GR (Phospho-Ser211)Colorimetric Cell-Based ELISA Kit (CBCAB01242)

Product Name:	GR (Phospho-Ser211)Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB01242
ELISA Type:	Cell-Based
Target:	GR (Phospho-Ser211)
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nm
Format:	2 x 96-Well Microplates

The GR (Phospho-Ser211) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect GR protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated GR in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on GR phosphorylation.

Qualitative determination of GR (Phospho-Ser211) concentration is achieved by an indirect ELISA format. In essence, GR (Phospho-Ser211) is captured by GR (Phospho-Ser211)-specific primary (1Ãƒ¸) antibodies while the HRP-conjugated secondary (2Ãƒ¸) antibodies bind the Fc region of the 1Ãƒ¸ antibody. Through this binding, the HRP enzyme conjugated to the 2Ãƒ¸ antibody can catalyze a colorimetric reaction upon substrate addition. Due to the qualitative nature of the Cell-Based ELISA, multiple normalization methods are needed:

1.	A monoclonal antibody specific for human GAPDH is included to serve as an internal positive control in normalizing the target absorbance values.
2.	Following the colorimetric measurement of HRP activity via substrate addition, the Crystal Violet whole-cell staining method may be used to determine cell density. After staining, the results can be analysed by normalizing the absorbance values to cell amounts, by which the plating difference can be adjusted.

Database Information:	Gene ID: 2908, UniProt ID: P04150, OMIM: 138040, Unigene: Hs.122926
Gene Symbol:	NR3C1
Sub Type:	Phospho

UniProt Protein Function:	GR: transcription factor of the nuclear receptor family. Receptor for glucocorticoids (GC). Binds to glucocorticoid response elements (GRE) and modulates other transcription factors. Affects inflammatory responses, cellular proliferation and differentiation in target tissues. Three alternatively spliced isoforms have been described.
UniProt Protein Details:	Protein type:Mitochondrial; DNA-binding; Transcription factor; Nuclear receptor Chromosomal Location of Human Ortholog: 5q31.3 Cellular Component: cytoplasm; cytosol; membrane; mitochondrial matrix; nucleoplasm; nucleus Molecular Function:glucocorticoid receptor activity; protein binding; protein dimerization activity; steroid binding; transcription factor activity; zinc ion binding Biological Process: adrenal gland development; chromatin modification; gene expression; glucocorticoid mediated signaling; glucocorticoid metabolic process; glucocorticoid receptor signaling pathway; maternal behavior; positive regulation of neuron apoptosis; positive regulation of transcription from RNA polymerase II promoter; regulation of glucocorticoid biosynthetic process; regulation of gluconeogenesis; regulation of transcription, DNA-dependent; signal transduction; transcription from RNA polymerase II promoter; transcription initiation from RNA polymerase II promoter; transcription, DNA-dependent Disease: Glucocorticoid Resistance, Generalized
NCBI Summary:	This gene encodes glucocorticoid receptor, which can function both as a transcription factor that binds to glucocorticoid response elements in the promoters of glucocorticoid responsive genes to activate their transcription, and as a regulator of other transcription factors. This receptor is typically found in the cytoplasm, but upon ligand binding, is transported into the nucleus. It is involved in inflammatory responses, cellular proliferation, and differentiation in target tissues. Mutations in this gene are associated with generalized glucocorticoid resistance. Alternative splicing of this gene results in transcript variants encoding either the same or different isoforms. Additional isoforms resulting from the use of alternate in-frame translation initiation sites have also been described, and shown to be functional, displaying diverse cytoplasm-to-nucleus trafficking patterns and distinct transcriptional activities (PMID:15866175). [provided by RefSeq, Feb 2011]
UniProt Code:	P04150
NCBI GenInfo Identifier:	121069
NCBI Gene ID:	2908
NCBI Accession:	P04150.1
UniProt Secondary Accession:	P04150,P04151, Q53EP5, Q6N0A4, A0ZXF9, B0LPG8, D3DQF4
UniProt Related Accession:	P04150
Molecular Weight:	82,904 Da
NCBI Full Name:	Glucocorticoid receptor
NCBI Synonym Full Names:	nuclear receptor subfamily 3 group C member 1
NCBI Official Symbol:	NR3C1
NCBI Official Synonym Symbols:	GR; GCR; GRL; GCCR; GCRST
NCBI Protein Information:	glucocorticoid receptor
UniProt Protein Name:	Glucocorticoid receptor
UniProt Synonym Protein Names:	Nuclear receptor subfamily 3 group C member 1
Protein Family:	Glucocorticoid receptor
UniProt Gene Name:	NR3C1
UniProt Entry Name:	GCR_HUMAN

