
Cytoplasm. Nucleus. Cytoplasm > cytoskeleton. Cell junction > adherens junction. Cell junction. Cell membrane. Cytoplasmic when it is unstabilized (high level of phosphorylation) or bound to CDH1. Translocates to the nucleus when it is stabilized (low level of phosphorylation). Interaction with GLIS2 and MUC1 promotes nuclear translocation. Interaction with EMD inhibits nuclear localization. - Information by UniProt
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KO cell lysates
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应用
The Abpromise guarantee
Abpromise™承诺保证使用ab273712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. Predicted molecular weight: 85 kDa.
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说明 |
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WB
Use at an assay dependent concentration. Predicted molecular weight: 85 kDa. |
图片
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All lanes : Anti-beta Catenin antibody [E247] - ChIP Grade (ab32572) at 1/5000 dilution
Lane 1 : Wild-type HCT 116 cell lysate
Lane 2 : CTNNB1 knockout HCT 116 cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 85 kDa
Observed band size: 95 kDa why is the actual band size different from the predicted?False colour image of Western blot: Anti-beta Catenin antibody [E247] - ChIP Grade staining at 1/5000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab32572 was shown to bind specifically to beta Catenin. A band was observed at 95 kDa in wild-type HCT 116 cell lysates with no signal observed at this size in CTNNB1 knockout cell line ab273712 (knockout cell lysate ab275247). The band observed in the knockout lysate lane below 95 kDa is likely to represent a truncated form of beta Catenin. This has not been investigated further and the functional properties of the gene product have not been determined. To generate this image, wild-type and CTNNB1 knockout HCT 116 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-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-beta Catenin antibody [IGX4794R-3] (ab223075) at 1 µg/ml
Lane 1 : Wild-type HCT 116 cell lysate
Lane 2 : CTNNB1 knockout HCT 116 cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 85 kDa
Observed band size: 95 kDa why is the actual band size different from the predicted?False colour image of Western blot: Anti-beta Catenin antibody [IGX4794R-3] staining at 1 ug/ml, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab223075 was shown to bind specifically to beta Catenin. A band was observed at 95 kDa in wild-type HCT 116 cell lysates with no signal observed at this size in CTNNB1 knockout cell line ab273712 (knockout cell lysate ab275247). The band observed in the knockout lysate lane below 95 kDa is likely to represent a truncated form of beta Catenin. This has not been investigated further and the functional properties of the gene product have not been determined. To generate this image, wild-type and CTNNB1 knockout HCT 116 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-beta Catenin antibody [IGX4794R-3] (ab223075) at 1 µg/ml
Lane 1 : Wild-type HCT116 cell lysate
Lane 2 : CTNNB1 knockout HCT116 cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 85 kDa
Observed band size: 95 kDa why is the actual band size different from the predicted?Lanes 1 - 2: Merged signal (red and green). Green - ab223075 observed at 95 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.
ab223075 was shown to react with Anti-beta Catenin in wild-type HCT 116 cells in western blot with loss of signal observed in CTNNB1 knockout cell line ab273712 (CTNNB1 knockout cell lysate ab275247). Wild-type and CTNNB1 knockout HCT 116 cell lysates were subjected to SDS-PAGE. Membranes were blocked in fluoroscent western blot (TBS-based) blocking solution 50% (v/v) in TBS-T (0.1% Tween®) before incubation with ab223075 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at 1 µg/ml and a 1 in 20000 dilution respectively. Blots were incubated 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 in 20000 dilution for 1 hour at room temperature before imaging.
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Allele-1: 7 bp insertion and 14 bp deletion in exon 3.
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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