Human KRT14 (Cytokeratin 14) knockout A-431 cell line (ab261897)
Overview
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Product name
Human KRT14 (Cytokeratin 14) knockout A-431 cell line -
Parental Cell Line
A431 -
Organism
Human -
Mutation description
Knockout achieved by CRISPR/Cas9; X = 13 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/IFmore 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.
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
The nonhelical tail domain is involved in promoting KRT5-KRT14 filaments to self-organize into large bundles and enhances the mechanical properties involved in resilience of keratin intermediate filaments in vitro. -
Tissue specificity
Detected in the basal layer, lowered within the more apically located layers specifically in the stratum spinosum, stratum granulosum but is not detected in stratum corneum. Strongly expressed in the outer root sheath of anagen follicles but not in the germinative matrix, inner root sheath or hair. Found in keratinocytes surrounding the club hair during telogen. -
Involvement in disease
Defects in KRT14 are a cause of epidermolysis bullosa simplex Dowling-Meara type (DM-EBS) [MIM:131760]. DM-EBS is a severe form of intraepidermal epidermolysis bullosa characterized by generalized herpetiform blistering, milia formation, dystrophic nails, and mucous membrane involvement.
Defects in KRT14 are a cause of epidermolysis bullosa simplex Weber-Cockayne type (WC-EBS) [MIM:131800]. WC-EBS is a form of intraepidermal epidermolysis bullosa characterized by blistering limited to palmar and plantar areas of the skin.
Defects in KRT14 are a cause of epidermolysis bullosa simplex Koebner type (K-EBS) [MIM:131900]. K-EBS is a form of intraepidermal epidermolysis bullosa characterized by generalized skin blistering. The phenotype is not fundamentally distinct from the Dowling-Meara type, although it is less severe.
Defects in KRT14 are the cause of epidermolysis bullosa simplex autosomal recessive (AREBS) [MIM:601001]. AREBS is an intraepidermal epidermolysis bullosa characterized by localized blistering on the dorsal, lateral and plantar surfaces of the feet.
Defects in KRT14 are the cause of Naegeli-Franceschetti-Jadassohn syndrome (NFJS) [MIM:161000]; also known as Naegeli syndrome. NFJS is a rare autosomal dominant form of ectodermal dysplasia. The cardinal features are absence of dermatoglyphics (fingerprints), reticular cutaneous hyperpigmentation (starting at about the age of 2 years without a preceding inflammatory stage), palmoplantar keratoderma, hypohidrosis with diminished sweat gland function and discomfort provoked by heat, nail dystrophy, and tooth enamel defects.
Defects in KRT14 are the cause of dermatopathia pigmentosa reticularis (DPR) [MIM:125595]. DPR is a rare ectodermal dysplasia characterized by lifelong persistent reticulate hyperpigmentation, noncicatricial alopecia, and nail dystrophy. -
Sequence similarities
Belongs to the intermediate filament family. -
Cellular localization
Cytoplasm. Nucleus. Expressed in both as a filamentous pattern. - Information by UniProt
Associated products
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KO cell lysates
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Related Products
- Anti-Cytokeratin 14 antibody [SP53] (ab119695)
- Alexa Fluor® 488 Anti-Cytokeratin 14 antibody [EP1612Y] (ab192055)
- Alexa Fluor® 647 Anti-Cytokeratin 14 antibody [EP1612Y] (ab192056)
- Anti-Cytokeratin 14 antibody [EPR17336] (ab197893)
- Anti-Cytokeratin 19 antibody [EPR1579Y] - BSA and Azide free (ab232566)
- Anti-Cytokeratin 14 antibody [SP53] - BSA and Azide free (ab236439)
- Anti-Lin28B antibody [EPR18717] - BSA and Azide free (ab240326)
- Anti-Cytokeratin 14 antibody [EP1612Y] - BSA and Azide free (ab243907)
- Anti-Cytokeratin 14 antibody [EPR17336] - BSA and Azide free (ab251249)
- Anti-Cytokeratin 14 antibody [LL002] - BSA and Azide free (ab264498)
- Anti-Cytokeratin 14 antibody [EP1612Y] (ab51054)
- Anti-Cytokeratin 14 antibody [LL002] (ab7800)
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab261897 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. Predicted molecular weight: 52 kDa.