Component	Quantity
96-Well Cell Culture Clear-Bottom Microplate	2 plates
10X TBS	24 mL
Quenching Buffer	24 mL
Blocking Buffer	50 mL
15X Wash Buffer	50 mL
Primary Antibody Diluent	12 mL
100x Anti-Phospho Target Antibody	60 µL
100x Anti-Target Antibody	60 µL
Anti-GAPDH Antibody	60 µL
HRP-Conjugated Anti-Rabbit IgG Antibody	12 mL
HRP-Conjugated Anti-Mouse IgG Antibody	12 mL
SDS Solution	12 mL
Stop Solution	24 mL
Ready-to-Use Substrate	12 mL
Crystal Violet Solution	12 mL
Adhesive Plate Seals	2 seals

The following materials and/or equipment are NOT provided in this kit but are necessary to successfully conduct the experiment:

Microplate reader able to measure absorbance at 450 nm and/or 595 nm for Crystal Violet Cell Staining (Optional)
Micropipettes with capability of measuring volumes ranging from 1 µL to 1 ml
37% formaldehyde (Sigma Cat# F-8775) or formaldehyde from other sources
Squirt bottle, manifold dispenser, multichannel pipette reservoir or automated microplate washer
Graph paper or computer software capable of generating or displaying logarithmic functions
Absorbent papers or vacuum aspirator
Test tubes or microfuge tubes capable of storing ≥1 ml
Poly-L-Lysine (Sigma Cat# P4832 for suspension cells)
Orbital shaker (optional)
Deionized or sterile water

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Step	Procedure
1.	Seed 200 µL of 20,000 adherent cells in culture medium in each well of a 96-well plate. The plates included in the kit are sterile and treated for cell culture. For suspension cells and loosely attached cells, coat the plates with 100 µL of 10 µg/ml Poly-L-Lysine (not included) to each well of a 96-well plate for 30 minutes at 37 °C prior to adding cells.
2.	Incubate the cells for overnight at 37 °C, 5% CO2.
3.	Treat the cells as desired.
4.	Remove the cell culture medium and rinse with 200 µL of 1x TBS, twice.
5.	Fix the cells by incubating with 100 µL of Fixing Solution for 20 minutes at room temperature. The 4% formaldehyde is used for adherent cells and 8% formaldehyde is used for suspension cells and loosely attached cells.
6.	Remove the Fixing Solution and wash the plate 3 times with 200 µL 1x Wash Buffer for five minutes each time with gentle shaking on the orbital shaker. The plate can be stored at 4 °C for a week.
7.	Add 100 µL of Quenching Buffer and incubate for 20 minutes at room temperature.
8.	Wash the plate 3 times with 1x Wash Buffer for 5 minutes each time.
9.	Add 200 µL of Blocking Buffer and incubate for 1 hour at room temperature.
10.	Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
11.	Add 50 µL of 1x primary antibodies Anti-GR (Phospho-Ser211) Antibody, Anti-GR Antibody and/or Anti-GAPDH Antibody) to the corresponding wells, cover with Parafilm and incubate for 16 hours (overnight) at 4 °C. If the target expression is known to be high, incubate for 2 hours at room temperature.
12.	Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
13.	Add 50 µL of 1x secondary antibodies (HRP-Conjugated AntiRabbit IgG Antibody or HRP-Conjugated Anti-Mouse IgG Antibody) to corresponding wells and incubate for 1.5 hours at room temperature.
14.	Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
15.	Add 50 µL of Ready-to-Use Substrate to each well and incubate for 30 minutes at room temperature in the dark.
16.	Add 50 µL of Stop Solution to each well and read OD at 450 nm immediately using the microplate reader.

(Additional Crystal Violet staining may be performed if desired – details of this may be found in the kit technical manual.)