Human NCSTN (Nicastrin) knockout HEK-293T cell line
- Advanced Validation
- What is this?
Be the first to review this product! Submit a review
|
(0 Publication)
NCSTN KO cell line available to order. KO validated by Western blot. Free of charge wild type control provided. Knockout achieved by using CRISPR/Cas9, Homozygous: 1 bp insertion in exon 1.
View Alternative Names
APH 2, ATAG1874, Anterior pharynx defective 2, KIAA0253, NCT, NICA_HUMAN, Ncstn, Nicastrin, RP11 517F10.1, RP11517F101
- WB
Lab
Western blot - Human NCSTN (Nicastrin) knockout HEK-293T cell line (AB266602)
Lanes 1-4 : Merged signal (red and green). Green - ab68145 observed at 100-125 kDa. Red - loading control ab8245 observed at 36 kDa.
ab68145 Anti-Nicastrin antibody [EPR2575Y] was shown to specifically react with Nicastrin in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab266602 (knockout cell lysate ab258061) was used. Wild-type and Nicastrin knockout samples were subjected to SDS-PAGE. ab68145 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:
Western blot - Anti-Nicastrin antibody [EPR2575Y] (<a href='/en-us/products/primary-antibodies/nicastrin-antibody-epr2575y-ab68145'>ab68145</a>) at 1/1000 dilution
Lane 1:
Wild-type HEK293T cell lysate at 20 µg
Lane 2:
NCSTN knockout HEK293T cell lysate at 20 µg
Lane 2:
Western blot - Human NCSTN (Nicastrin) knockout HEK-293T cell line (ab266602)
Lane 3:
HeLa cell lysate at 20 µg
Lane 4:
HL-60 cell lysate at 20 µg
Secondary
All lanes:
Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/10000 dilution
Predicted band size: 78 kDa
Observed band size: 100-125 kDa
false
- Cell Culture
Unknown
Cell Culture - Human NCSTN (Nicastrin) knockout HEK-293T cell line (AB266602)
Representative images of NCSTN 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 an EVOS M5000 microscope.
- Sanger seq
Unknown
Sanger Sequencing - Human NCSTN (Nicastrin) knockout HEK-293T cell line (AB266602)
Homozygous : 1 bp insertion in exon 1
Reactivity data
Product 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.
We will provide viable cells that proliferate on revival.
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.
What's included?
Properties and storage information
Gene name
Gene editing type
Gene editing method
Knockout validation
Zygosity
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
DMEM (High Glucose) + 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
Nicastrin functions as a part of the gamma-secretase complex which also includes presenilin anterior pharynx defective 1 (APH-1) and presenilin enhancer 2 (PEN-2). This complex is essential for intramembrane proteolysis of type I membrane proteins such as amyloid precursor protein (APP) and Notch. The activity of nicastrin influences processes like synaptic organization and neuronal development.
Pathways
Gamma-secretase activity facilitated by nicastrin is central in the Notch signaling pathway and the amyloidogenic pathway. In the Notch pathway it helps release the Notch intracellular domain important for T-cell development. In the amyloidogenic pathway the complex processes APP contributing to amyloid-beta peptide formation. Beta-secretase (BACE1) also plays an important role in this pathway acting upstream of gamma-secretase.
Quality control
STR analysis
CSF1PO, D13S317, D7S820, D5S818, TH01, D16S539, TPOX
Cell culture
Biosafety level
EU: 2 US: 2
Adherent/suspension
Adherent
Gender
Female
Target data
Complementary Products
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