人LDLR (LDL Receptor) knockout HeLa cell line (ab273838)
概述
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产品名称
人LDLR (LDL Receptor) knockout HeLa cell line -
Parental Cell Line
HeLa -
Organism
Human -
Mutation description
Knockout achieved by CRISPR/Cas9; X = 17 bp deletion, 8 bp deletion, 2 bp deletion; Frameshift: 99% -
Passage number
<20 -
Knockout validation
Next Generation Sequencing (NGS) -
经测试应用
适用于: Next Generation Sequencing, WBmore details -
Biosafety level
2 -
常规说明
Recommended control: Human wild-type HeLa cell line (ab271142). 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 -
Adherent /Suspension
Adherent -
Tissue
Cervix -
Cell type
epithelial -
Disease
Adenocarcinoma -
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
- Metabolism
- Pathways and Processes
- Metabolic signaling pathways
- Lipid and lipoprotein metabolism
- Lipid metabolism
- Metabolism
- Pathways and Processes
- Metabolic signaling pathways
- Lipid and lipoprotein metabolism
- Cholesterol Metabolism
靶标
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功能
Binds LDL, the major cholesterol-carrying lipoprotein of plasma, and transports it into cells by endocytosis. In order to be internalized, the receptor-ligand complexes must first cluster into clathrin-coated pits. In case of HIV-1 infection, functions as a receptor for extracellular Tat in neurons, mediating its internalization in uninfected cells. -
疾病相关
Defects in LDLR are the cause of familial hypercholesterolemia (FH) [MIM:143890]; a common autosomal semi-dominant disease that affects about 1 in 500 individuals. The receptor defect impairs the catabolism of LDL, and the resultant elevation in plasma LDL-cholesterol promotes deposition of cholesterol in the skin (xanthelasma), tendons (xanthomas), and coronary arteries (atherosclerosis). -
序列相似性
Belongs to the LDLR family.
Contains 3 EGF-like domains.
Contains 7 LDL-receptor class A domains.
Contains 6 LDL-receptor class B repeats. -
翻译后修饰
N- and O-glycosylated.
Ubiquitinated by MYLIP leading to degradation. -
细胞定位
Cell membrane. Endomembrane system. Membrane > clathrin-coated pit. Found distributed from the plasma membrane to intracellular compartments. - Information by UniProt
应用
The Abpromise guarantee
Abpromise™承诺保证使用ab273838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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Next Generation Sequencing |
Use at an assay dependent concentration.
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WB |
Use at an assay dependent concentration. Predicted molecular weight: 95 kDa.
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说明 |
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Next Generation Sequencing
Use at an assay dependent concentration. |
WB
Use at an assay dependent concentration. Predicted molecular weight: 95 kDa. |
图片
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All lanes : Anti-LDL Receptor antibody [EPR24874-56] (ab271189) at 1/1000 dilution
Lane 1 : Wild-type HeLa cell lysate
Lane 2 : LDLR CRISPR-Cas9 edited HeLa cell lysate
Lane 3 : HepG2 cell lysate
Lane 4 : A431 cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 95 kDa
Observed band size: 120,150 kDa why is the actual band size different from the predicted?Anti-LDLR antibody [EPR24874-56] (ab271189) staining at 1/1000 dilution, shown in green; Mouse anti-CANX [CANX/1543] (ab238078) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab271189 was shown to bind specifically to LDLR. A band was observed at 120, 150 kDa in wild-type HeLa cell lysates with no signal observed at this size in LDLR CRISPR-Cas9 edited cell line ab273838 (CRISPR-Cas9 edited cell lysate ab273792). The band observed in the CRISPR-Cas9 edited lysate lane below 120, 150 kDa is likely to represent a possible truncated form of LDLR. 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 LDLR CRISPR-Cas9 edited HeLa 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 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.
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Knockout achieved by CRISPR/Cas9; X = 17 bp deletion, 8 bp deletion, 2 bp deletion; Frameshift: 99%
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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