Human SIPA1 (Spa-1) knockout HeLa cell line
- Advanced Validation
- What is this?
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(0 Publication)
- WB
Lab
Western blot - Human SIPA1 (Spa-1) knockout HeLa cell line (AB265434)
Lanes 1-4 : Merged signal (red and green). Green - ab189929 observed at 130 kDa. Red - loading control ab7291 observed at 50 kDa.
ab189929 Anti-Spa-1 antibody [EPR14134] was shown to specifically react with Spa-1 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265434 (knockout cell lysate ab258189) was used. Wild-type and Spa-1 knockout samples were subjected to SDS-PAGE. ab189929 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) were incubated overnight at 4° at 1 in 5000 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-Spa-1 antibody [EPR14134] (<a href='/en-us/products/primary-antibodies/spa-1-antibody-epr14134-ab189929'>ab189929</a>) at 1/5000 dilution
Lane 1:
Wild-type HeLa cell lysate at 20 µg
Lane 2:
Spa-1 knockout HeLa cell lysate at 20 µg
Lane 2:
Western blot - Human SIPA1 (Spa-1) knockout HeLa cell line (ab265434)
Lane 3:
Raji cell lysate at 20 µg
Lane 4:
HEK-293 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 1/20000 dilution
Predicted band size: 112 kDa
Observed band size: 130 kDa
false
- Sanger seq
Unknown
Sanger Sequencing - Human SIPA1 (Spa-1) knockout HeLa cell line (AB265434)
Allele-2 : 1 bp deletion in exon 5.
- Sanger seq
Unknown
Sanger Sequencing - Human SIPA1 (Spa-1) knockout HeLa cell line (AB265434)
Allele-3 : 182 bp deletion in exon 5.
- Sanger seq
Unknown
Sanger Sequencing - Human SIPA1 (Spa-1) knockout HeLa cell line (AB265434)
Allele-1 : 4 bp deletion in exon 5.
Complementary Products
![Flow Cytometry (Intracellular) - Anti-Spa-1 antibody [EPR14134] (AB189929)](https://content.abcam.com/products/images/spa-1-antibody-epr14134-ab189929--flow-cytometry-intracellular-img59536.png)
Reactivity data
Product details
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.
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
Spa-1 engages in the control of cell adhesion and migration through its interaction with the Ras-related protein Rap1. As part of larger signal transduction complexes Spa-1 influences the organization of actin cytoskeleton and impacts integrin-mediated cell adhesion. Through its GAP activity it maintains cellular responsiveness to external stimuli impacting processes like immune cell trafficking and neuronal growth. Studies have noted its significance in maintaining homeostasis in tissues where it is expressed.
Pathways
Spa-1 integrates into key signaling cascades impacting the Ras superfamily of GTPases. Within the Rap1 signaling pathway Spa-1 works alongside proteins like RapGEF to finely regulate signal transduction processes affecting cell adhesion and cytoskeletal dynamics. In the context of the integrin signaling pathway Spa-1 modulates the actions of B-Raf in connection with Rap1 dictating responses necessary for cellular motility and positioning.
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
Product promise
Please note: All products are 'FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES'.
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