Product nameHuman EIF2AK4 (GCN2) knockout HEK293T cell line
See all GCN2 lysates
Parental Cell LineHEK293T
Mutation descriptionKnockout achieved by using CRISPR/Cas9, 1 bp deletion in exon 9 and 1 bp insertion in exon 9
Knockout validationSanger Sequencing, Western Blot (WB)
Tested applicationsSuitable for: WBmore details
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.
- 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.
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.
Number of cells1 x 106 cells/vial, 1 mL
STR AnalysisAmelogenin 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 resistancePuromycin 1.00µg/ml
Storage instructionsShipped on Dry Ice. Store in liquid nitrogen.
Storage bufferConstituents: 8.7% DMSO, 2% Cellulose, methyl ether
PurityImmunogen affinity purified
FunctionCan phosphorylate the alpha subunit of EIF2 and may mediate translational control.
Tissue specificityWidely expressed.
Sequence similaritiesBelongs to the protein kinase superfamily. Ser/Thr protein kinase family. GCN2 subfamily.
Contains 2 protein kinase domains.
Contains 1 RWD domain.
DomainKinase domain 1 is a degenerate kinase domain.
RWD domain is reported to interact with GCN1L1.
modificationsAutophosphorylated on threonine residues.
- Information by UniProt
KO cell lysates
Our Abpromise guarantee covers the use of ab267247 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|WB||Use at an assay dependent concentration. Predicted molecular weight: 187 kDa.|
Allel-1: 1 bp deletion in exon9
All lanes : Anti-GCN2 antibody [EPR5970(2)] (ab134053) at 1/1000 dilution
Lane 1 : Wild-type HEK-293T cell lysate
Lane 2 : EIF2AK4 knockout HEK-293T cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 187 kDa
Observed band size: 187 kDa
ab134053 was shown to react with GCN2 in wild-type HEK-293T cells in western blot. Loss of signal was observed when knockout cell line ab267247 (knockout cell lysate ab256903) was used. Wild-type HEK-293T and EIF2AK4 knockout HEK-293T cell lysates were subjected to SDS-PAGE. ab134053 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) overnight at 4°C at a 1 in 1000 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.
Allel-2: 1 bp insertion in exon 9.
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.
ab267247 has not yet been referenced specifically in any publications.