Human MMP14 knockout A-431 cell line (ab261890)
Overview
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Product name
Human MMP14 knockout A-431 cell line -
Parental Cell Line
A431 -
Organism
Human -
Mutation description
Knockout achieved by CRISPR/Cas9; X = 10 bp deletion; Frameshift = 99.9% -
Passage number
<20 -
Knockout validation
Immunocytochemistry (ICC), Next Generation Sequencing (NGS), Western Blot (WB) -
Tested applications
Suitable for: WB, ICC/IF, Next Generation Sequencingmore 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 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.
We will provide viable cells that proliferate on revival.
Properties
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Number of cells
1 x 106 cells/vial, 1 mL -
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
Seems to specifically activate progelatinase A. May thus trigger invasion by tumor cells by activating progelatinase A on the tumor cell surface. May be involved in actin cytoskeleton reorganization by cleaving PTK7. -
Tissue specificity
Expressed in stromal cells of colon, breast, and head and neck. Expressed in lung tumors. -
Sequence similarities
Belongs to the peptidase M10A family.
Contains 4 hemopexin-like domains. -
Domain
The conserved cysteine present in the cysteine-switch motif binds the catalytic zinc ion, thus inhibiting the enzyme. The dissociation of the cysteine from the zinc ion upon the activation-peptide release activates the enzyme. -
Post-translational
modificationsThe precursor is cleaved by a furin endopeptidase. -
Cellular localization
Membrane. Melanosome. Identified by mass spectrometry in melanosome fractions from stage I to stage IV. - 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 ab261890 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 |
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WB |
Use at an assay dependent concentration.
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ICC/IF |
Use at an assay dependent concentration.
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Next Generation Sequencing |
Use at an assay dependent concentration.
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Notes |
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WB
Use at an assay dependent concentration. |
ICC/IF
Use at an assay dependent concentration. |
Next Generation Sequencing
Use at an assay dependent concentration. |
Images
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10 bp deletion after Pro190 of the WT protein
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All lanes : Anti-MMP14 antibody [EP1264Y] (ab51074) at 1/2000 dilution
Lane 1 : Wild-type A-431 (Human epidermoid carcinoma cell line) whole cell lysate
Lane 2 : MMP14 knockout A-431 (Human epidermoid carcinoma cell line) whole cell lysate
Lane 3 : Caco-2 (Human colorectal adenocarcinoma cell line) whole cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.Lanes 1 - 3: Merged signal (red and green). Green - ab51074 observed at 54 kDa. Red - loading control, ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.
ab51074 was shown to react with MMP14 in wild-type A-431 cells in Western blot Loss of signal was observed when MMP14 knockout cell line ab261890 (knockout cell lysate ab261699) was used. Wild-type A-431 and MMP14 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBS-T (0.1% Tween®) before incubation with ab51074 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at a 1 in 2000 dilution and a 1 in 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 in 20000 dilution for 1 hour at room temperature before imaging.
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ab51074 staining MMP14 in wild-type A431 cells (top panel) and MMP14 knockout A431 cells (bottom panel) (ab261890). The cells were fixed with 4% paraformaldehyde (10 min) then permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab51074 at 0.2μg/ml concentration and ab7291 (Mouse monoclonal to alpha Tubulin) at 1/1000 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor® 594) (ab150120) at 2 μg/ml (shown in red). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8). -
X = 10 bp deletion
Protocols
Datasheets and documents
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SDS download
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Datasheet download
References (0)
ab261890 has not yet been referenced specifically in any publications.