Cytochrome P450 24A1 Colorimetric Cell-Based ELISA (CBCAB01187)

Product Name:	Cytochrome P450 24A1 Colorimetric Cell-Based ELISA
Product Code:	CBCAB01187
ELISA Type:	Cell-Based
Target:	Cytochrome P450 24A1
Reactivity:	Human
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:	96-Well Microplate

The Cytochrome P450 24A1 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect Cytochrome P450 24A1 protein expression profile in cells. The kit can be used for measuring the relative amounts of Cytochrome P450 24A1 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on Cytochrome P450 24A1.

Qualitative determination of Cytochrome P450 24A1 concentration is achieved by an indirect ELISA format. In essence, Cytochrome P450 24A1 is captured by Cytochrome P450 24A1-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: 1591, UniProt ID: Q07973, OMIM: 126065, Unigene: Hs.89663
Gene Symbol:	CYP24A1
Sub Type:	None

UniProt Protein Function:	CYP24A1: Has a role in maintaining calcium homeostasis. Catalyzes the NADPH-dependent 24-hydroxylation of calcidiol (25- hydroxyvitamin D(3)) and calcitriol (1-alpha,25-dihydroxyvitamin D(3)). The enzyme can perform up to 6 rounds of hydroxylation of calcitriol leading to calcitroic acid. It also shows 23- hydroxylating activity leading to 1-alpha,25-dihydroxyvitamin D(3)-26,23-lactone as end product. Defects in CYP24A1 are the cause of hypercalcemia infantile (HCAI). HCAI is a disorder characterized by abnormally high level of calcium in the blood, failure to thrive, vomiting, dehydration, and nephrocalcinosis. Belongs to the cytochrome P450 family. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:EC 1.14.13.126; Mitochondrial; Oxidoreductase Chromosomal Location of Human Ortholog: 20q13 Cellular Component: mitochondrial inner membrane Molecular Function:iron ion binding; heme binding; 25-hydroxycholecalciferol-24-hydroxylase activity; oxidoreductase activity; 1-alpha,25-dihydroxyvitamin D3 (1,25-(OH)2D3) 24-hydroxylase activity Biological Process: osteoblast differentiation; steroid metabolic process; response to vitamin D; vitamin metabolic process; xenobiotic metabolic process; vitamin D metabolic process; vitamin D catabolic process Disease: Hypercalcemia, Infantile
NCBI Summary:	This gene encodes a member of the cytochrome P450 superfamily of enzymes. The cytochrome P450 proteins are monooxygenases which catalyze many reactions involved in drug metabolism and synthesis of cholesterol, steroids and other lipids. This mitochondrial protein initiates the degradation of 1,25-dihydroxyvitamin D3, the physiologically active form of vitamin D3, by hydroxylation of the side chain. In regulating the level of vitamin D3, this enzyme plays a role in calcium homeostasis and the vitamin D endocrine system. Alternatively spliced transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Jul 2008]
UniProt Code:	Q07973
NCBI GenInfo Identifier:	19862747
NCBI Gene ID:	1591
NCBI Accession:	Q07973.2
UniProt Secondary Accession:	Q07973,Q15807, Q32ML3, Q5I2W7,
UniProt Related Accession:	Q07973
Molecular Weight:	59kDa
NCBI Full Name:	1,25-dihydroxyvitamin D(3) 24-hydroxylase, mitochondrial
NCBI Synonym Full Names:	cytochrome P450, family 24, subfamily A, polypeptide 1
NCBI Official Symbol:	CYP24A1
NCBI Official Synonym Symbols:	CP24; HCAI; CYP24; P450-CC24
NCBI Protein Information:	1,25-dihydroxyvitamin D(3) 24-hydroxylase, mitochondrial; 1,25-dihydroxyvitamin D(3) 24-hydroxylase, mitochondrial; 24-OHase; cytochrome P450 24A1; cytochrome P450-CC24; vitamin D 24-hydroxylase; exo-mitochondrial protein; vitamin D(3) 24-hydroxylase; 1,25-@dihydroxyvitamin D3 24-hydroxylase; cytochrome P450, subfamily XXIV (vitamin D 24-hydroxylase)
UniProt Protein Name:	1,25-dihydroxyvitamin D(3) 24-hydroxylase, mitochondrial
UniProt Synonym Protein Names:	Cytochrome P450 24A1; Cytochrome P450-CC24
Protein Family:	1,25-dihydroxyvitamin D(3) 24-hydroxylase
UniProt Gene Name:	CYP24A1
UniProt Entry Name:	CP24A_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-Cytochrome P450 24A1 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.)