人TLE1 knockout HEK-293T cell line (ab265059)
概述
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产品名称
人TLE1 knockout HEK-293T cell line
参阅全部 TLE 1 细胞裂解液 -
Parental Cell Line
HEK293T -
Organism
Human -
Mutation description
Knockout achieved by using CRISPR/Cas9, 13 bp deletion in exon 12 and Insertion of the selection cassette in exon 12 -
Passage number
<20 -
Knockout validation
Immunocytochemistry (ICC), Sanger Sequencing, Western Blot (WB) -
经测试应用
适用于: ICC/IF, IHC-P, WBmore details -
Biosafety level
2 -
常规说明
Recommended control: Human wild-type HEK293T cell line (ab255593). 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, 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
Kidney -
Cell type
epithelial -
STR Analysis
Amelogenin X D5S818: 8, 9 D13S317: 11, 12, 14 D7S820: 11 D16S539: 9, 13 vWA: 15, 20 TH01: 7, 9.3 TPOX: 11, 12 CSF1PO: 12 -
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
Transcriptional corepressor that binds to a number of transcription factors. Inhibits NF-kappa-B-regulated gene expression. Inhibits the transcriptional activation mediated by FOXA2, and by CTNNB1 and TCF family members in Wnt signaling. The effects of full-length TLE family members may be modulated by association with dominant-negative AES. Unusual function as coactivator for ESRRG. -
组织特异性
In all tissues examined, mostly in brain, liver and muscle. -
序列相似性
Belongs to the WD repeat Groucho/TLE family.
Contains 6 WD repeats. -
结构域
WD repeat Groucho/TLE family members are characterized by 5 regions, a glutamine-rich Q domain, a glycine/proline-rich GP domain, a central CcN domain, containing a nuclear localization signal, and a serine/proline-rich SP domain. The most highly conserved are the N-terminal Q domain and the C-terminal WD-repeat domain. -
翻译后修饰
Phosphorylated, probably by CDK1. The degree of phosphorylation varies throughout the cell cycle, and is highest at the G2/M transition. Becomes hyperphosphorylated in response to cell differentiation and interaction with HES1 or RUNX1.
Ubiquitinated by XIAP/BIRC4. -
细胞定位
Nucleus. Nuclear and chromatin-associated, depending on isoforms and phosphorylation status. Hyperphosphorylation decreases the affinity for nuclear components. - Information by UniProt
相关产品
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KO cell lysates
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Related Products
应用
The Abpromise guarantee
Abpromise™承诺保证使用ab265059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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ICC/IF |
Use at an assay dependent concentration.
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IHC-P |
Use at an assay dependent concentration. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
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WB |
Use at an assay dependent concentration. Predicted molecular weight: 83 kDa.
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说明 |
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ICC/IF
Use at an assay dependent concentration. |
IHC-P
Use at an assay dependent concentration. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol. |
WB
Use at an assay dependent concentration. Predicted molecular weight: 83 kDa. |
图片
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All lanes : Anti-TLE 1 antibody [OTI1F5] (ab131648) at 1/500 dilution
Lane 1 : Wild-type HEK293T cell lysate
Lane 2 : TLE1 knockout HEK293T cell lysate
Lane 3 : HepG2 cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 83 kDa
Observed band size: 83 kDaLanes 1-3: Merged signal (red and green). Green - ab131648 observed at 83 kDa. Red - loading control, ab181602 observed at 37 kDa.
ab131648 Anti-TLE 1 antibody [OTI1F5] was shown to specifically react with TLE 1 in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab265059 (knockout cell lysate ab257240) was used. Wild-type and TLE 1 knockout samples were subjected to SDS-PAGE. ab131648 and Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) were incubated overnight at 4°C at 1 in 500 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed (ab216777) secondary antibodies at 1 in 10000 dilution for 1 hour at room temperature before imaging.
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Immunohistochemical analysis of paraffin-embedded fixed (A) Parental HEK293 (Human embryonic kidney epithelial cell) cell pellet (B) TLE1 knockout HEK293 (ab265059) cell pellet, staining TLE 1 with ab183742 at 1/250 dilution for 30 mins at room temperature. LeicaDS9800 (Bond™ Polymer Refine Detection) used as secondary antibody. Counter-stained using hematoxylin.
Positive staining on Wild-type HEK293T cell pellet and no staining on TLE1 knockout HEK293 cell pellet.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument.Heat mediated antigen retrieval was performed with Citrate buffer (pH 6.0, Epitope Retrieval Solution 1) for 20 mins.
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Immunofluorescence analysis of 4% paraformaldehyde-fixed, 0.1% TritonX-100 permeabilised wildtype HEK293T cells and TLE1 knockout HEK293T cells (ab265059) with ab183742 (green) at 1/50 dilution. Alexa Fluor® 488 Goat Anti-Rabbit IgG H&L (ab150081) was used as a secondary antibody, presabsorbed at 1/1000 dilution. Alexa Fluor® 594 Anti-alpha Tubulin mouse monoclonal antibody (ab195889) used as microtubule marker counterstain (red). Nuclei were counterstained with DAPI (blue).
Confocal image showing nuclear staining in wildtype HEK293T cells and showing no staining in TLE1 knockout HEK293T cells.
Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8). -
All lanes : Anti-TLE 1 antibody [EPR9386(2)] (ab183742) at 1/1000 dilution
Lane 1 : Wild-type HEK293T cell lysate
Lane 2 : TLE1 knockout HEK293T cell lysate
Lane 3 : HepG2 cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 83 kDa
Observed band size: 83 kDaLanes 1-3: Merged signal (red and green). Green - ab183742 observed at 83 kDa. Red - loading control, ab8245 observed at 37 kDa.
ab183742 Anti-TLE 1 antibody [EPR9386(2)] was shown to specifically react with TLE 1 in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab265059 (knockout cell lysate ab257240) was used. Wild-type and TLE 1 knockout samples were subjected to SDS-PAGE. ab183742 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 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 10000 dilution for 1 hour at room temperature before imaging.
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Allele-1: 13 bp deletion in exon 12.
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Allele-2: Insertion of the selection cassette in exon 12.
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Representative images of TLE1 knockout HEK293T cells, low and high confluency examples (top left and right respectively) and wild-type HEK293T cells, low and high confluency (bottom left and right respectively) showing typical adherent, epithelial-like morphology. Images were captured at 10X magnification using a EVOS XL Core microscope.
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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