Human PXN (Paxillin) knockout A-431 cell line (ab261892)
Overview
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Product name
Human PXN (Paxillin) knockout A-431 cell line
See all Paxillin lysates -
Parental Cell Line
A431 -
Organism
Human -
Mutation description
Knockout achieved by CRISPR/Cas9; X = 7 bp deletion, 1 bp insertion; Frameshift = 99.7% -
Passage number
<20 -
Knockout validation
Next Generation Sequencing (NGS), Western Blot (WB) -
Tested applications
Suitable for: WBmore details -
Biosafety level
1 -
General notes
Recommended control: Human wild-type A-431 cell line (ab263975). 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.
Cryopreservation cell medium: Cell Freezing Medium-DMSO Serum free media, contains 8.7% DMSO in MEM supplemented with methyl cellulose.
Culture medium: DMEM (High Glucose) + 10% FBS
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. Based on cell count, seed cells in an appropriate cell culture 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.
4. Incubate the culture at 37°C incubator with 5% CO2. 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.
- A partial media change 24 hours prior to subculture may be helpful to encourage growth, if required.
- Cells should be passaged when they have achieved 80-90% confluence.
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.
Properties
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Number of cells
1 x 106 cells/vial, 1 mL -
Viability
~80% -
Adherent /Suspension
Adherent -
Tissue
Skin -
Cell type
epithelial -
Disease
Epidermoid Carcinoma -
Gender
Female -
Mycoplasma free
Yes -
Storage instructions
Shipped on Dry Ice. Store in liquid nitrogen. -
Storage buffer
Constituents: 8.7% Dimethylsulfoxide, 2% Cellulose, methyl ether -
Research areas
Target
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Function
Cytoskeletal protein involved in actin-membrane attachment at sites of cell adhesion to the extracellular matrix (focal adhesion). -
Sequence similarities
Belongs to the paxillin family.
Contains 4 LIM zinc-binding domains. -
Post-translational
modificationsPhosphorylated on tyrosine residues during integrin-mediated cell adhesion, embryonic development, fibroblast transformation and following stimulation of cells by mitogens. -
Cellular localization
Cytoplasm > cytoskeleton. Cell junction > focal adhesion. - Information by UniProt
Associated products
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KO cell lysates
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Related Products
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab261892 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Application | Abreviews | Notes |
---|---|---|
WB |
Use at an assay dependent concentration.
|
Notes |
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WB
Use at an assay dependent concentration. |
Images
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All lanes : Anti-Paxillin antibody [E228] (ab32115) at 1/10000 dilution
Lane 1 : PXN knockout A-431 (Human epidermoid carcinoma cell line) whole cell lysate
Lane 3 : U-87 MG (Human glioblastoma-astrocytoma epithelial cell line) whole cell lysate
Lane 4 : PC3 (Human prostate adenocarcinoma cell line) whole cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Observed band size: 65 kDa why is the actual band size different from the predicted?Lanes 1 - 4: Merged signal (red and green). Green - ab32115 observed at 65 kDa. Red - loading control, ab8245, observed at 37 kDa.
ab32115 was shown to specifically react with PXN in wild-type A-431 cells as signal was lost in PXN knockout cell line ab261892 (knockout cell lysate ab261701). Wild-type and PXN knockout samples were subjected to SDS-PAGE. The membrane was blocked with 3% milk. Ab32115 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1/10000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.
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All lanes : Anti-Paxillin antibody [M107] (ab23510) at 1/1000 dilution
Lane 1 : Wild-type A-431 (Human epidermoid carcinoma cell line) whole cell lysate
Lane 2 : PXN knockout A-431 (Human epidermoid carcinoma cell line) whole cell lysate
Lane 3 : U-87 MG (Human glioblastoma-astrocytoma epithelial cell line) whole cell lysate
Lane 4 : PC3 (Human prostate adenocarcinoma cell line) whole cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.Lanes 1 - 4: Merged signal (red and green). Green - ab23510 observed at 65 kDa. Red - loading control, ab181602, observed at 37 kDa.
ab23510 was shown to recognize PXN in wild-type A-431 cells as signal was lost at the expected MW in PXN knockout cell line ab261892 (knockout cell lysate ab261701). Additional cross-reactive bands were observed in the wild-type and knockout samples. Wild-type and PXN knockout samples were subjected to SDS-PAGE. Ab23510 and ab181602 (Rabbit anti-GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye® 800CW) preabsorbed ab216772 and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preabsorbed ab216777 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.
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X = 7 bp deletion, 1 bp insertion
Protocols
Datasheets and documents
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SDS download
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Datasheet download
References (0)
ab261892 has not yet been referenced specifically in any publications.