Human NFATC2 knockout Raji cell line (ab280906)
Overview
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Product name
Human NFATC2 knockout Raji cell line -
Parental Cell Line
Raji -
Organism
Human -
Mutation description
Knockout achieved by using CRISPR/Cas9, Homozygous: 122 bp deletion and 3 bp insertion in exon 2 -
Passage number
<20 -
Knockout validation
Sanger Sequencing -
Tested applications
Suitable for: WBmore details -
Biosafety level
2 -
General notes
Western blot data indicates that the CRISPR gene edit may have resulted in a truncation of the protein of interest. Please see data images.
Recommended Control: Human wild-type Raji (ab275473). 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: RPMI + 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 for bath 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 4x105 cells/mL. 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 4x105 cells/mL is recommended.
•A maximum of 3x106 viable cells/ml is obtainableThis 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.
Properties
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Number of cells
1 x 106 cells/vial, 1 mL -
Viability
~80 -
Adherent /Suspension
Suspension -
Tissue
Lymphatic -
Cell type
Burkitt's lymphoma -
Disease
Lymphoma -
Gender
Male -
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
Plays a role in the inducible expression of cytokine genes in T-cells, especially in the induction of the IL-2, IL-3, IL-4, TNF-alpha or GM-CSF. -
Tissue specificity
Expressed in thymus, spleen, heart, testis, brain, placenta, muscle and pancreas. -
Sequence similarities
Contains 1 RHD (Rel-like) domain. -
Domain
Rel Similarity Domain (RSD) allows DNA-binding and cooperative interactions with AP1 factors. -
Post-translational
modificationsIn resting cells, phosphorylated by NFATC-kinase on at least 18 sites in the 99-363 region. Upon cell stimulation, all these sites except Ser-243 are dephosphorylated by calcineurin. Dephosphorylation induces a conformational change that simultaneously exposes an NLS and masks an NES, which results in nuclear localization. Simultaneously, Ser-53 or Ser-56 is phosphorylated; which is required for full transcriptional activity. -
Cellular localization
Cytoplasm. Nucleus. Cytoplasmic for the phosphorylated form and nuclear after activation that is controlled by calcineurin-mediated dephosphorylation. Rapid nuclear exit of NFATC is thought to be one mechanism by which cells distinguish between sustained and transient calcium signals. The subcellular localization of NFATC plays a key role in the regulation of gene transcription. - 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 ab280906 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.
Western blot data indicates that the CRISPR gene edit may have resulted in a truncation of the protein of interest. Please see data images. |
| Notes |
|---|
|
WB
Use at an assay dependent concentration. Western blot data indicates that the CRISPR gene edit may have resulted in a truncation of the protein of interest. Please see data images. |
Images
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Western blot - Human NFATC2 knockout Raji cell line (ab280906)All lanes : Anti-NFAT1 antibody [EPR24658-43] (ab283691) at 1/1000 dilution
Lane 1 : Wild-type Raji cell lysate
Lane 2 : NFATC2 knockout Raji cell lysate
Lane 3 : Jurkat cell lysate
Lane 4 : SH-SY5Y cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Observed band size: 100 kDa why is the actual band size different from the predicted?False colour image of Western blot: Anti-NFAT1 antibody [EPR24658-43] staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab283691 was shown to bind specifically to NFAT1. A band was observed at 100 kDa in wild-type Raji cell lysates with no signal observed at this size in NFATC2 knockout cell line ab280906 (knockout cell lysate ab282940). The band observed in the knockout lysate lane below 100 kDa is likely to represent a truncated form of NFAT1. This has not been investigated further and the functional properties of the gene product have not been determined. To generate this image, wild-type and NFATC2 knockout Raji cell lysates were analysed.First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/20000 dilution.
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Sanger Sequencing - Human NFATC2 knockout Raji cell line (ab280906)122 bp deletion and 3 bp insertion in exon 2
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Western blot - Human NFATC2 knockout Raji cell line (ab280906)All lanes : Anti-NFAT1 antibody [EPR24658-149] (ab283649) at 1/1000 dilution
Lane 1 : Wild-type Raji cell lysate
Lane 2 : NFATC2 knockout Raji cell lysate
Lane 3 : Jurkat cell lysate
Lane 4 : SH-SY5Y cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Observed band size: 100 kDa why is the actual band size different from the predicted?False colour image of Western blot: Anti-NFAT1 antibody [EPR24658-149] staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab283649 was shown to bind specifically to NFAT1. A band was observed at 100 kDa in wild-type Raji cell lysates with no signal observed at this size in NFATC2 knockout cell line ab280906 (knockout cell lysate ab282940). The band observed in the knockout lysate lane below 100 kDa is likely to represent a truncated form of NFAT1. This has not been investigated further and the functional properties of the gene product have not been determined. To generate this image, wild-type and NFATC2 knockout Raji cell lysates were analysed.First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/20000 dilution.
Protocols
To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.
Datasheets and documents
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SDS download
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Datasheet download
References (0)
ab280906 has not yet been referenced specifically in any publications.