Human MGMT knockout HCT116 cell line
- Advanced Validation
- What is this?
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Human MGMT knockout HCT116 cell line available to order. Although we aim to provide customers with a homozygous clone, feasibility will be dependent on the biology of the protein.
View Alternative Names
6-O-methylguanine-DNA methyltransferase, AI267024, Agat, EC 2.1.1.63, MGC107020, MGMT_HUMAN, Methylated-DNA--protein-cysteine methyltransferase, Methylguanine DNA methyltransferase, O-6-methylguanine-DNA methyltransferase, O-6-methylguanine-DNA-alkyltransferase
- WB
Lab
Western blot - Human MGMT knockout HCT116 cell line (AB286541)
Western blot : Anti-MGMT antibody [MT3.1] (ab39253) staining at 1/500 dilution, shown in green; Rabbit anti-alpha Tubulin antibody [EP1332Y] (ab52866) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab39253 was shown to bind specifically to MGMT. A band was observed at 22 kDa in wild-type HCT 116 cell lysates with no signal observed at this size in MGMT knockout cell line ab286541. The band observed in the knockout lysate lane below 22 kDa is likely to represent a truncated form of MGMT. 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 MGMT knockout HCT 116 cell lysates were analysed.First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % 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-Mouse IgG H&L 800CW and Goat anti-Rabbit IgG H&L 680RD at 1/20000 dilution.
All lanes:
Western blot - Anti-MGMT antibody [MT3.1] (<a href='/en-us/products/primary-antibodies/mgmt-antibody-mt31-ab39253'>ab39253</a>) at 1/500 dilution
Lane 1:
Wild-type HCT 116 cell lysate at 20 µg
Lane 2:
Western blot - Human MGMT knockout HCT116 cell line (ab286541)
Lane 2:
MGMT knockout HCT 116 cell lysate at 20 µg
Secondary
Lanes 1 - 2:
Goat anti-Mouse IgG H&L 800CW at 1/20000 dilution
Lanes 1 - 2:
Goat anti-Rabbit IgG H&L 680RD at 1/20000 dilution
false
- WB
Lab
Western blot - Human MGMT knockout HCT116 cell line (AB286541)
Western blot : Anti-MGMT antibody [EPR4398] (ab108989) staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab108989 was shown to bind specifically to MGMT. A band was observed at 22 kDa in wild-type HCT 116 cell lysates with no signal observed at this size in MGMT CRISPR-Cas9 edited cell line ab286541. The band observed in the CRISPR-Cas9 edited lysate lane below 22 kDa is likely to represent a truncated form of MGMT. 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 MGMT CRISPR-Cas9 edited HCT 116 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % 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.
All lanes:
Western blot - Anti-MGMT antibody [EPR4398] (<a href='/en-us/products/primary-antibodies/mgmt-antibody-epr4398-ab108989'>ab108989</a>) at 1/1000 dilution
Lane 1:
Wild-type HCT 116 cell lysate at 20 µg
Lane 2:
Western blot - Human MGMT knockout HCT116 cell line (ab286541)
Lane 2:
MGMT CRISPR-Cas9 edited HCT 116 cell lysate at 20 µg
Secondary
Lanes 1 - 2:
Goat anti-Rabbit IgG H&L 800CW at 1/20000 dilution
Lanes 1 - 2:
Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution
false
- WB
Lab
Western blot - Human MGMT knockout HCT116 cell line (AB286541)
Western blot : Anti-MGMT antibody [EPR4397] (ab108630) staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab108630 was shown to bind specifically to MGMT. A band was observed at 22 kDa in wild-type HCT 116 cell lysates with no signal observed at this size in MGMT CRISPR-Cas9 edited cell line ab286541. The band observed in the CRISPR-Cas9 edited lysate lane below 22 kDa is likely to represent a truncated form of MGMT. 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 MGMT CRISPR-Cas9 edited HCT 116 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % 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.
All lanes:
Western blot - Anti-MGMT antibody [EPR4397] (<a href='/en-us/products/primary-antibodies/mgmt-antibody-epr4397-ab108630'>ab108630</a>) at 1/1000 dilution
Lane 1:
Wild-type HCT 116 cell lysate at 20 µg
Lane 2:
Western blot - Human MGMT knockout HCT116 cell line (ab286541)
Lane 2:
MGMT CRISPR-Cas9 edited HCT 116 cell lysate at 20 µg
Secondary
Lanes 1 - 2:
Goat anti-Rabbit IgG H&L 800CW at 1/20000 dilution
Lanes 1 - 2:
Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution
false
- NGS
Supplier Data
Next Generation Sequencing - Human MGMT knockout HCT116 cell line (AB286541)
92 bp deletion after Ala81
Reactivity data
Product details
Although we aim to provide customers with a homozygous clone, feasibility will be dependent on the biology of the protein. Should only heterozygous edits be achieved, you will be notified of the outcome and be asked to confirm whether the cell line is acceptable. All clones will be accompanied with DNA sequencing data, and the mutation description.
Recommended control: Human wild-type HCT116 cell line (ab288559). 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.
We will provide viable cells that proliferate on revival.
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.
What's included?
Properties and storage information
Gene name
Gene editing type
Gene editing method
Knockout validation
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
McCoY5a + 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
MGMT prevents mutations by reversing alkylation damage playing a significant role in preserving DNA integrity. It acts independently and does not form part of any larger protein complexes. This enzyme-mediated repair is central to cellular defense against mutagenic threats such as alkylating agents. By curbing DNA damage MGMT can thwart the onset of harmful genetic mutations that might lead to issues like cancer development.
Pathways
MGMT is intimately involved in DNA damage response and repair pathways. Notably it interacts with base excision repair and direct reversal repair pathways. MGMT's function is distinct but complementary to the activities of other DNA repair proteins like XRCC1 and APE1 which are involved in the base excision repair pathway. The presence of MGMT bolsters cellular resilience by ensuring that DNA lesions caused by alkylating agents are addressed promptly and effectively.
Quality control
STR analysis
CSF1PO, D13S317, D7S820, D5S818, TH01, D16S539, TPOX
Cell culture
Biosafety level
EU: 1 US: 1
Adherent/suspension
Adherent
Gender
Male
Product protocols
- Visit the General protocols
- Visit the Troubleshooting
Target data
Product promise
Please note: All products are 'FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES'.
For licensing inquiries, please contact partnerships@abcam.com