SNCA KO cell line available to order. KO validated by Western blot. Free of charge wild type control provided. Knockout achieved by using CRISPR/Cas9, 1 bp insertion in exon 2 and Insertion of the selection cassette in exon 2.
Alpha-synuclein, Alpha-synuclein, isoform NACP140, MGC105443, MGC110988, MGC127560, MGC64356, NACP, Non A4 component of amyloid, Non-A beta component of AD amyloid, Non-A-beta component of alzheimers disease amyloid , precursor of, Non-A4 component of amyloid precursor, Non-A4 component of amyloid, precursor of, OTTHUMP00000218549, OTTHUMP00000218551, OTTHUMP00000218552, OTTHUMP00000218553, OTTHUMP00000218554, PARK 1, PARK 4, Parkinson disease (autosomal dominant, Lewy body) 4, Parkinson disease familial 1, SNCA, SYN, SYUA_HUMAN, Snca synuclein, Snca synuclein, alpha (non A4 component of amyloid precursor), Synuclein alpha, Synuclein alpha 140, Synuclein, alpha (non A4 component of amyloid precursor), alphaSYN
SNCA KO cell line available to order. KO validated by Western blot. Free of charge wild type control provided. Knockout achieved by using CRISPR/Cas9, 1 bp insertion in exon 2 and Insertion of the selection cassette in exon 2.
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.
Alpha-synuclein often referred to by alternate names such as SNCA is a protein of around 14 kDa mass. It mainly expresses in the brain particularly in presynaptic nerve terminals. This protein functions mechanically by stabilizing synaptic vesicles and maintaining synaptic function. It exists both in soluble monomer forms and as aggregates in protein filaments. Antibodies like 4D6 and EP1536Y target monomer forms of protein for more detailed studies.
The alpha-synuclein protein plays critical roles in neuronal activity. It contributes to neurotransmitter release regulation by acting in the formation and plasticity of the presynaptic neuronal network. Alpha-synuclein doesn't usually form parts of large protein complexes but it may associate transiently with membranes and vesicular structures. The protein's monomer form has also been observed in alpha lines and related neuronal processes operating alongside various cellular functions.
Synaptic vesicle trafficking and dopamine neurotransmitter release are significant areas involving the alpha-synuclein protein. In these pathways alpha-synuclein interacts with other proteins like synaptophysin and protein monomer monomerizations are intrinsic to these processes. Altered function or aggregation of alpha-synuclein disrupts these pathways influencing broader neurological functions.
Alterations or accumulations of alpha-synuclein are strongly linked to Parkinson's disease and Lewy body dementia. In these conditions alpha-synuclein forms abnormal protein filaments known as Lewy bodies within neurons. These formations disrupt cellular processes and neuron health. Synucleinopathies such as these show connections with proteins like parkin and DJ-1 which also have key roles in these neurodegenerative diseases.
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** Lanes 1- 2:** Merged signal (red and green). Green - Anti-Alpha-synuclein antibody [MJFR1] ab138501 observed at 18 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (Anti-GAPDH antibody [6C5] - Loading Control ab8245) observed at 37 kDa.
Anti-Alpha-synuclein antibody [MJFR1] ab138501 was shown to react with SNCA in wild-type HEK-293T cells in western blot. Loss of signal was observed when knockout cell line ab255433 (knockout cell lysate Human SNCA (Alpha-synuclein) knockout HEK-293T cell lysate ab263769) was used. Wild-type HEK-293T and SNCA knockout HEK-293T cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. Anti-Alpha-synuclein antibody [MJFR1] ab138501 and Anti-GAPDH antibody [6C5] - Loading Control (Anti-GAPDH antibody [6C5] - Loading Control ab8245) overnight at 4°C at a 1 in 10000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye®800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye®680RD) preadsorbed (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.
Lanes 1 - 2: Western blot - Anti-Alpha-synuclein antibody [MJFR1] (Anti-Alpha-synuclein antibody [MJFR1] ab138501) at 1/10000 dilution
Lanes 1 - 2: Anti-Alpha-synuclein antibody [MJFR1] - BSA and Azide free (ab156369) at 1/10000 dilution
Lane 1: Wild-type HEK-293T cell lysate at 20 µg
Lane 2: SNCA knockout HEK-293T cell lysate at 20 µg
Lane 2: Western blot - Human SNCA (Alpha-synuclein) knockout HEK-293T cell line (ab255433)
Performed under reducing conditions.
Predicted band size: 14 kDa
Observed band size: 18 kDa
Allele-1: Insertion of the selection cassette in exon2
Allele-2: 1 bp insertion in exon 2.
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