Human PPP2R5D knockout A-431 cell line (ab270476)
Overview
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Product name
Human PPP2R5D knockout A-431 cell line -
Parental Cell Line
A431 -
Organism
Human -
Mutation description
Knockout achieved by CRISPR/Cas9; X = 7 bp deletion; Frameshift = 99.91% -
Passage number
<20 -
Knockout validation
Next Generation Sequencing (NGS), Western Blot (WB) -
Tested applications
Suitable for: WB, 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
The B regulatory subunit might modulate substrate selectivity and catalytic activity, and also might direct the localization of the catalytic enzyme to a particular subcellular compartment. -
Tissue specificity
Isoform Delta-2 is widely expressed. Isoform Delta-1 is highly expressed in brain. -
Sequence similarities
Belongs to the phosphatase 2A regulatory subunit B56 family. -
Cellular localization
Cytoplasm. Nucleus. Nuclear in interphase, nuclear during mitosis. - 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 ab270476 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.
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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. |
Next Generation Sequencing
Use at an assay dependent concentration. |
Images
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7 bp deletion after Gly147 of the WT protein
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All lanes : Anti-PPP2R5D antibody [EPR15617-50] (ab188325) at 1/10000 dilution
Lane 1 : Wild-type A-431 (Human epidermoid carcinoma cell line) whole cell lysate
Lane 2 : PPP2R5D knockout A-431 (Human epidermoid carcinoma cell line) whole cell lysate
Lane 3 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 4 : MCF7 (Human breast adenocarcinoma cell line) whole cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Observed band size: 60-65 kDa why is the actual band size different from the predicted?Lanes 1 - 4: Merged signal (red and green). Green - ab188325 observed at 60-65 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.
ab188325 was shown to react with PPP2R5D in wild-type A-431 cells in western blot with loss of signal observed in PPP2R5D knockout cell line ab270476 (knockout cell lysate ab270499). Wild-type and PPP2R5D knockout A-431 cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween®) before incubation with ab188325 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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Knockout achieved by CRISPR/Cas9; X = 7 bp deletion; Frameshift = 99.91%
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All lanes : Anti-PPP2R5D antibody [EPR15617] (ab188323) at 1/10000 dilution
Lane 1 : Wild-type A-431 (Human epidermoid carcinoma cell line) whole cell lysate
Lane 2 : PPP2R5D knockout A-431 (Human epidermoid carcinoma cell line) whole cell lysate
Lane 3 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 4 : MCF7 (Human breast adenocarcinoma cell line) whole cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Observed band size: 60-65 kDa why is the actual band size different from the predicted?Lanes 1 - 4: Merged signal (red and green). Green - ab188323 observed at 60-65 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.
ab188323 was shown to react with PPP2R5D in wild-type A-431 cells in western blot with loss of signal observed in PPP2R5D knockout cell line ab270476 (knockout cell lysate ab270499). Wild-type and PPP2R5D knockout A-431 cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween®) before incubation with ab188323 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.
Protocols
Datasheets and documents
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SDS download
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Datasheet download
References (0)
ab270476 has not yet been referenced specifically in any publications.