Human CAPN2 (Calpain 2) knockout HEK-293T cell line (ab266628)
Overview
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Product name
Human CAPN2 (Calpain 2) knockout HEK-293T cell line -
Parental Cell Line
HEK293T -
Organism
Human -
Mutation description
Knockout achieved by using CRISPR/Cas9, 17 bp deletion in exon 4 and 1 bp insertion in exon 4 and 47 bp deletion in exon 4 and 8 bp deletion in exon 4 -
Passage number
<20 -
Knockout validation
Sanger Sequencing, Western Blot (WB) -
Tested applications
Suitable for: WBmore details -
Biosafety level
2 -
General notes
Recommended control: Human wild-type HEK293T cell line (ab255449). 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.
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
Kidney -
Cell type
epithelial -
STR Analysis
Amelogenin X D5S818: 8, 9 D13S317: 12, 14 D7S820: 11 D16S539: 9, 13 vWA: 16, 19 TH01: 7, 9.3 TPOX: 11 CSF1PO: 11, 12 -
Antibiotic resistance
Puromycin 1.00µg/ml -
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
Calcium-regulated non-lysosomal thiol-protease which catalyze limited proteolysis of substrates involved in cytoskeletal remodeling and signal transduction. -
Tissue specificity
Ubiquitous. -
Sequence similarities
Belongs to the peptidase C2 family.
Contains 1 calpain catalytic domain.
Contains 3 EF-hand domains. -
Cellular localization
Cytoplasm. Cell membrane. Translocates to the plasma membrane upon Ca(2+) binding. - 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 ab266628 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. Predicted molecular weight: 80 kDa.
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Notes |
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WB
Use at an assay dependent concentration. Predicted molecular weight: 80 kDa. |
Images
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All lanes : Anti-Calpain 2 antibody [EPR2562Y] (ab75994) at 1/2000 dilution
Lane 1 : Wild-type HEK-293T cell lysate at 40 µg
Lane 2 : CAPN2 knockout HEK-293T cell lysate at 40 µg
Lane 3 : A431 cell lysate at 20 µg
Lane 4 : LNCaP cell lysate at 20 µg
Performed under reducing conditions.
Predicted band size: 80 kDa
Observed band size: 75 kDa why is the actual band size different from the predicted?Lanes 1 - 4: Merged signal (red and green). Green - ab75994 observed at 75 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37 kDa.
ab75994 was shown to react with Calpain 2 in wild-type HEK-293T cells in Western blot with loss of signal observed in CAPN2 knockout cell line ab266628 (CAPN2 knockout cell lysate ab257379). Wild-type HEK-293T and CAPN2 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween®) before incubation with ab75994 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 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 h at room temperature before imaging.
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All lanes : Anti-Calpain 2 antibody [EPR5977] (ab126600) at 1/1000 dilution
Lane 1 : Wild-type HEK293T cell lysate
Lane 2 : CAPN2 knockout HEK293T cell lysate
Lane 3 : A431 cell lysate
Lane 4 : LNCaP cell lysate
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution
Predicted band size: 80 kDa
Observed band size: 80 kDaLanes 1-4: Merged signal (red and green). Green - ab126600 observed at 80 kDa. Red - loading control ab8245 observed at 36 kDa.
ab126600 Anti-Calpain 2 antibody [EPR5977] was shown to specifically react with Calpain 2 in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab266628 (knockout cell lysate ab257379) was used. Wild-type and Calpain 2 knockout samples were subjected to SDS-PAGE. ab126600 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated at room temperature for 2. 5 hours at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.
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Allele-1: 47 bp deletion in exon 4
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Allele-2: 17 bp deletion in exon 4.
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Allele-3: 8 bp deletion in exon 4.
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Allele-4: 1 bp insertion in exon 4.
Protocols
Datasheets and documents
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SDS download
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Datasheet download
References (0)
ab266628 has not yet been referenced specifically in any publications.