人IRS2 knockout HEK-293 cell line (ab264013)
概述
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产品名称
人IRS2 knockout HEK-293 cell line -
Parental Cell Line
HEK-293 -
Organism
Human -
Mutation description
Knockout achieved by CRISPR/Cas9 -
Passage number
<20 -
Knockout validation
Immunocytochemistry (ICC), Next Generation Sequencing (NGS), Western Blot (WB) -
经测试应用
适用于: WB, ICCmore details -
Biosafety level
2 -
常规说明
Recommended control: Human wild-type HEK-293 cell line (ab259776). 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
Kidney -
Cell type
epithelial -
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
May mediate the control of various cellular processes by insulin. -
序列相似性
Contains 1 IRS-type PTB domain.
Contains 1 PH domain. -
翻译后修饰
Phosphorylated upon DNA damage, probably by ATM or ATR. -
细胞定位
Cytoplasm > cytosol. - 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ab264013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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| ICC |
Use at an assay dependent concentration.
|
| 说明 |
|---|
|
WB
Use at an assay dependent concentration. |
|
ICC
Use at an assay dependent concentration. |
图片
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Western blot - Human IRS2 knockout HEK293 cell line (ab264013)All lanes : Anti-IRS2 antibody [EPR904(2)] (ab134101) at 1/1000 dilution
Lane 1 : Wild-type HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate
Lane 2 : IRS2 knockout HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate
Lane 3 : A-375 cell lysate
Lane 4 : A549 (Human lung carcinoma cell line) whole cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Observed band size: 160 kDa why is the actual band size different from the predicted?Lanes 1 - 4: Merged signal (red and green). Green - ab134101 observed at 160 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55kDa.
ab134101 was shown to react with IRS2 in wild-type HEK-293 cells in western blot with loss of signal observed in IRS2 knockout cell line ab264013 (knockout cell lysate ab264506). Wild-type and IRS2 knockout HEK-293 cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween®) before incubation with ab134101 and ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4°C at a 1 in 1000 dilution 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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Immunocytochemistry/ Immunofluorescence - Human IRS2 knockout HEK-293 cell line (ab264013)ab134101 staining IRS2 in wild-type HEK-293 cells (top panel) and IRS2 knockout HEK-293 cells (ab264013) (bottom panel). The cells were fixed with 4% paraformaldehyde (10 min) permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab134101 at 1/500 and ab7291 (Mouse monoclonal to alpha Tubulin) at 1/1000 dilution overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor® 594) (ab150120) at 2 μg/ml (shown in red). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).
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Western blot - Human IRS2 knockout HEK293 cell line (ab264013)All lanes : Anti-IRS2 antibody [EPR1650(2)] (ab133276) at 1/1000 dilution
Lane 1 : Wild-type HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate
Lane 2 : IRS2 knockout HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate
Lane 3 : A-375 cell lysate
Lane 4 : A549 (Human lung carcinoma cell line) whole cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Observed band size: 160 kDa why is the actual band size different from the predicted?Lanes 1 - 4: Merged signal (red and green). Green - ab133276 observed at 160 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55kDa.
ab133276 was shown to react with IRS2 in wild-type HEK-293 cells in western blot with loss of signal observed in IRS2 knockout cell line ab264013 (knockout cell lysate ab264506). Wild-type and IRS2 knockout HEK-293 cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween®) before incubation with ab133276 and ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4°C at a 1 in 1000 dilution 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.
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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