Progesterone Receptor (Phospho-Ser294) Colorimetric Cell-Based ELISA Kit

Product Name:	Progesterone Receptor (Phospho-Ser294) Colorimetric Cell-Based ELISA
Product Code:	CBCAB01312
ELISA Type:	Cell-Based
Target:	Progesterone Receptor (Phospho-Ser294)
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nm
Format:	2 x 96-Well Microplates

The Progesterone Receptor (Phospho-Ser294) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect Progesterone Receptor protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated Progesterone Receptor in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on Progesterone Receptor phosphorylation.

Qualitative determination of Progesterone Receptor (Phospho-Ser294) concentration is achieved by an indirect ELISA format. In essence, Progesterone Receptor (Phospho-Ser294) is captured by Progesterone Receptor (Phospho-Ser294)-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: 5241, UniProt ID: P06401, OMIM: 607311, Unigene: Hs.32405
Gene Symbol:	PGR
Sub Type:	Phospho

UniProt Protein Function:	Function: The steroid hormones and their receptors are involved in the regulation of eukaryotic gene expression and affect cellular proliferation and differentiation in target tissues. Progesterone receptor isoform B (PRB) is involved activation of c-SRC/MAPK signaling on hormone stimulation. Ref.12 Ref.20 Ref.21 Ref.23 Ref.24 Ref.25 Ref.26 Ref.27Isoform A:inactive in stimulating c-Src/MAPK signaling on hormone stimulation. Ref.12 Ref.20 Ref.21 Ref.23 Ref.24 Ref.25 Ref.26 Ref.27Isoform 4:Increases mitochondrial membrane potential and cellular respiration upon stimulation by progesterone. Ref.12 Ref.20 Ref.21 Ref.23 Ref.24 Ref.25 Ref.26 Ref.27
UniProt Protein Details:	Subunit structure: Interacts with SMARD1 and UNC45A. Interacts with CUEDC2; the interaction promotes ubiquitination, decreases sumoylation, and repesses transcriptional activity. Interacts with PIAS3; the interaction promotes sumoylation of PR in a hormone-dependent manner, inhibits DNA-binding, and alters nuclear export. Interacts with SP1; the interaction requires ligand-induced phosphorylation on Ser-345 by ERK1/2 MAPK. Interacts with PRMT2. Ref.18 Ref.19 Ref.22 Ref.23 Ref.24 Ref.27 Subcellular location: Nucleus. Cytoplasm. Note: Nucleoplasmic shuttling is both homone- and cell cycle-dependent. On hormone stimulation, retained in the cytoplasm in the G1 and G2/M phases. Ref.12 Ref.20 Ref.21 Ref.26Isoform A: Nucleus. Cytoplasm. Note: Mainly nuclear. Ref.12 Ref.20 Ref.21 Ref.26Isoform 4: Mitochondrion outer membrane Ref.12 Ref.20 Ref.21 Ref.26. Domain: Composed of three domains: a modulating N-terminal domain, a DNA-binding domain and a C-terminal ligand-binding domain. Post-translational modification: Phosphorylated on multiple serine sites. Several of these sites are hormone-dependent. Phosphorylation on Ser-294 occurs preferentially on isoform B, is highly hormone-dependent and modulates ubiquitination and sumoylation on Lys-388. Phosphorylation on Ser-102 and Ser-345 also requires induction by hormone. Basal phosphorylation on Ser-81, Ser-162, Ser-190 and Ser-400 is increased in response to progesterone and can be phosphorylated in vitro by the CDK2-A1 complex. Increased levels of phosphorylation on Ser-400 also in the presence of EGF, heregulin, IGF, PMA and FBS. Phosphorylation at this site by CDK2 is ligand-independent, and increases nuclear translocation and transcriptional activity. Phosphorylation at Ser-162 and Ser-294, but not at Ser-190, is impaired during the G2/M phase of the cell cycle. Phosphorylation on Ser-345 by ERK1/2 MAPK is required for interaction with SP1. Ref.11 Ref.13 Ref.14 Ref.15 Ref.16 Ref.17 Ref.20 Ref.21 Ref.25 Ref.26 Ref.27Sumoylation is hormone-dependent and represses transcriptional activity. Sumoylation on all three sites is enhanced by PIAS3. Desumoylated by SENP1. Sumoylation on Lys-388, the main site of sumoylation, is repressed by ubiquitination on the same site, and modulated by phosphorylation at Ser-294. Ref.23 Ref.24 Ref.25Ubiquitination is hormone-dependent and represses sumoylation on the same site. Promoted by MAPK-mediated phosphorylation on Ser-294. Ref.23 Ref.24 Ref.25Palmitoylated by ZDHHC7 and ZDHHC21. Palmitoylation is required for plasma membrane targeting and for rapid intracellular signaling via ERK and AKT kinases and cAMP generation. Ref.28 Sequence similarities: Belongs to the nuclear hormone receptor family. NR3 subfamily.Contains 1 nuclear receptor DNA-binding domain.
NCBI Summary:	This gene encodes a member of the steroid receptor superfamily. The encoded protein mediates the physiological effects of progesterone, which plays a central role in reproductive events associated with the establishment and maintenance of pregnancy. This gene uses two distinct promotors and translation start sites in the first exon to produce two isoforms, A and B. The two isoforms are identical except for the additional 165 amino acids found in the N-terminus of isoform B and mediate their own response genes and physiologic effects with little overlap. [provided by RefSeq, Jan 2011]
UniProt Code:	P06401
NCBI GenInfo Identifier:	90110048
NCBI Gene ID:	5241
NCBI Accession:	P06401.4
UniProt Secondary Accession:	P06401,Q8TDS3, Q9UPF7, A7LQ08, A7X8B0, B4E3T0,
UniProt Related Accession:	P06401
Molecular Weight:
NCBI Full Name:	Progesterone receptor
NCBI Synonym Full Names:	progesterone receptor
NCBI Official Symbol:	PGR
NCBI Official Synonym Symbols:	PR; NR3C3
NCBI Protein Information:	progesterone receptor; nuclear receptor subfamily 3 group C member 3
UniProt Protein Name:	Progesterone receptor
UniProt Synonym Protein Names:	Nuclear receptor subfamily 3 group C member 3
Protein Family:	Perakine reductase
UniProt Gene Name:	PGR
UniProt Entry Name:	PRGR_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-Progesterone Receptor (Phospho-Ser294) Antibody, Anti-Progesterone Receptor 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.)

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Progesterone Receptor (Phospho-Ser294) Colorimetric Cell-Based ELISA Kit

Description