Human NUDT1 (MTH1) knockout HEK293T cell line (ab266400)
Overview
-
Product name
Human NUDT1 (MTH1) knockout HEK293T cell line
See all MTH1 lysates -
Parental Cell Line
HEK293T -
Organism
Human -
Mutation description
Knockout achieved by using CRISPR/Cas9, Homozygous: 1 bp deletion 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 -80ºC. Storage at -80ºC may result in loss of viability.
1. Thaw the vial in 37ºC water bath 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 (Click here to view haemocytometer protocol) or alternative cell counting method. Based on cell count, seed cells in an appropriate cell culture flask at a density of 2x104 cells/cm2. This should allow for confluency within 48 hours. Seeding density is given as a guide only and should be scaled to align with individual lab schedules.
4. Incubate the culture at 37ºC 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 for confluency (80-90% confluence) within 48 hours.
- 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.
Click here to view the Mammalian cell tissue culture protocol
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.
Properties
-
Number of cells
1 x 106 cells/vial, 1 mL -
Viability
~90% -
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% DMSO, 2% Cellulose, methyl ether -
Research areas
Target
-
Function
Antimutagenic. Acts as a sanitizing enzyme for oxidized nucleotide pools, thus suppressing cell dysfunction and death induced by oxidative stress. Hydrolyzes 8-oxo-dGTP, 8-oxo-dATP and 2-OH-dATP, thus preventing misincorporation of oxidized purine nucleoside triphosphates into DNA and subsequently preventing A:T to C:G and G:C to T:A transversions. Able to hydrolyze also the corresponding ribonucleotides, 2-OH-ATP, 8-oxo-GTP and 8-oxo-ATP. Does not play a role in U8 snoRNA decapping activity. Binds U8 snoRNA. -
Tissue specificity
Widely expressed with highest expression in thymus, testis, embryo and proliferating blood lymphocytes. -
Sequence similarities
Belongs to the Nudix hydrolase family.
Contains 1 nudix hydrolase domain. -
Developmental stage
In peripheral blood lymphocytes, expressed at much higher levels in proliferating cells than in resting cells. -
Post-translational
modificationsThe N-terminus is blocked. -
Cellular localization
Cytoplasm. Mitochondrion matrix and Cytoplasm. Mitochondrion matrix. Nucleus. Mostly present in cytoplasm. Variant Met-124 has decreased efficiency in translocation to mitochondria. - Information by UniProt
Associated products
-
KO cell lysates
-
Related Products
Applications
Our Abpromise guarantee covers the use of ab266400 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: 23 kDa. |
Images
-
All lanes : Anti-MTH1 antibody [EPR15934-50] (ab200832) at 1/5000 dilution
Lane 1 : Wild-type HEK-293T cell lysate
Lane 2 : NUDT1 knockout HEK-293T cell lysate
Lane 3 : HAP1 cell lysate
Lane 4 : HeLa cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 23 kDa
Observed band size: 18 kDa why is the actual band size different from the predicted?Lanes 1- 4: Merged signal (red and green). Green - ab200832 observed at 18 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.
ab200832 was shown to react with MTH1 in wild-type HEK-293T cells in western blot. Loss of signal was observed when knockout cell line ab266400 (knockout cell lysate ab257565) was used. Wild-type HEK-293T and NUDT1 knockout HEK-293T cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab200832 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 5000 dilution and a 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 : Anti-MTH1 antibody [EPR15934] (ab197028) at 1/2000 dilution
Lane 1 : Wild-type HEK-293T cell lysate
Lane 2 : NUDT1 knockout HEK-293T cell lysate
Lane 3 : HAP1 cell lysate
Lane 4 : HeLa cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 23 kDa
Observed band size: 18 kDa why is the actual band size different from the predicted?Lanes 1- 4: Merged signal (red and green). Green - ab197028 observed at 18 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.
ab197028 was shown to react with MTH1 in wild-type HEK-293T cells in western blot. Loss of signal was observed when knockout cell line ab266400 (knockout cell lysate ab257565) was used. Wild-type HEK-293T and NUDT1 knockout HEK-293T cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab197028 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 2000 dilution and a 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.
-
Homozygous: 1 bp deletion in exon 3
-
Representative images NUDT1 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 M5000 microscope.
Protocols
To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.
References (0)
ab266400 has not yet been referenced specifically in any publications.