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| ICC/IF |
Use at an assay dependent concentration.
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| Notes |
|---|
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WB
Use at an assay dependent concentration. Predicted molecular weight: 52 kDa. |
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ICC/IF
Use at an assay dependent concentration. |
Images
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Next Generation Sequencing - Human KRT14 (Cytokeratin 14) knockout A-431 cell line (ab261897)
Knockout achieved by CRISPR/Cas9; X = 13 bp deletion; Frameshift = 99.9%
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Western blot - Human KRT14 (Cytokeratin 14) knockout A-431 cell line (ab261897)All lanes : Anti-Cytokeratin 14 antibody [EP1612Y] (ab51054) at 1/10000 dilution
Lane 1 : Wild-type A431 cell lysate
Lane 2 : KRT14 knockout A431 cell lysate
Lane 3 : Human skin cell lysate
Lane 4 : MCF7 (Human breast adenocarcinoma cell line) whole cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 52 kDa
Observed band size: 49 kDa why is the actual band size different from the predicted?Lanes 1 - 4: Merged signal (red and green). Green - ab51054 observed at 49 kDa. Red - loading control, ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.
ab51054 was shown to react with Cytokeratin 14 in wild-type A-431 cells in western blot Loss of signal was observed when KRT14 knockout cell line ab261897 (knockout cell lysate ab261706) was used. Wild-type A-431 and KRT14 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween®) before incubation with ab51054 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at a 1 in 10000 dilution and a 1 in 20000 dilution respectively. Blots were incubated 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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Immunocytochemistry/ Immunofluorescence - Human KRT14 (Cytokeratin 14) knockout A-431 cell line (ab261897)ab181595 staining Cytokeratin 14 in wild-type A431 cells (top panel) and KRT14 knockout A431 cells (bottom panel). The cells were fixed with 100% methanol (5 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 ab181595 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). -
Immunocytochemistry/ Immunofluorescence - Human KRT14 (Cytokeratin 14) knockout A-431 cell line (ab261897)ab192056 staining KRT14 in wild-type A-431 cells (top panel) and KRT14 knockout A-431 cells (ab261897) (bottom panel). The cells were fixed with 100% methanol (5 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 ab192056 at 1/100 dilution and ab195887 (Mouse monoclonal to alpha Tubulin - Alexa Fluor® 488) at 1/250 dilution overnight at 4oC. Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8). -
Western blot - Human KRT14 (Cytokeratin 14) knockout A-431 cell line (ab261897)All lanes : Anti-Cytokeratin 14 antibody [EPR17336] (ab197893) at 1/50000 dilution
Lane 1 : Wild-type A-431 (Human epidermoid carcinoma cell line) whole cell lysate
Lane 2 : KRT14 knockout A-431 (Human epidermoid carcinoma cell line) whole cell lysate
Lane 3 : Human skin whole tissue lysate
Lane 4 : MCF7 (Human breast adenocarcinoma cell line) whole cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 52 kDa
Observed band size: 52 kDaLanes 1 - 4: Merged signal (red and green). Green - ab197893 observed at 52 kDa. Red - loading control, ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.
ab197893 was shown to react with KRT14 in wild-type A-431 cells in Western blot Loss of signal was observed when KRT14 knockout cell line ab261897 (knockout cell lysate ab261706) was used. Wild-type A-431 and KRT14 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween®) before incubation with ab197893 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at a 1 in 50000 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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Western blot - Human KRT14 (Cytokeratin 14) knockout A-431 cell line (ab261897)All lanes : Anti-Cytokeratin 14 antibody [SP53] (ab119695) at 1/93 dilution
Lane 1 : Wild-type A-431 (Human epidermoid carcinoma cell line) whole cell lysate
Lane 2 : KRT14 knockout A-431 (Human epidermoid carcinoma cell line) whole cell lysate
Lane 3 : Human skin whole tissue lysate
Lane 4 : MCF7 (Human breast adenocarcinoma cell line) whole cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 52 kDa
Observed band size: 52 kDaLanes 1 - 4: Merged signal (red and green). Green - ab119695 observed at 52 kDa. Red - loading control, ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.
