Human CTSD knockout A-431 cell line (ab261891)
Overview
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Product name
Human CTSD knockout A-431 cell line -
Parental Cell Line
A431 -
Organism
Human -
Mutation description
Knockout achieved by CRISPR/Cas9; X = 22 bp deletion; Frameshift = 100% -
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 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
Acid protease active in intracellular protein breakdown. Involved in the pathogenesis of several diseases such as breast cancer and possibly Alzheimer disease. -
Tissue specificity
Expressed in the aorta extrcellular space (at protein level). -
Involvement in disease
Ceroid lipofuscinosis, neuronal, 10 -
Sequence similarities
Belongs to the peptidase A1 family.
Contains 1 peptidase A1 domain. -
Post-translational
modificationsN- and O-glycosylated. -
Cellular localization
Lysosome. Melanosome. Secreted, extracellular space. Identified by mass spectrometry in melanosome fractions from stage I to stage IV. In aortic samples, detected as an extracellular protein loosely bound to the matrix (PubMed:20551380). - 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 ab261891 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 |
|---|
|
WB
Use at an assay dependent concentration. |
Images
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Western blot - Human CTSD knockout A-431 cell line (ab261891)All lanes : Anti-Cathepsin D antibody (ab72915) at 1 µg/ml
Lane 1 : Wild-type A-431 (Human epidermoid carcinoma cell line) whole cell lysate
Lane 2 : CTSD knockout A-431 (Human epidermoid carcinoma cell line) whole cell lysate
Lane 3 : MCF7 (Human breast adenocarcinoma cell line) whole cell lysate
Lane 4 : Hep G2 (Human liver hepatocellular carcinoma 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 - ab72915 observed at 28, 43, 46 kDa. Red - loading control, ab8245, observed at 37 kDa.
ab72915 was shown to specifically react with CTSD in wild-type A-431 cells as signal was lost in CTSD knockout cell line ab261891 (knockout cell lysate ab261700). Wild-type and CTSD knockout samples were subjected to SDS-PAGE. The membrane was blocked with 3% milk. Ab72915 and ab8245 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1 µg/ml 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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Western blot - Human CTSD knockout A-431 cell line (ab261891)All lanes : Anti-proCathepsin D antibody [EPR3054] (ab134169) at 1/2000 dilution
Lane 1 : Wild-type A-431 (Human epidermoid carcinoma cell line) whole cell lysate
Lane 2 : Cathepsin D knockout A-431 (Human epidermoid carcinoma cell line) whole cell lysate
Lane 3 : Hep G2 (Human liver hepatocellular carcinoma cell line) whole cell lysate
Lane 4 : MCF7 (Human breast adenocarcinoma cell line) whole cell lysate
Lysates/proteins at 40 µg per lane.
Performed under reducing conditions.
Observed band size: 46 kDa why is the actual band size different from the predicted?Lanes 1 - 4: Merged signal (red and green). Green - ab134169 observed at 46 kDa. Red - loading control, ab7291 (mouse anti-tubulin), observed at 50 kDa.
ab134169 was shown to recognize in wild-type A-431 cells as signal was lost at the expected MW in CTSD knockout cell line ab261891 (knockout cell lysate ab261700). Additional cross-reactive bands were observed in the wild-type and knockout samples. Wild-type and CTSD knockout samples were subjected to SDS-PAGE. The membrane was blocked with 3% NF milk. Ab134169 and ab7291 (Mouse anti-tubulin loading control) were incubated overnight at 4°C at 1/2000 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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Western blot - Human CTSD knockout A-431 cell line (ab261891)All lanes : Anti-Cathepsin D antibody [EPR3056Y] (ab75811) at 1/1000 dilution (Unpurified)
Lane 1 : Wild-type A-431 (Human epidermoid carcinoma cell line) whole cell lysate
Lane 2 : CTSD knockout A-431 (Human epidermoid carcinoma cell line) whole cell lysate
Lane 3 : Hep G2 (Human liver hepatocellular carcinoma cell line) whole cell lysate
Lane 4 : MCF7 (Human breast adenocarcinoma cell line) whole cell lysate
Lysates/proteins at 40 µg per lane.
Performed under reducing conditions.
Observed band size: 44 kDa why is the actual band size different from the predicted?Lanes 1 - 4: Merged signal (red and green). Green - ab75811 observed at 44 kDa. Red - loading control, ab7291, observed at 55 kDa.
ab75811 was shown to specifically react with in wild-type A-431 cells as signal was lost in CTSD knockout cell line ab261891 (knockout cell lysate ab261700). Wild-type and CTSD knockout samples were subjected to SDS-PAGE. The membrane was blocked with 3% NF milk. Ab75811 and ab7291 (Mouse anti Tubulin loading control) were incubated overnight at 4°C at 1/1000 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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Next Generation Sequencing - Human CTSD knockout A-431 cell line (ab261891)X = 22 bp deletion
Protocols
Datasheets and documents
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SDS download
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Datasheet download
References (0)
ab261891 has not yet been referenced specifically in any publications.