人LEF1 knockout Jurkat cell line (ab274898)
概述
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产品名称
人LEF1 knockout Jurkat cell line -
Parental Cell Line
Jurkat -
Organism
Human -
Mutation description
Knockout achieved by CRISPR/Cas9; X = 4 bp insertion, 10 bp insertion, 4 bp deletion; Frameshift: 100% -
Passage number
<20 -
Knockout validation
Next Generation Sequencing (NGS) -
经测试应用
适用于: WB, Next Generation Sequencingmore details -
Biosafety level
1 -
常规说明
Recommended control: Human wild-type Jurkat cell line (ab271143). 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: RPMI + 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 for bath 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 1x105 cells/mL. 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 1x105 cells/mL is recommended.
- Do not allow the cell density to exceed 3x106.
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
Suspension -
Tissue
Blood -
Cell type
T cell lymphoblast-like -
Disease
Non-Hodgkin Lymphoma -
Gender
Male -
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
Participates in the Wnt signaling pathway. Activates transcription of target genes in the presence of CTNNB1 and EP300. May play a role in hair cell differentiation and follicle morphogenesis. TLE1, TLE2, TLE3 and TLE4 repress transactivation mediated by LEF1 and CTNNB1. Regulates T-cell receptor alpha enhancer function. Binds DNA in a sequence-specific manner. PIAG antagonizes both Wnt-dependent and Wnt-independent activation by LEF1 (By similarity). Isoform 3 lacks the CTNNB1 interaction domain and may be an antagonist for Wnt signaling. Isoform 5 transcriptionally activates the fibronectin promoter, binds to and represses transcription from the E-cadherin promoter in a CTNNB1-independent manner, and is involved in reducing cellular aggregation and increasing cell migration of pancreatic cancer cells. Isoform 1 transcriptionally activates MYC and CCND1 expression and enhances proliferation of pancreatic tumor cells. -
组织特异性
Detected in thymus. Not detected in normal colon, but highly expressed in colon cancer biopsies and colon cancer cell lines. Expressed in several pancreatic tumors and weakly expressed in normal pancreatic tissue. Isoforms 1 and 5 are detected in several pancreatic cell lines. -
序列相似性
Belongs to the TCF/LEF family.
Contains 1 HMG box DNA-binding domain. -
结构域
Proline-rich and acidic regions are implicated in the activation functions of RNA polymerase II transcription factors. -
细胞定位
Nucleus. Found in nuclear bodies upon PIASG binding. - Information by UniProt
应用
The Abpromise guarantee
Abpromise™承诺保证使用ab274898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.
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| Next Generation Sequencing |
Use at an assay dependent concentration.
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| 说明 |
|---|
|
WB
Use at an assay dependent concentration. |
|
Next Generation Sequencing
Use at an assay dependent concentration. |
图片
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Next Generation Sequencing - Human LEF1 knockout Jurkat cell line (ab274898)4 bp deletion after Pro133 (allele 1) and 4 bp deletion after Ile134 (allele 2) of the WT protein
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Western blot - Human LEF1 knockout Jurkat cell line (ab274898)All lanes : Anti-LEF1 antibody [EPR2029Y] (ab137872) at 1/1000 dilution
Lane 1 : Wild-type Jurkat cell lysate at 40 µg
Lane 2 : Lef1 knockout Jurkat cell lysate at 40 µg
Lane 3 : Jurkat cell lysate at 20 µg
Performed under reducing conditions.
Observed band size: 40 kDa why is the actual band size different from the predicted?False colour image of Western blot: Anti-LEF1 antibody [EPR2029Y] staining at 1/1000 dilution, shown in black; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab137872 was shown to bind specifically to LEF1. A band was observed at 40/53 kDa in wild-type Jurkat cell lysates with no signal observed at this size in Lef1 knockout cell line ab274898 (knockout cell lysate ab274956). To generate this image, wild-type and Lef1 knockout Jurkat 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 before development with Optiblot (ECL reagent ab133456) and imaged with 4 minutes exposure time. Secondary antibodies used were HRP conjugated Goat anti-Rabbit (H+L) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/20000 dilution.
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Western blot - Human LEF1 knockout Jurkat cell line (ab274898)All lanes : Anti-LEF1 antibody [EP2030Y] (ab53293) at 1/5000 dilution
Lane 1 : Wild-type Jurkat cell lysate at 40 µg
Lane 2 : Lef1 knockout Jurkat cell lysate at 40 µg
Lane 3 : Jurkat cell lysate at 20 µg
Performed under reducing conditions.
Observed band size: 40 kDa why is the actual band size different from the predicted?False colour image of Western blot: Anti-LEF1 antibody [EP2030Y] staining at 1/5000 dilution, shown in black; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab53293 was shown to bind specifically to LEF1. A band was observed at 40/53 kDa in wild-type Jurkat cell lysates with no signal observed at this size in Lef1 knockout cell line ab274898 (knockout cell lysate ab274956). To generate this image, wild-type and Lef1 knockout Jurkat 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 before development with Optiblot (ECL reagent ab133456) and imaged with 8 minutes exposure time. Secondary antibodies used were HRP conjugated Goat anti-Rabbit (H+L) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/20000 dilution.
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Next Generation Sequencing - Human LEF1 knockout Jurkat cell line (ab274898)Knockout achieved by CRISPR/Cas9; X = 4 bp insertion, 10 bp insertion, 4 bp deletion; Frameshift: 100%
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)
ab274898 尚未被引用在任何文献中。