ab119695 was shown to react with KRT14 in wild-type A-431 cells in Western blot Loss of signal was observed when KRT14 knockout cell line ab261897 (knockout cell lysate ab261706) was used. Wild-type A-431 and KRT14 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween®) before incubation with ab119695 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at a 1 in 93 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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Immunocytochemistry/ Immunofluorescence - Human KRT14 (Cytokeratin 14) knockout A-431 cell line (ab261897)ab192055 staining KRT14 in wild-type A-431 cells (top panel) and KRT14 knockout A-431 cells (ab261897) (bottom panel). The cells were fixed with 100% methanol (5 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 ab192055 at 1/100 dilution and ab190573 (Rabbit monoclonal to alpha Tubulin - Alexa Fluor® 647) at 1/250 dilution overnight at 4oC. Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8). -
Immunocytochemistry/ Immunofluorescence - Human KRT14 (Cytokeratin 14) knockout A-431 cell line (ab261897)ab119695 staining KRT14 in wild-type A-431 cells (top panel) and KRT14 knockout A-431 cells (ab261897) (bottom panel). The cells were fixed with 100% methanol (5 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 ab119695 at 5?g/ml concentration and ab7291 (Mouse monoclonal to alpha Tubulin) at 1/1000 dilution overnight at 4oC followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 ?g/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor® 594) (ab150120) at 2 ?g/ml (shown in red). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8). -
Immunocytochemistry/ Immunofluorescence - Human KRT14 (Cytokeratin 14) knockout A-431 cell line (ab261897)ab108417 staining KRT14 in wild-type A-431 cells (top panel) and KRT14 knockout A-431 cells (ab261897) (bottom panel). The cells were fixed with 100% methanol (5 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 ab108417 at 1/100 dilution and ab7291 (Mouse monoclonal to alpha Tubulin) at 1/1000 dilution overnight at 4oC followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 ?g/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor® 594) (ab150120) at 2 ?g/ml (shown in red). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8). -
Immunocytochemistry/ Immunofluorescence - Human KRT14 (Cytokeratin 14) knockout A-431 cell line (ab261897)ab51054 staining KRT14 in wild-type A-431 cells (top panel) and KRT14 knockout A-431 cells (ab261897) (bottom panel). The cells were fixed with 100% methanol (5 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 ab51054 at 1/100 dilution and ab7291 (Mouse monoclonal to alpha Tubulin) at 1/1000 dilution overnight at 4oC followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 ug/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor® 594) (ab150120) at 2 ug/ml (shown in red). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8). -
Immunocytochemistry/ Immunofluorescence - Human KRT14 (Cytokeratin 14) knockout A-431 cell line (ab261897)ab7800 staining KRT14 in wild-type A-431 cells (top panel) and KRT14 knockout A-431 cells (ab261897) (bottom panel). The cells were fixed with 100% methanol (5 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 ab7800 at 1ug/ml concentration and ab6046 (Rabbit polyclonal to beta Tubulin) at 1/1000 dilution overnight at 4oC followed by a further incubation at room temperature for 1h with a goat secondary antibody to mouse IgG (Alexa Fluor® 488) (ab150117) at 2 ug/ml (shown in green) and a goat secondary antibody to rabbit IgG (Alexa Fluor® 594) (ab150080) at 2 ug/ml (shown in red). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8).
Protocols
Datasheets and documents
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SDS download
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Datasheet download
References (0)
ab261897 has not yet been referenced specifically in any publications.