Metabolism
Cytochrome P450 3A4/5 Colorimetric Cell-Based ELISA
- SKU:
- CBCAB01198
- Product Type:
- ELISA Kit
- ELISA Type:
- Cell Based
- Research Area:
- Metabolism
- Reactivity:
- Human
- Detection Method:
- Colorimetric
Description
| Product Name: | Cytochrome P450 3A4/5 Colorimetric Cell-Based ELISA |
| Product Code: | CBCAB01198 |
| ELISA Type: | Cell-Based |
| Target: | Cytochrome P450 3A4/5 |
| Reactivity: | Human |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nmStorage/Stability:4°C/6 Months |
| Format: | 96-Well Microplate |
The Cytochrome P450 3A4/5 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect Cytochrome P450 3A4/5 protein expression profile in cells. The kit can be used for measuring the relative amounts of Cytochrome P450 3A4/5 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on Cytochrome P450 3A4/5.
Qualitative determination of Cytochrome P450 3A4/5 concentration is achieved by an indirect ELISA format. In essence, Cytochrome P450 3A4/5 is captured by Cytochrome P450 3A4/5-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: 1576/1577, UniProt ID: P08684/P20815, OMIM: 124010/605325, Unigene: Hs.111944/Hs.695915 |
| Gene Symbol: | CYP3A4/5 |
| Sub Type: | None |
| UniProt Protein Function: | CYP3A4: Cytochromes P450 are a group of heme-thiolate monooxygenases. In liver microsomes, this enzyme is involved in an NADPH-dependent electron transport pathway. It performs a variety of oxidation reactions (e.g. caffeine 8-oxidation, omeprazole sulphoxidation, midazolam 1'-hydroxylation and midazolam 4- hydroxylation) of structurally unrelated compounds, including steroids, fatty acids, and xenobiotics. Acts as a 1,8-cineole 2- exo-monooxygenase. The enzyme also hydroxylates etoposide. Belongs to the cytochrome P450 family. |
| UniProt Protein Details: | Protein type:EC 1.14.14.1; Xenobiotic Metabolism - drug metabolism - cytochrome P450; Cell surface; Oxidoreductase; Membrane protein, integral; Xenobiotic Metabolism - metabolism by cytochrome P450; Xenobiotic Metabolism - drug metabolism - other enzymes; Lipid Metabolism - linoleic acid; Cofactor and Vitamin Metabolism - retinol Chromosomal Location of Human Ortholog: 7q21.1 Cellular Component: endoplasmic reticulum membrane; intracellular membrane-bound organelle; cytoplasm; integral to membrane Molecular Function:quinine 3-monooxygenase activity; albendazole monooxygenase activity; taurochenodeoxycholate 6alpha-hydroxylase activity; 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; oxidoreductase activity; oxygen binding; vitamin D3 25-hydroxylase activity; testosterone 6-beta-hydroxylase activity; steroid binding; enzyme binding; iron ion binding; heme binding; steroid hydroxylase activity; monooxygenase activity Biological Process: steroid metabolic process; drug catabolic process; xenobiotic metabolic process; androgen metabolic process; exogenous drug catabolic process; monoterpenoid metabolic process; alkaloid catabolic process; heterocycle metabolic process; lipid metabolic process; drug metabolic process; vitamin D metabolic process; steroid catabolic process |
| NCBI Summary: | This gene encodes a member of the cytochrome P450 superfamily of enzymes. The cytochrome P450 proteins are monooxygenases that catalyze many reactions involved in drug metabolism and synthesis of cholesterol, steroids and other lipids. This protein localizes to the endoplasmic reticulum and its expression is induced by glucocorticoids and some pharmacological agents. This enzyme is involved in the metabolism of approximately half the drugs in use today, including acetaminophen, codeine, cyclosporin A, diazepam and erythromycin. The enzyme also metabolizes some steroids and carcinogens. This gene is part of a cluster of cytochrome P450 genes on chromosome 7q21.1. Previously another CYP3A gene, CYP3A3, was thought to exist; however, it is now thought that this sequence represents a transcript variant of CYP3A4. Alternatively spliced transcript variants encoding different isoforms have been identified. [provided by RefSeq, Feb 2011] |
| UniProt Code: | P08684 |
| NCBI GenInfo Identifier: | 116241312 |
| NCBI Gene ID: | 1576 |
| NCBI Accession: | P08684.4 |
| UniProt Secondary Accession: | P08684,P05184, Q16757, Q9UK50, |
| UniProt Related Accession: | P08684 |
| Molecular Weight: | 503 |
| NCBI Full Name: | Cytochrome P450 3A4 |
| NCBI Synonym Full Names: | cytochrome P450, family 3, subfamily A, polypeptide 4 |
| NCBI Official Symbol: | CYP3A4 |
| NCBI Official Synonym Symbols: | HLP; CP33; CP34; CYP3A; NF-25; CYP3A3; P450C3; CYPIIIA3; CYPIIIA4; P450PCN1 |
| NCBI Protein Information: | cytochrome P450 3A4; nifedipine oxidase; cytochrome P450 3A3; cytochrome P450 HLp; cytochrome P450-PCN1; cytochrome P450 NF-25; albendazole sulfoxidase; quinine 3-monooxygenase; albendazole monooxygenase; P450-III, steroid inducible; glucocorticoid-inducible P450; 1,8-cineole 2-exo-monooxygenase; taurochenodeoxycholate 6-alpha-hydroxylase; cytochrome P450, subfamily IIIA (niphedipine oxidase), polypeptide 3; cytochrome P450, subfamily IIIA (niphedipine oxidase), polypeptide 4 |
| UniProt Protein Name: | Cytochrome P450 3A4 |
| UniProt Synonym Protein Names: | 1,8-cineole 2-exo-monooxygenase (EC:1.14.13.157); Albendazole monooxygenase (EC:1.14.13.32); Albendazole sulfoxidase; CYPIIIA3; CYPIIIA4; Cytochrome P450 3A3; Cytochrome P450 HLp; Cytochrome P450 NF-25; Cytochrome P450-PCN1; Nifedipine oxidase; Quinine 3-monooxygenase (EC:1.14.13.67); Taurochenodeoxycholate 6-alpha-hydroxylase (EC:1.14.13.97) |
| Protein Family: | Cytochrome |
| UniProt Gene Name: | CYP3A4 |
| UniProt Entry Name: | CP3A4_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 3A4/5 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.)
