Vitamin D Receptor (Phospho-Ser208)Colorimetric Cell-Based ELISA Kit (CBCAB00457)

Product Name:	Vitamin D Receptor (Phospho-Ser208)Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB00457
ELISA Type:	Cell-Based
Target:	Vitamin D Receptor (Phospho-Ser208)
Reactivity:	Human
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nm
Format:	2 x 96-Well Microplates

The Vitamin D Receptor (Phospho-Ser208) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect Vitamin D Receptor protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated Vitamin D Receptor in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on Vitamin D Receptor phosphorylation.

Qualitative determination of Vitamin D Receptor (Phospho-Ser208) concentration is achieved by an indirect ELISA format. In essence, Vitamin D Receptor (Phospho-Ser208) is captured by Vitamin D Receptor (Phospho-Ser208)-specific primary antibodies while the HRP-conjugated secondary antibodies bind the Fc region of the primary antibody. Through this binding, the HRP enzyme conjugated to the secondary 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: 7421, UniProt ID: P11473, OMIM: 277440/601769/607948, Unigene: Hs.524368
Gene Symbol:	VDR
Sub Type:	Phospho

UniProt Protein Function:	VDR: Nuclear hormone receptor. Transcription factor that mediates the action of vitamin D3 by controlling the expression of hormone sensitive genes. Regulates transcription of hormone sensitive genes via its association with the WINAC complex, a chromatin-remodeling complex. Recruited to promoters via its interaction with the WINAC complex subunit BAZ1B/WSTF, which mediates the interaction with acetylated histones, an essential step for VDR-promoter association. Plays a central role in calcium homeostasis. Defects in VDR are the cause of rickets vitamin D- dependent type 2A (VDDR2A). A disorder of vitamin D metabolism resulting in severe rickets, hypocalcemia and secondary hyperparathyroidism. Most patients have total alopecia in addition to rickets. Belongs to the nuclear hormone receptor family. NR1 subfamily.
UniProt Protein Details:	Protein type:DNA-binding; Transcription factor; Nuclear receptor Chromosomal Location of Human Ortholog: 12q13.11 Cellular Component: nucleoplasm; nucleus; receptor complex Molecular Function:retinoid X receptor binding; protein binding; DNA binding; zinc ion binding; sequence-specific DNA binding; steroid hormone receptor activity; vitamin D3 receptor activity; transcription factor activity Biological Process: lactation; transcription initiation from RNA polymerase II promoter; positive regulation of apoptosis involved in mammary gland involution; cell morphogenesis; decidualization; negative regulation of transcription from RNA polymerase II promoter; signal transduction; intestinal absorption; cellular calcium ion homeostasis; negative regulation of cell proliferation; calcium ion transport; steroid hormone mediated signaling; positive regulation of transcription from RNA polymerase II promoter; gene expression; negative regulation of transcription, DNA-dependent; skeletal development; positive regulation of keratinocyte differentiation Disease: Vitamin D-dependent Rickets, Type 2a; Osteoporosis
NCBI Summary:	This gene encodes the nuclear hormone receptor for vitamin D3. This receptor also functions as a receptor for the secondary bile acid lithocholic acid. The receptor belongs to the family of trans-acting transcriptional regulatory factors and shows sequence similarity to the steroid and thyroid hormone receptors. Downstream targets of this nuclear hormone receptor are principally involved in mineral metabolism though the receptor regulates a variety of other metabolic pathways, such as those involved in the immune response and cancer. Mutations in this gene are associated with type II vitamin D-resistant rickets. A single nucleotide polymorphism in the initiation codon results in an alternate translation start site three codons downstream. Alternative splicing results in multiple transcript variants encoding different proteins. [provided by RefSeq, Feb 2011]
UniProt Code:	P11473
NCBI GenInfo Identifier:	137617
NCBI Gene ID:	7421
NCBI Accession:	P11473.1
UniProt Secondary Accession:	P11473,Q5PSV3, B2R5Q1, G3V1V9,
UniProt Related Accession:	P11473
Molecular Weight:
NCBI Full Name:	Vitamin D3 receptor
NCBI Synonym Full Names:	vitamin D (1,25- dihydroxyvitamin D3) receptor
NCBI Official Symbol:	VDR
NCBI Official Synonym Symbols:	NR1I1; PPP1R163
NCBI Protein Information:	vitamin D3 receptor; 1,25-dihydroxyvitamin D3 receptor; vitamin D nuclear receptor variant 1; nuclear receptor subfamily 1 group I member 1; protein phosphatase 1, regulatory subunit 163
UniProt Protein Name:	Vitamin D3 receptor
UniProt Synonym Protein Names:	1,25-dihydroxyvitamin D3 receptor; Nuclear receptor subfamily 1 group I member 1
Protein Family:	Vitamin D3 receptor
UniProt Gene Name:	VDR
UniProt Entry Name:	VDR_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-Vitamin D Receptor (Phospho-Ser208) Antibody, Anti-Vitamin D 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.)