APP KO cell line available to order. KO validated by Immunocytochemistry, Western blot. Free of charge wild type control provided. Knockout achieved by using CRISPR/Cas9, Homozygous: 1 bp insertion in exon 5.
A BETA, A4 amyloid protein, A4_HUMAN, AAA, ABPP, AD1, AICD-50, AICD-57, AICD-59, AID(50), AID(57), AID(59), APP, APPI, Alzheimer disease amyloid protein, Amyloid beta (A4) precursor protein, Amyloid beta A4 protein, Amyloid beta protein, Amyloid intracellular domain 50, Amyloid intracellular domain 57, Amyloid intracellular domain 59, Amyloid precursor protein, Beta-APP40, Beta-APP42, Beta-amyloid precursor protein, C31, CTFgamma, CVAP, Cerebral vascular amyloid peptide, Gamma-CTF(50), Gamma-CTF(57), Gamma-CTF(59), PN 2, PN-II, PreA4, Protease nexin-II, S-APP-alpha, S-APP-beta, beta-amyloid peptide, peptidase nexin-II, sAPP
APP KO cell line available to order. KO validated by Immunocytochemistry, Western blot. Free of charge wild type control provided. Knockout achieved by using CRISPR/Cas9, Homozygous: 1 bp insertion in exon 5.
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.
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.
The Amyloid Precursor Protein (APP) also known as amyloid protein is a transmembrane protein that is approximately 695 to 770 amino acids in length depending on the isoform. The molecular mass of APP can vary but typically falls around 100 to 140 kDa. It is heavily expressed in the central nervous system particularly in neurons but also in other tissues like muscle and kidney. The APP undergoes proteolytic processing which leads to the generation of various fragments including beta-amyloid peptides.
The processing of APP plays a fundamental role in neuronal growth survival and repair. APP is cleaved into fragments that can regulate synaptic function and plasticity. It does not operate as a part of a complex but interacts with various cellular components. The protein participates in signaling pathways influencing cellular adhesion motility and neurite outgrowth. APP’s numerous interaction partners facilitate its involvement in different cellular processes highlighting its critical role in normal cell function.
The APP is a central component in the amyloidogenic pathway where its cleavage by beta-secretase and gamma-secretase yields beta-amyloid. This pathway is one of two primary metabolic routes for APP—alternative enzymatic processing through the non-amyloidogenic pathway precludes beta-amyloid formation releasing peptides that do not aggregate. Enzymes like BACE1 (beta-secretase 1) and presenilin are important in the amyloidogenic pathway directly resulting in the production of the neurotoxic amyloid beta-peptide.
APP is intensely linked to Alzheimer's disease and cerebral amyloid angiopathy. Accumulation of beta-amyloid peptides formed from APP cleavage is a hallmark of Alzheimer's disease leading to plaque formation in the brain. This aggregation impacts neuronal function and is associated with neurodegenerative processes. Interactions with proteins like tau are significant as tau also plays an essential role in Alzheimer's disease pathology. Misprocessing of APP and the resulting beta-amyloid aggregates are also contributors to cerebral amyloid angiopathy where deposits within cerebrovascular walls compromise vascular integrity.
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Lanes 2 and 6: SH-SY5Y cell lysate (20 µg)
Lanes 3 and 7: Wild-type HEK-293T cell lysate (20 µg)
Lanes 4 and 8: APP knockout HEK-293T cell lysate (20 µg)
ab12266 was shown to react with Amyloid Precursor Protein in wild-type HEK-293T cells. Loss of signal was observed when knockout cell line ab255362 (knockout cell lysate Human APP (Amyloid Precursor Protein) knockout HEK-293T cell lysate ab263777) was used. Wild-type and Amyloid Precursor Protein knockout samples were subjected to SDS-PAGE. ab12266 and Anti-GAPDH antibody [EPR16891] - Loading Control (Anti-GAPDH antibody [EPR16891] - Loading Control ab181602) were incubated overnight at 4°C at 1 in 500 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed ab216772) and Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed (Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed ab216777) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.
All lanes: Anti-Amyloid Precursor Protein antibody [DE2B4] (ab12266) at 1/500 dilution
Lane 1: Wild-type HeLa cell lysate at 20 µg
Lane 2: SH-SY5Y cell lysate at 20 µg
Lane 3: Wild-type HEK-293T cell lysate at 20 µg
Lane 4: APP knockout HEK-293T cell lysate at 20 µg
Lane 4: Western blot - Human APP (Amyloid Precursor Protein) knockout HEK-293T cell line (ab255362)
All lanes: Western blot - Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed ab216772) at 1/20000 dilution
Performed under reducing conditions.
Anti-Amyloid Precursor Protein antibody [Y188] ab32136 staining Amyloid Precursor Protein in wild-type HEK293 cells (top panel) and APP knockout HEK293 cells (ab255362) (bottom panel). The cells were fixed with 100% methanol (5 min) then permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with Anti-Amyloid Precursor Protein antibody [Y188] ab32136 at 1/500 dilution and Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (Mouse monoclonal to alpha Tubulin) at 1/1000 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor® 488) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor® 594) (Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120) at 2 μg/ml (shown in red). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8).
Homozygous: 1 bp insertion in exon 5
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