Human NR3C1 (Glucocorticoid Receptor) knockout HeLa cell line (ab261766)
Overview
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Product name
Human NR3C1 (Glucocorticoid Receptor) knockout HeLa cell line -
Parental Cell Line
HeLa -
Organism
Human -
Mutation description
Knockout achieved by using CRISPR/Cas9, Homozygous: 1 bp insertion in exon 2. The cells can still express the functionally active isoforms GRα -D1, D2, D3 from alternative start codons. -
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 HeLa cell line (ab255448). 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
Cervix -
Cell type
epithelial -
Disease
Adenocarcinoma -
Gender
Female -
STR Analysis
Amelogenin X D5S818: 11, 12 D13S317: 12, 13.3 D7S820: 8, 12 D16S539: 9, 10 vWA: 16, 18 TH01: 7 TPOX: 8, 12 CSF1PO: 9, 10 -
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
Receptor for glucocorticoids (GC). Has a dual mode of action: as a transcription factor that binds to glucocorticoid response elements (GRE) and as a modulator of other transcription factors. Affects inflammatory responses, cellular proliferation and differentiation in target tissues. Could act as a coactivator for STAT5-dependent transcription upon growth hormone (GH) stimulation and could reveal an essential role of hepatic GR in the control of body growth. Involved in chromatin remodeling. Plays a significant role in transactivation. Involved in nuclear translocation. -
Tissue specificity
Widely expressed. In the heart, detected in left and right atria, left and right ventricles, aorta, apex, intraventricular septum, and atrioventricular node as well as whole adult and fetal heart. -
Involvement in disease
Defects in NR3C1 are a cause of glucocorticoid resistance (GCRES) [MIM:138040]; also known as cortisol resistance. It is a hypertensive, hyperandrogenic disorder characterized by increased serum cortisol concentrations. Inheritance is autosomal dominant. -
Sequence similarities
Belongs to the nuclear hormone receptor family. NR3 subfamily.
Contains 1 nuclear receptor DNA-binding domain. -
Domain
Composed of three domains: a modulating N-terminal domain, a DNA-binding domain and a C-terminal ligand-binding domain. -
Post-translational
modificationsIncreased proteasome-mediated degradation in response to glucocorticoids.
Phosphorylated in the absence of hormone; becomes hyperphosphorylated in the presence of glucocorticoid. The Ser-203-phosphorylated form is mainly cytoplasmic, and the Ser-211-phosphorylated form is nuclear. Transcriptional activity correlates with the amount of phosphorylation at Ser-211.
Sumoylated; this reduces transcription transactivation.
Ubiquitinated; restricts glucocorticoid-mediated transcriptional signaling. -
Cellular localization
Cytoplasm. Nucleus. Cytoplasmic in the absence of ligand, nuclear after ligand-binding and Nucleus. Localized largely in the nucleus. - 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 ab261766 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: 85 kDa.
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Notes |
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WB
Use at an assay dependent concentration. Predicted molecular weight: 85 kDa. |
Images
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All lanes : Anti-Glucocorticoid Receptor antibody [EPR19621] (ab183127) at 1/1000 dilution
Lane 1 : Wild-type HeLa cell lysate
Lane 2 : NR3C1 knockout HeLa cell lysate
Lane 3 : Wild-type A549 cell lysate
Lane 4 : NR3C1 knockout A549 cell lysate
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/20000 dilution
Performed under reducing conditions.
Predicted band size: 85 kDa
Observed band size: 90-100 kDa why is the actual band size different from the predicted?Lanes 1-4: Merged signal (red and green). Green - ab183127 observed at 90-100 kDa. Red - loading control ab8245 observed at 37 kDa.
ab183127 Anti-Glucocorticoid Receptor antibody [EPR19621] was shown to specifically react with Glucocorticoid Receptor in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab261766 (knockout cell lysate ab257009) was used. Wild-type and Glucocorticoid Receptor knockout samples were subjected to SDS-PAGE. ab183127 and Anti-alpha Tubulin antibody [EP1332Y] - Microtubule Marker (ab52866) were incubated overnight at 4°C 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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Homozygous: 1 bp insertion in exon 2.
Protocols
Datasheets and documents
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SDS download
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Datasheet download
References (0)
ab261766 has not yet been referenced specifically in any publications.