Human DEGS1 (MLD) knockout HEK-293T cell line (ab266481)
Overview
-
Product name
Human DEGS1 (MLD) knockout HEK-293T cell line -
Parental Cell Line
HEK293T -
Organism
Human -
Mutation description
Knockout achieved by using CRISPR/Cas9, 14 bp deletion in exon 1 and 1 bp insertion in exon 1 -
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 -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. Based on cell count, seed cells in an appropriate cell culture 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.
4. Incubate the culture at 37°C 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.
- 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.
We will provide viable cells that proliferate on revival.
Properties
-
Number of cells
1 x 106 cells/vial, 1 mL -
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 -
Mycoplasma free
Yes -
Storage instructions
Shipped on Dry Ice. Store in liquid nitrogen. -
Storage buffer
Constituents: 8.7% Dimethylsulfoxide, 2% Cellulose, methyl ether -
Research areas
Target
-
Relevance
DEGS1 is a member of the membrane fatty acid desaturase family which is responsible for inserting double bonds into specific positions in fatty acids. It contains three His containing consensus motifs that are characteristic of a group of membrane fatty acid desaturases. It has sphingolipid-delta-4-desaturase activity and converts D-erythro-sphinganine to D-erythro-sphingosine (E-sphing-4-enine). -
Cellular localization
Endoplasmic reticulum and Mitochondrial
Associated products
-
KO cell lysates
-
Related Products
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab266481 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: 38 kDa.
|
Notes |
---|
WB
Use at an assay dependent concentration. Predicted molecular weight: 38 kDa. |
Images
-
All lanes : Anti-MLD antibody [EPR9681] (ab167169) 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 : DEGS1 knockout HEK-293T (Human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate
Lane 3 : U-87 MG (Human glioblastoma-astrocytoma epithelial cell line) whole cell lysate
Lane 4 : T-47D (Human ductal breast epithelial tumor cell line) whole cell lysate
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution
Predicted band size: 38 kDa
Observed band size: 38 kDaLanes 1-4: Merged signal (red and green). Green - ab167169 observed at 38 kDa. Red - loading control ab8245 observed at 36 kDa.
ab167169 Anti-MLD antibody [EPR9681] was shown to specifically react with MLD in wild-type HEK-293T cells. Loss of signal was observed when knockout cell line ab266481 (knockout cell lysate ab257918) was used. Wild-type and MLD knockout samples were subjected to SDS-PAGE. ab167169 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.
-
All lanes : Anti-MLD antibody [EPR9680] (ab185237) 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 : DEGS1 knockout HEK-293T (Human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate
Lane 3 : U-87 MG (Human glioblastoma-astrocytoma epithelial cell line) whole cell lysate
Lane 4 : T-47D (Human ductal breast epithelial tumor cell line) whole cell lysate
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution
Predicted band size: 38 kDa
Observed band size: 38 kDaLanes 1-4: Merged signal (red and green). Green - ab185237 observed at 38 kDa. Red - loading control ab8245 observed at 36 kDa.
ab185237 Anti-MLD antibody [EPR9680] was shown to specifically react with MLD in wild-type HEK-293T cells. Loss of signal was observed when knockout cell line ab266481 (knockout cell lysate ab257918) was used. Wild-type and MLD knockout samples were subjected to SDS-PAGE. ab185237 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: 14 bp deletion in exon1
-
Allele-2: 1 bp insertion in exon 1.
Protocols
Datasheets and documents
-
SDS download
-
Datasheet download
References (1)
ab266481 has been referenced in 1 publication.
- Hayashi Y et al. N-(4-Hydroxyphenyl) Retinamide Suppresses SARS-CoV-2 Spike Protein-Mediated Cell-Cell Fusion by a Dihydroceramide ?4-Desaturase 1-Independent Mechanism. J Virol 95:e0080721 (2021). PubMed: 34106748