Human COMT knockout HEK-293T cell line (ab266537)
Overview
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Product name
Human COMT knockout HEK-293T cell line -
Parental Cell Line
HEK293T -
Organism
Human -
Mutation description
Knockout achieved by using CRISPR/Cas9, Homozygous: 1 bp insertion in exon 3 -
Passage number
<20 -
Knockout validation
Sanger Sequencing, Western Blot (WB) -
Tested applications
Suitable for: WBmore details -
Biosafety level
2 -
General notes
Recommended control: Human wild-type HEK293T cell line (ab255449). 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.
Properties
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Number of cells
1 x 106 cells/vial, 1 mL -
Viability
~80% -
Adherent /Suspension
Adherent -
Tissue
Kidney -
Cell type
epithelial -
STR Analysis
Amelogenin X D5S818: 8, 9 D13S317: 12, 14 D7S820: 11 D16S539: 9, 13 vWA: 16, 19 TH01: 7, 9.3 TPOX: 11 CSF1PO: 11, 12 -
Antibiotic resistance
Puromycin 1.00µg/ml -
Mycoplasma free
Yes -
Storage instructions
Shipped on Dry Ice. Store in liquid nitrogen. -
Storage buffer
Constituents: 8.7% Dimethylsulfoxide, 2% Cellulose, methyl ether -
Research areas
Target
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Function
Catalyzes the O-methylation, and thereby the inactivation, of catecholamine neurotransmitters and catechol hormones. Also shortens the biological half-lives of certain neuroactive drugs, like L-DOPA, alpha-methyl DOPA and isoproterenol. -
Tissue specificity
Brain, liver, placenta, lymphocytes and erythrocytes. -
Sequence similarities
Belongs to the mammalian catechol-O-methyltransferase family. -
Post-translational
modificationsThe N-terminus is blocked. -
Cellular localization
Cytoplasm and Cell membrane. - Information by UniProt
Associated products
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KO cell lysates
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Related Products
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab266537 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Application | Abreviews | Notes |
---|---|---|
WB |
Use at an assay dependent concentration. Predicted molecular weight: 30 kDa.
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Notes |
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WB
Use at an assay dependent concentration. Predicted molecular weight: 30 kDa. |
Images
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All lanes : Anti-COMT antibody [EPR6490] (ab126618) at 1/1000 dilution
Lane 1 : Wild-type HEK-293T cell lysate
Lane 2 : COMT knockout HEK-293T cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 30 kDa
Observed band size: 24-28 kDa why is the actual band size different from the predicted?Lanes 1-2: Merged signal (red and green). Green - ab126618 observed at 24-28 kDa. Red - loading control ab8245 observed at 37 kDa.
ab126618 Anti-COMT antibody [EPR6490] was shown to specifically react with COMT in wild-type HEK-293T cells. Loss of signal was observed when knockout cell line ab266537 (knockout cell lysate ab257396) was used. Wild-type and COMT knockout samples were subjected to SDS-PAGE. ab126618 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 20000 dilution for 1 hour at room temperature before imaging.
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All lanes : Anti-COMT antibody [EPR6491(B)] (ab124813) at 1/1000 dilution
Lane 1 : Wild-type HEK-293T cell lysate
Lane 2 : COMT knockout HEK-293T cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 30 kDa
Observed band size: 28 kDa why is the actual band size different from the predicted?Lanes 1-2: Merged signal (red and green). Green - ab124813 observed at 28 kDa. Red - loading control ab8245 observed at 37 kDa.
ab124813 Anti-COMT antibody [EPR6491(B)] was shown to specifically react with COMT in wild-type HEK-293T cells. Loss of signal was observed when knockout cell line ab266537 (knockout cell lysate ab257396) was used. Wild-type and COMT knockout samples were subjected to SDS-PAGE. ab124813 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 20000 dilution for 1 hour at room temperature before imaging.
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Homozygous: 1 bp insertion in exon 3
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Representative images of COMT 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.
Protocols
Datasheets and documents
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SDS download
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Datasheet download
References (0)
ab266537 has not yet been referenced specifically in any publications.