Human COMT knockout HEK-293T cell line
- Advanced Validation
- What is this?
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COMT KO cell line available to order. KO validated by Western blot. Free of charge wild type control available. Knockout achieved by using CRISPR/Cas9, Homozygous: 1 bp insertion in exon 3. To order both knockout and wild-type control cells: select 2 x 1000000Cells/vial. To order only knockout cells: select 1000000Cells/vial.
View Alternative Names
COMT_HUMAN, Catechol O-methyltransferase, EC 2.1.1.6
- WB
Lab
Western blot - Human COMT knockout HEK-293T cell line (AB266537)
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° 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.
All lanes:
Western blot - Anti-COMT antibody [EPR6491(B)] (<a href='/en-us/products/primary-antibodies/comt-antibody-epr6491b-ab124813'>ab124813</a>) at 1/1000 dilution
Lane 1:
Wild-type HEK-293T cell lysate at 20 µg
Lane 2:
COMT knockout HEK-293T cell lysate at 20 µg
Lane 2:
Western blot - Human COMT knockout HEK-293T cell line (ab266537)
Predicted band size: 30 kDa
Observed band size: 28 kDa
false
- WB
Lab
Western blot - Human COMT knockout HEK-293T cell line (AB266537)
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° 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.
All lanes:
Western blot - Anti-COMT antibody [EPR6490] (<a href='/en-us/products/primary-antibodies/comt-antibody-epr6490-ab126618'>ab126618</a>) at 1/1000 dilution
Lane 1:
Wild-type HEK-293T cell lysate at 20 µg
Lane 2:
COMT knockout HEK-293T cell lysate at 20 µg
Lane 2:
Western blot - Human COMT knockout HEK-293T cell line (ab266537)
Predicted band size: 30 kDa
Observed band size: 24-28 kDa
false
- Cell Culture
Unknown
Cell Culture - Human COMT knockout HEK-293T cell line (AB266537)
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.
- Sanger seq
Unknown
Sanger Sequencing - Human COMT knockout HEK-293T cell line (AB266537)
Homozygous : 1 bp insertion in exon 3
- ICC/IF
Supplier Data
Immunocytochemistry/ Immunofluorescence - Human COMT knockout HEK-293T cell line (AB266537)
Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized COMT KO HEK293T ( COMT knockout human embryonic kidney epithelial cell) (ab266537) cells labelling COMT with ab321813 at 1/50 (10.46 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2 ug/ml dilution (Green).
Confocal image showing cytoplasmic staining in parental HEK293 cell line (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).
ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Magenta). The Nuclear counterstain was DAPI (Blue).
Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/ml dilution.
Reactivity data
Product details
We will provide viable cells that proliferate on revival.
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.
What's included?
Properties and storage information
Gene name
Gene editing type
Gene editing method
Knockout validation
Zygosity
Shipped at conditions
Appropriate short-term storage conditions
Appropriate long-term storage conditions
Handling procedures
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 seed all remaining cells into a T25.
4. Incubate the culture at 37°C incubator with 5% CO2. Check the culture one day after revival and continue to check until 80% confluent. Media change can be given if needed.
5. Once confluent passage into an appropriate 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. 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.
- Cells should be passaged when they have achieved 80-90% confluence.
Culture medium
DMEM (High Glucose) + 10% FBS
Cryopreservation medium
Cell Freezing Medium-DMSO Serum free media, contains 8.7% DMSO in MEM supplemented with methyl cellulose.
Supplementary information
This supplementary information is collated from multiple sources and compiled automatically.
Biological function summary
Catechol-O-methyltransferase serves as a regulator of neurotransmitter levels in the central nervous system and peripheral tissues. It does not appear to form part of a larger protein complex functioning mainly as a singular entity for metabolizing catechols. Through its action COMT impacts processes such as mood regulation cognition and stress response. Its role in degrading dopamine is especially significant in regions like the prefrontal cortex where dopamine plays an important role in executive functions.
Pathways
COMT participates in the catecholamine degradation pathway and impacts dopaminergic signaling. It works alongside enzymes such as monoamine oxidase (MAO) to regulate levels of neurotransmitters. COMT's activity influences the synaptic presence of dopamine and its interaction in signaling pathways. Moreover it indirectly affects the balance within the dopaminergic system which is important for normal neurological function and behavior.
Quality control
STR analysis
CSF1PO, D13S317, D7S820, D5S818, TH01, D16S539, TPOX
Cell culture
Biosafety level
EU: 2 US: 2
Adherent/suspension
Adherent
Gender
Female
Complementary Products
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Product promise
Please note: All products are 'FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES'.
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