Rat CYP1B1 (Cytochrome P450, family 1, subfamily B, polypeptide 1) ELISA Kit (RTES01088)
- SKU:
- RTES01088
Description
Rat CYP1B1 (Cytochrome P450, family 1, subfamily B, polypeptide 1) ELISA Kit
This ELISA kit uses Sandwich-ELISA as the method. The micro ELISA plate provided in this kit has been pre-coated with an antibody specific to Rat CYP1B1 . Standards or samples are added to the appropriate micro ELISA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Rat CYP1B1 and Avidin-Horseradish Peroxidase (HRP) conjugate are added to each micro plate well successively and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain Rat CYP1B1, biotinylated detection antibody and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by adding Stop Solution and the color turns yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The OD value is proportional to the concentration of Rat CYP1B1. The concentration of Rat CYP1B1 in samples can be calculated by comparing the OD of the samples to the standard curve.
| Assay type | Sandwich |
| Format | 96T |
| Assay time | 4.5h |
| Reactivity | Rat |
| Detection Method | Colormetric |
| Detection Range | 0.78-50 ng/mL |
| Sensitivity | 0.47 ng/mL |
| Sample Volume Required Per Well | 100uL |
| Sample Type | Serum, plasma and other biological fluids |
Specificity
This kit recognizes Rat CYP1B1 in samples. No significant cross-reactivity or interference between Rat CYP1B1 and analogues was observed.
Typical Data
As the OD values of the standard curve may vary according to the conditions of the actual assay performance (e.g. operator, pipetting technique, washing technique or temperature effects), the operator should establish a standard curve for each test. Typical standard curve and data is provided below for reference only.
| Concentration (ng/mL) | O.D | Average | Corrected |
|---|---|---|---|
| 50 | 2.441 2.449 | 2.445 | 2.391 |
| 25 | 1.523 1.559 | 1.541 | 1.487 |
| 12.5 | 0.91 0.874 | 0.892 | 0.838 |
| 6.25 | 0.413 0.443 | 0.428 | 0.374 |
| 3.13 | 0.223 0.213 | 0.218 | 0.164 |
| 1.57 | 0.163 0.147 | 0.155 | 0.101 |
| 0.78 | 0.096 0.114 | 0.105 | 0.051 |
| 0 | 0.049 0.059 | 0.054 | -- |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Rat CYP1B1 were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Rat CYP1B1 were tested on 3 different plates, 20 replicates in each plate.
| Intra-assay Precision | Inter-assay Precision | |||||
|---|---|---|---|---|---|---|
| Sample | 1 | 2 | 3 | 1 | 2 | 3 |
| n | 20 | 20 | 20 | 20 | 20 | 20 |
| Mean (ng/mL) | 2.72 | 7.32 | 19.70 | 2.75 | 7.45 | 18.14 |
| Standard deviation | 0.18 | 0.39 | 0.62 | 0.15 | 0.40 | 0.59 |
| C V (%) | 6.62 | 5.33 | 3.15 | 5.45 | 5.37 | 3.25 |
Recovery
The recovery of Rat CYP1B1 spiked at three different levels in samples throughout the range of the assay was evaluated in various matrices.
| Sample Type | Range (%) | Average Recovery (%) |
|---|---|---|
| Serum (n=5) | 90-101 | 96 |
| EDTA plasma (n=5) | 94-111 | 101 |
| Cell culture media (n=5) | 96-110 | 101 |
Linearity
Samples were spiked with high concentrations of Rat CYP1B1 and diluted with Reference Standard & Sample Diluent to produce samples with values within the range of the assay.
| Serum (n=5) | EDTA plasma (n=5) | Cell culture media (n=5) | ||
|---|---|---|---|---|
| 1:2 | Range (%) | 90-104 | 87-101 | 88-100 |
| Average (%) | 95 | 93 | 93 | |
| 1:4 | Range (%) | 92-105 | 81-95 | 87-101 |
| Average (%) | 99 | 87 | 92 | |
| 1:8 | Range (%) | 89-102 | 84-94 | 87-103 |
| Average (%) | 95 | 89 | 94 | |
| 1:16 | Range (%) | 88-104 | 83-95 | 85-97 |
| Average (%) | 95 | 89 | 92 |
Kit Components & Storage
An unopened kit can be stored at 4'C for 1 month. If the kit is not used within 1 month, store the items separately according to the following conditions once the kit is received.
| Item | Specifications | Storage |
|---|---|---|
| Micro ELISA Plate(Dismountable) | 8 wells X12 strips | -20'C, 6 months |
| Reference Standard | 2 vials | |
| Concentrated Biotinylated Detection Ab (100X) | 1 vial, 120 uL | |
| Concentrated HRP Conjugate (100X) | 1 vial, 120 uL | -20'C(shading light), 6 months |
| