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Cytochrome P450 2R1 Colorimetric Cell-Based ELISA

SKU:
CBCAB00217
Product Type:
ELISA Kit
ELISA Type:
Cell Based
Reactivity:
Human
Mouse
Detection Method:
Colorimetric
€599
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Description

system_update_altDatasheetMSDS
Product Name:Cytochrome P450 2R1 Colorimetric Cell-Based ELISA
Product Code:CBCAB00217
ELISA Type:Cell-Based
Target:Cytochrome P450 2R1
Reactivity:Human, Mouse
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:96-Well Microplate

The Cytochrome P450 2R1 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect Cytochrome P450 2R1 protein expression profile in cells. The kit can be used for measuring the relative amounts of Cytochrome P450 2R1 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on Cytochrome P450 2R1.

Qualitative determination of Cytochrome P450 2R1 concentration is achieved by an indirect ELISA format. In essence, Cytochrome P450 2R1 is captured by Cytochrome P450 2R1-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: 120227, UniProt ID: Q6VVX0, OMIM: 600081/608713, Unigene: Hs.371427
Gene Symbol:CYP2R1
Sub Type:None
UniProt Protein Function:CYP2R1: Has a D-25-hydroxylase activity on both forms of vitamin D, vitamin D(2) and D(3). Defects in CYP2R1 are the cause of rickets vitamin D- dependent type 1B (VDDR1B); also known as pseudovitamin D(3) deficiency rickets due to 25-hydroxylase deficiency. A disorder caused by a selective deficiency of the active form of vitamin D (1,25-dihydroxyvitamin D3) and resulting in defective bone mineralization and clinical features of rickets. The patients sera have low calcium concentrations, low phosphate concentrations, elevated alkaline phosphatase activityand low levels of 25-hydroxyvitamin D. Belongs to the cytochrome P450 family.
UniProt Protein Details:

Protein type:Oxidoreductase; EC 1.14.13.15

Chromosomal Location of Human Ortholog: 11p15.2

Cellular Component: endoplasmic reticulum membrane; intracellular membrane-bound organelle; cytoplasm

Molecular Function:iron ion binding; oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen, reduced flavin or flavoprotein as one donor, and incorporation of one atom of oxygen; heme binding; steroid hydroxylase activity; oxygen binding; vitamin D3 25-hydroxylase activity

Biological Process: steroid metabolic process; organic acid metabolic process; vitamin metabolic process; xenobiotic metabolic process; exogenous drug catabolic process; vitamin D metabolic process

Disease: Vitamin D Hydroxylation-deficient Rickets, Type 1b

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 enzyme is a microsomal vitamin D hydroxylase that converts vitamin D into the active ligand for the vitamin D receptor. A mutation in this gene has been associated with selective 25-hydroxyvitamin D deficiency. [provided by RefSeq, Jul 2008]
UniProt Code:Q6VVX0
NCBI GenInfo Identifier:62286619
NCBI Gene ID:120227
NCBI Accession:Q6VVX0.1
UniProt Secondary Accession:Q6VVX0,Q2M3H3, Q5RT65,
UniProt Related Accession:Q6VVX0
Molecular Weight:57,359 Da
NCBI Full Name:Vitamin D 25-hydroxylase
NCBI Synonym Full Names:cytochrome P450, family 2, subfamily R, polypeptide 1
NCBI Official Symbol:CYP2R1  
NCBI Protein Information:vitamin D 25-hydroxylase; cytochrome P450 2R1; cytochrome P450, family 2, R1
UniProt Protein Name:Vitamin D 25-hydroxylase
UniProt Synonym Protein Names:Cytochrome P450 2R1
UniProt Gene Name:CYP2R1  
UniProt Entry Name:CP2R1_HUMAN
ComponentQuantity
96-Well Cell Culture Clear-Bottom Microplate2 plates
10X TBS24 mL
Quenching Buffer24 mL
Blocking Buffer50 mL
15X Wash Buffer50 mL
Primary Antibody Diluent12 mL
100x Anti-Phospho Target Antibody 60 µL
100x Anti-Target Antibody60 µL
Anti-GAPDH Antibody60 µL
HRP-Conjugated Anti-Rabbit IgG Antibody12 mL
HRP-Conjugated Anti-Mouse IgG Antibody12 mL
SDS Solution12 mL
Stop Solution24 mL
Ready-to-Use Substrate12 mL
Crystal Violet Solution12 mL
Adhesive Plate Seals2 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.

StepProcedure
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 2R1 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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