Human SPAST (Spastin) knockout HEK-293T cell line
- Advanced Validation
- What is this?
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SPAST 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 3 and 4 bp deletion in exon 3.
View Alternative Names
ADPSP, FSP 2, KIAA1083, SPG 4, SPG4 gene, Spastic paraplegia 4, spastic paraplegia 4 (autosomal dominant; spastin), spastin
- WB
Lab
Western blot - Human SPAST (Spastin) knockout HEK-293T cell line (AB267238)
False colour image of Western blot : Anti-Spastin antibody [Sp 3G11/1] staining at 1/500 dilution shown in green; Rabbit Anti-GAPDH antibody [EPR16891] (ab181602) loading control staining at 1/20000 dilution shown in red. In Western blot ab31850 was shown to bind specifically to Spastin. A band was observed at 55 kDa in wild-type HEK-293T cell lysates with no signal observed at this size in SPAST knockout cell line ab267238 (knockout cell lysate ab258698). To generate this image wild-type and SPAST knockout HEK-293T 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-Mouse IgG H&L (IRDye® 800CW) preabsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preabsorbed (ab216777) at 1/20000 dilution.
All lanes:
Western blot - Anti-Spastin antibody [Sp 3G11/1] (<a href='/en-us/products/primary-antibodies/spastin-antibody-sp-3g11-1-ab31850'>ab31850</a>) at 1/500 dilution
Lane 1:
Wild-type HEK-293T cell lysate at 20 µg
Lane 2:
Spastin knockout HEK-293T cell lysate at 20 µg
Lane 2:
Western blot - Human SPAST (Spastin) knockout HEK-293T cell line (ab267238)
Lane 3:
SH-SY5Y cell lysate at 20 µg
Lane 4:
HeLa cell lysate at 20 µg
Predicted band size: 68 kDa
Observed band size: 55 kDa
false
- WB
Lab
Western blot - Human SPAST (Spastin) knockout HEK-293T cell line (AB267238)
ab31850 was shown to react with SPAST in wild-type HEK293T cells in Western blot with loss of signal observed in SPAST knockout cell line ab267238 (SPAST knockout cell lysate ab258698). Wild-type HEK293T and SPAST knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBST for 1 hr before incubation with ab31850 overnight at 4 ° at a 1/500 dilution. Blots were incubated with goat anti-rabbit HRP secondary antibodies at 0.2μg/mL before imaging.
These data were provided by YCharOS Inc. an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.
All lanes:
Western blot - Anti-Spastin antibody [Sp 3G11/1] (<a href='/en-us/products/primary-antibodies/spastin-antibody-sp-3g11-1-ab31850'>ab31850</a>) at 1/500 dilution
Lane 1:
Wild-type HEK-293T cell lysate at 20 µg
Lane 2:
SPAST knockout HEK293T cell lysate at 20 µg
Lane 2:
Western blot - Human SPAST (Spastin) knockout HEK-293T cell line (ab267238)
Lane 3:
Wild-type HAP1 cell lysate at 20 µg
Lane 4:
SPAST knockout HAP1 cell lysate at 20 µg
Secondary
All lanes:
goat anti-rabbit HRP at 0.2 µg/mL
Predicted band size: 68 kDa
false
- WB
Lab
Western blot - Human SPAST (Spastin) knockout HEK-293T cell line (AB267238)
Lanes 1-3 : Merged signal (red and green). Green - ab77144 observed at 52 kDa. Red - loading control ab181602 observed at 36 kDa.
ab77144 Anti-Spastin antibody [Sp 6C6] was shown to specifically react with Spastin in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab267238 (knockout cell lysate ab258698) was used. Wild-type and Spastin knockout samples were subjected to SDS-PAGE. ab77144 and Anti-GAPDH antibody[EPR16891] - Loading Control (ab181602) were incubated overnight at 4° at 1 in 500 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed (ab216777) and Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed (ab216772) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.
All lanes:
Western blot - Anti-Spastin antibody [Sp 6C6] (<a href='/en-us/products/primary-antibodies/spastin-antibody-sp-6c6-ab77144'>ab77144</a>) at 1/500 dilution
Lane 1:
Wild-type HEK-293T cell lysate at 20 µg
Lane 2:
SPAST knockout HEK293T cell lysate at 20 µg
Lane 2:
Western blot - Human SPAST (Spastin) knockout HEK-293T cell line (ab267238)
Lane 3:
Rat brain tissue lysate at 20 µg
Secondary
All lanes:
Western blot - Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-680rd-preadsorbed-ab216777'>ab216777</a>) at 1/10000 dilution
Predicted band size: 68 kDa
Observed band size: 52 kDa
false
- Sanger seq
Unknown
Sanger Sequencing - Human SPAST (Spastin) knockout HEK-293T cell line (AB267238)
Allele-1 : 4 bp deletion in exon3
- Sanger seq
Unknown
Sanger Sequencing - Human SPAST (Spastin) knockout HEK-293T cell line (AB267238)
Allele-2 : 1 bp insertion in exon 3.
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
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
Spastin participates in regulating microtubule dynamics. It plays a critical role in axonal growth and maintenance by modulating microtubule lengths and branching. Spastin does not function alone; it forms part of the AAA protein family functioning in significant processes like neurogenesis and neural maintenance. Its activity affects neural cell structure and transport by influencing cytoskeletal rearrangement.
Pathways
Any disturbances in Spastin function can impact the microtubule dynamics pathway and the axonal transport pathway. Spastin interacts closely with proteins such as Tubulin and Katanin which are also involved in microtubule severing and organization. Proper Spastin function is necessary to maintain intracellular transport and signal transduction critical for neural cell health and function.
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
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Product promise
Please note: All products are 'FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES'.
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