人CBS knockout HeLa cell line (ab264950)
概述
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产品名称
人CBS knockout HeLa cell line -
Parental Cell Line
HeLa -
Organism
Human -
Mutation description
Knockout achieved by using CRISPR/Cas9, 11 bp deletion in exon 5 and 1 bp insertion in exon 5 -
Passage number
<20 -
Knockout validation
Sanger Sequencing, Western Blot (WB) -
经测试应用
适用于: WBmore details -
Biosafety level
2 -
常规说明
Recommended control: Human wild-type HeLa cell line (ab255448). 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
Cervix -
Cell type
epithelial -
Disease
Adenocarcinoma -
Gender
Female -
STR Analysis
Amelogenin X D5S818: 11, 12 D13S317: 12, 13.3 D7S820: 8, 12 D16S539: 9, 10 vWA: 16, 18 TH01: 7 TPOX: 8, 12 CSF1PO: 9, 10 -
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
Only known pyridoxal phosphate-dependent enzyme that contains heme. Important regulator of hydrogen sulfide, especially in the brain, utilizing cysteine instead of serine to catalyze the formation of hydrogen sulfide. Hydrogen sulfide is a gastratransmitter with signaling and cytoprotective effects such as acting as a neuromodulator in the brain to protect neurons against hypoxic injury. -
组织特异性
In the adult strongly expressed in liver and pancreas, some expression in heart and brain, weak expression in lung and kidney. In the fetus, expressed in brain, liver and kidney. -
通路
Amino-acid biosynthesis; L-cysteine biosynthesis; L-cysteine from L-homocysteine and L-serine: step 1/2. -
疾病相关
Defects in CBS are the cause of cystathionine beta-synthase deficiency (CBSD) [MIM:236200]. CBSD is an enzymatic deficiency resulting in altered sulfur metabolism and homocystinuria. The clinical features of untreated homocystinuria due to CBS deficiency include myopia, ectopia lentis, mental retardation, skeletal anomalies resembling Marfan syndrome, and thromboembolic events. Light skin and hair can also be present. Biochemical features include increased urinary homocystine and methionine. -
序列相似性
Belongs to the cysteine synthase/cystathionine beta-synthase family.
Contains 1 CBS domain. -
细胞定位
Cytoplasm. Nucleus. - 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ab264950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: 61 kDa.
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说明 |
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WB
Use at an assay dependent concentration. Predicted molecular weight: 61 kDa. |
图片
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All lanes : Anti-CBS antibody [EPR8579] (ab140600) at 1/1000 dilution
Lane 1 : Wild-type HeLa cell lysate
Lane 2 : CBS knockout HeLa cell lysate
Lane 3 : Raji cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 61 kDa
Observed band size: 70 kDa why is the actual band size different from the predicted?Lanes 1-3: Merged signal (red and green). Green - ab140600 observed at 70 kDa. Red - loading control, ab8245 observed at 37 kDa.
ab140600 Anti-CBS antibody [EPR8579] was shown to specifically react with CBS in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab264950 (knockout cell lysate ab257203) was used. Wild-type and CBS knockout samples were subjected to SDS-PAGE. ab140600 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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All lanes : Anti-CBS antibody [EPR8578] (ab131155) at 1/1000 dilution
Lane 1 : Wild-type HeLa cell lysate
Lane 2 : CBS knockout HeLa cell lysate
Lane 3 : Raji cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 61 kDa
Observed band size: 70 kDa why is the actual band size different from the predicted?Lanes 1-3: Merged signal (red and green). Green - ab131155 observed at 70 kDa. Red - loading control, ab8245 observed at 37 kDa.
ab131155 Anti-CBS antibody [EPR8578] was shown to specifically react with CBS in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab264950 (knockout cell lysate ab257203) was used. Wild-type and CBS knockout samples were subjected to SDS-PAGE. ab131155 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: 11 bp deletion in exon 5.
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Allele-2: 1 bp insertion in exon 5.
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Representative images of CBS knockout HeLa cells, low and high confluency examples (top left and right respectively) and wild-type HeLa 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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