Cytochrome P450 7B1 Colorimetric Cell-Based ELISA (CBCAB01200)

Product Name:	Cytochrome P450 7B1 Colorimetric Cell-Based ELISA
Product Code:	CBCAB01200
ELISA Type:	Cell-Based
Target:	Cytochrome P450 7B1
Reactivity:	Human
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:	96-Well Microplate

The Cytochrome P450 7B1 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect Cytochrome P450 7B1 protein expression profile in cells. The kit can be used for measuring the relative amounts of Cytochrome P450 7B1 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on Cytochrome P450 7B1.

Qualitative determination of Cytochrome P450 7B1 concentration is achieved by an indirect ELISA format. In essence, Cytochrome P450 7B1 is captured by Cytochrome P450 7B1-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: 9420, UniProt ID: O75881, OMIM: 270800/603711, Unigene: Hs.667720
Gene Symbol:	CYP7B1
Sub Type:	None

UniProt Protein Function:	CYP7B1: Defects in CYP7B1 are the cause of spastic paraplegia autosomal recessive type 5A (SPG5A). Spastic paraplegia is a neurodegenerative disorder characterized by a slow, gradual, progressive weakness and spasticity of the lower limbs. Rate of progression and the severity of symptoms are quite variable. Initial symptoms may include difficulty with balance, weakness and stiffness in the legs, muscle spasms, and dragging the toes when walking. In some forms of the disorder, bladder symptoms (such as incontinence) may appear, or the weakness and stiffness may spread to other parts of the body. Defects in CYP7B1 are the cause of congenital bile acid synthesis defect type 3 (CBAS3). Clinical features include severe cholestasis, cirrhosis and liver synthetic failure. Hepatic microsomal oxysterol 7-alpha-hydroxylase activity is undetectable. Belongs to the cytochrome P450 family.
UniProt Protein Details:	Protein type:EC 1.14.13.100; Oxidoreductase; Endoplasmic reticulum; Lipid Metabolism - primary bile acid biosynthesis Chromosomal Location of Human Ortholog: 8q21.3 Cellular Component: endoplasmic reticulum membrane Molecular Function:oxysterol 7-alpha-hydroxylase activity Biological Process: bile acid biosynthetic process; sterol metabolic process Disease: Bile Acid Synthesis Defect, Congenital, 3; Spastic Paraplegia 5a, Autosomal Recessive
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 endoplasmic reticulum membrane protein catalyzes the first reaction in the cholesterol catabolic pathway of extrahepatic tissues, which converts cholesterol to bile acids. This enzyme likely plays a minor role in total bile acid synthesis, but may also be involved in the development of atherosclerosis, neurosteroid metabolism and sex hormone synthesis. Mutations in this gene have been associated with hereditary spastic paraplegia (SPG5 or HSP), an autosomal recessive disorder. [provided by RefSeq, Apr 2016]
UniProt Code:	O75881
NCBI GenInfo Identifier:	20141320
NCBI Gene ID:	9420
NCBI Accession:	O75881.2
UniProt Secondary Accession:	O75881,Q9UNF5, B2RN07,
UniProt Related Accession:	O75881
Molecular Weight:	58,256 Da
NCBI Full Name:	25-hydroxycholesterol 7-alpha-hydroxylase
NCBI Synonym Full Names:	cytochrome P450 family 7 subfamily B member 1
NCBI Official Symbol:	CYP7B1
NCBI Official Synonym Symbols:	CP7B; CBAS3; SPG5A
NCBI Protein Information:	25-hydroxycholesterol 7-alpha-hydroxylase
UniProt Protein Name:	25-hydroxycholesterol 7-alpha-hydroxylase
UniProt Synonym Protein Names:	Cytochrome P450 7B1; Oxysterol 7-alpha-hydroxylase
Protein Family:	25-hydroxycholesterol 7-alpha-hydroxylase
UniProt Gene Name:	CYP7B1
UniProt Entry Name:	CP7B1_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 7B1 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.)