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SNCA KO cell line available to order. Free of charge wild type control provided. Knockout achieved by using CRISPR/Cas9, Homozygous: 42 bp deletion in exon 2 and intron 2-3, destroy donor splicing site and lead to stop codon.

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Images

Western blot - Human SNCA (Alpha-synuclein) knockout U-87 MG cell line (AB282333), expandable thumbnail
  • Sanger Sequencing - Human SNCA (Alpha-synuclein) knockout U-87 MG cell line (AB282333), expandable thumbnail

Key facts

Cell type

U-87 MG

Species or organism

Human

Tissue

Brain

Form

Liquid

Knockout validation

Sanger Sequencing

Mutation description

Knockout achieved by using CRISPR/Cas9, Homozygous: 42 bp deletion in exon 2 and intron 2-3, destroy donor splicing site and lead to stop codon

Alternative names

Recommended products

SNCA KO cell line available to order. Free of charge wild type control provided. Knockout achieved by using CRISPR/Cas9, Homozygous: 42 bp deletion in exon 2 and intron 2-3, destroy donor splicing site and lead to stop codon.

Key facts

Cell type

U-87 MG

Form

Liquid

Mutation description

Knockout achieved by using CRISPR/Cas9, Homozygous: 42 bp deletion in exon 2 and intron 2-3, destroy donor splicing site and lead to stop codon

Disease

Glioblastoma

Concentration
Loading...

Properties

Gene name

SNCA

Gene editing type

Knockout

Gene editing method

CRISPR technology

Knockout validation

Sanger Sequencing

Zygosity

Homozygous

Quality control

STR analysis

CSF1PO, D13S317, D7S820, D5S818, TH01, D16S539, TPOX

Cell culture

Biosafety level

EU: 1 US: 1

Adherent/suspension

Adherent

Gender

Male

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

EMEM + 10% FBS

Cryopreservation medium

Cell Freezing Medium-DMSO Serum free media, contains 8.7% DMSO in MEM supplemented with methyl cellulose.

Storage

Shipped at conditions

Dry Ice

Appropriate short-term storage conditions

-196°C

Appropriate long-term storage conditions

-196°C

Notes

Recommended control: Human wild-type U-87 MG cell line (Human wild-type U-87 MG cell line ab278079). 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 and ERS Genomics Limited, and is developed with patented technology. For full details of the limited use licenses and relevant patents please refer to our limited use license and patent pages.

Supplementary info

This supplementary information is collated from multiple sources and compiled automatically.

Activity summary

Alpha-synuclein aggregates are critical in the study of neurobiology. These aggregates form by accumulation of alpha-synuclein a protein that exists mostly in neural tissues. Also known as SNCA this protein has a mass of approximately 14 kDa. Researchers frequently find alpha-synuclein in presynaptic terminals of neurons but expression also occurs in the nucleus and cytoplasm of neuronal cells. This protein has a notable interaction with lipid membranes indicating its role in synaptic vesicle regulation.

Biological function summary

Alpha-synuclein manages several cellular processes most notably in neurotransmitter release and vesicle recycling. It does not work alone; it often forms complexes with other proteins like synaptobrevin-2 of the SNARE complex. The formation of these complexes allows for the proper functioning of synaptic vesicles. Alteration in alpha-synuclein's normal function through aggregation disrupts these processes which underlies many neural complications.

Pathways

Many neuronal functions depend on alpha-synuclein's participation in synaptic vesicle pathways. Key pathways include the dopamine metabolic pathway where alpha-synuclein regulates dopamine release. The protein interacts with other synaptic proteins like the SNARE proteins potentially affecting protein kinase pathways. Abnormal aggregation impacts these pathways which can propagate cellular stress and contribute to cellular degeneration.

Associated diseases and disorders

Alpha-synuclein aggregation correlates strongly with Parkinson's disease and Lewy body dementia. In these conditions aggregates form Lewy bodies protein clusters within neurons that impair their function and encourage neural degradation. The protein interacts with structural proteins like ubiquitin. Ubiquitin involvement highlights the importance of degradation pathways in these disorders as impaired proteostasis due to aggregated alpha-synuclein exacerbates disease progression.

Product promise

We are dedicated to supporting your work with high quality reagents and we are here for you every step of the way should you need us.

In the unlikely event of one of our products not working as expected, you are covered by our product promise.

Full details and terms and conditions can be found here:
Terms & Conditions.

2 product images

  • Western blot - Human SNCA (Alpha-synuclein) knockout U-87 MG cell line (ab282333), expandable thumbnail

    Western blot - Human SNCA (Alpha-synuclein) knockout U-87 MG cell line (ab282333)

    False colour image of Western blot: Anti-Alpha-synuclein antibody [EPR20535] staining at 1/1000 dilution shown in green; Mouse anti-GAPDH antibody [6C5] (Anti-GAPDH antibody [6C5] - Loading Control ab8245) loading control staining at 1/20000 dilution shown in red. In Western blot Anti-Alpha-synuclein antibody [EPR20535] ab212184 was shown to bind specifically to Alpha-synuclein. A band was observed at 16 kDa in wild-type U-87 MG cell lysates with no signal observed at this size in SNCA knockout cell line ab282333 (knockout cell lysate Human SNCA (Alpha-synuclein) knockout U-87 MG cell lysate ab283006). To generate this image wild-type and SNCA knockout U-87 MG cell lysates were analysed. First samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3% milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4°. Blots were washed four times in TBS-T incubated with secondary antibodies for 1 h at room temperature washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) at 1/20000 dilution.

    All lanes: Western blot - Anti-Alpha-synuclein antibody [EPR20535] (Anti-Alpha-synuclein antibody [EPR20535] ab212184) at 1/1000 dilution

    Lane 1: Wild-type U-87 MG cell lysate at 20 µg

    Lane 2: SNCA knockout U-87 MG cell lysate at 20 µg

    Performed under reducing conditions.

    Predicted band size: 14 kDa

    Observed band size: 16 kDa

  • Sanger Sequencing - Human SNCA (Alpha-synuclein) knockout U-87 MG cell line (ab282333), expandable thumbnail

    Sanger Sequencing - Human SNCA (Alpha-synuclein) knockout U-87 MG cell line (ab282333)

    42 bp deletion in exon 2 and intron 2-3, destroy donor splicing site and lead to stop codon

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Product protocols

For this product, it's our understanding that no specific protocols are required. You can:

Please note: All products are 'FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES'.

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