Cytochrome P450 21A2 Colorimetric Cell-Based ELISA

Product Name:	Cytochrome P450 21A2 Colorimetric Cell-Based ELISA
Product Code:	CBCAB01186
ELISA Type:	Cell-Based
Target:	Cytochrome P450 21A2
Reactivity:	Human
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:	96-Well Microplate

The Cytochrome P450 21A2 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect Cytochrome P450 21A2 protein expression profile in cells. The kit can be used for measuring the relative amounts of Cytochrome P450 21A2 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on Cytochrome P450 21A2.

Qualitative determination of Cytochrome P450 21A2 concentration is achieved by an indirect ELISA format. In essence, Cytochrome P450 21A2 is captured by Cytochrome P450 21A2-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: 1589, UniProt ID: P08686, OMIM: 201910, Unigene: Hs.654479
Gene Symbol:	CYP21A2
Sub Type:	None

UniProt Protein Function:	CYP21A2: Specifically catalyzes the 21-hydroxylation of steroids. Required for the adrenal synthesis of mineralocorticoids and glucocorticoids. Defects in CYP21A2 are the cause of adrenal hyperplasia type 3 (AH3). AH3 is a form of congenital adrenal hyperplasia, a common recessive disease due to defective synthesis of cortisol. Congenital adrenal hyperplasia is characterized by androgen excess leading to ambiguous genitalia in affected females, rapid somatic growth during childhood in both sexes with premature closure of the epiphyses and short adult stature. Four clinical types: 'salt wasting' (SW, the most severe type), 'simple virilizing' (SV, less severely affected patients), with normal aldosterone biosynthesis, 'non-classic form' or late onset (NC or LOAH), and 'cryptic' (asymptomatic). Belongs to the cytochrome P450 family.
UniProt Protein Details:	Protein type:Oxidoreductase; Lipid Metabolism - C21-steroid hormone; EC 1.14.99.10 Chromosomal Location of Human Ortholog: 6p21.3 Cellular Component: endoplasmic reticulum membrane Molecular Function:heme binding; steroid 21-monooxygenase activity; steroid hydroxylase activity Biological Process: glucocorticoid biosynthetic process; mineralocorticoid biosynthetic process; steroid biosynthetic process; steroid metabolic process; sterol metabolic process Disease: Adrenal Hyperplasia, Congenital, Due To 21-hydroxylase Deficiency
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 protein localizes to the endoplasmic reticulum and hydroxylates steroids at the 21 position. Its activity is required for the synthesis of steroid hormones including cortisol and aldosterone. Mutations in this gene cause congenital adrenal hyperplasia. A related pseudogene is located near this gene; gene conversion events involving the functional gene and the pseudogene are thought to account for many cases of steroid 21-hydroxylase deficiency. Two transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Jul 2008]
UniProt Code:	P08686
NCBI GenInfo Identifier:	117275
NCBI Gene ID:	1589
NCBI Accession:	P08686.1
UniProt Secondary Accession:	P08686,P04033, Q01204, Q08AG8, Q16749, Q16806, Q5ST44 Q96NU8, A2BHY6,
UniProt Related Accession:	P08686
Molecular Weight:	52,597 Da
NCBI Full Name:	Steroid 21-hydroxylase
NCBI Synonym Full Names:	cytochrome P450 family 21 subfamily A member 2
NCBI Official Symbol:	CYP21A2
NCBI Official Synonym Symbols:	CAH1; CPS1; CA21H; CYP21; CYP21B; P450c21B
NCBI Protein Information:	steroid 21-hydroxylase
UniProt Protein Name:	Steroid 21-hydroxylase
UniProt Synonym Protein Names:	21-OHase; Cytochrome P-450c21; Cytochrome P450 21; Cytochrome P450 XXI; Cytochrome P450-C21; Cytochrome P450-C21B
UniProt Gene Name:	CYP21A2
UniProt Entry Name:	CP21A_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 21A2 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.)

슈반 세포: 신경 기능을 지원하는 특수 세포

신경면역학: CNS의 면역체계

바르덴부르크 증후군 및 클라인-바르덴부르크 증후군

Cytochrome P450 21A2 Colorimetric Cell-Based ELISA

Description