Product nameHuman ULK3 knockout HEK293T cell line
Parental Cell LineHEK293T
Mutation descriptionKnockout achieved by using CRISPR/Cas9, 1 bp deletion in exon 1 and 1 bp insertion in exon 1
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
Storage instructionsShipped on Dry Ice. Store in liquid nitrogen.
Storage bufferConstituents: 8.7% DMSO, 2% Cellulose, methyl ether
PurityImmunogen affinity purified
FunctionSerine/threonine protein kinase which enhances GLI1 and GLI2 transcriptional activity and consequently positively regulates GLI-dependent SHH signaling. May exert this function by promoting GLI1 nuclear localization. Phosphorylates in vitro GLI2, as well as GLI1 and GLI3, although less efficiently.
Tissue specificityWidely expressed. Highest levels observed in fetal brain. In adult tissues, high levels in brain, liver and kidney, moderate levels in testis and adrenal gland and low levels in heart, lung, stomach, thymus, prostate and placenta. In the brain, highest expression in the hippocampus, high levels also detected in the cerebellum, olfactory bulb and optice nerve. In the central nervous system, lowest levels in the spinal cord.
Sequence similaritiesBelongs to the protein kinase superfamily. Ser/Thr protein kinase family. APG1/unc-51/ULK1 subfamily.
Contains 2 MIT domains.
Contains 1 protein kinase domain.
modificationsAutophosphorylated in vitro.
- Information by UniProt
FormThere are 3 isoforms produced by alternative splicing.
KO cell lysates
Our Abpromise guarantee covers the use of ab266152 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: 53 kDa.|
All lanes : Anti-ULK3 antibody [EPR4888] (ab124947) at 1/1000 dilution
Lane 1 : Wild-type HEK-293T (Human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate
Lane 2 : ULK3 knockout HEK-293T (Human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate
Lane 3 : HAP1 whole cell lyate
Lane 4 : LNCaP (Human prostate cancer cell line) whole cell lysate
Lysates/proteins at 20 µg per lane.
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution
Predicted band size: 53 kDa
Observed band size: 55 kDa why is the actual band size different from the predicted?
ab124947 Anti-ULK3 antibody [EPR4888] was shown to specifically react with ULK3 in wild-type HEK-293T cells. Loss of signal was observed when knockout cell line ab266152 (knockout cell lysate ab258271) was used. Wild-type and ULK3 knockout samples were subjected to SDS-PAGE. ab124947 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.
Allele-1: 1 bp deletion in exon1
Allele-2: 1 bp insertion in exon 1.
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.
ab266152 has not yet been referenced specifically in any publications.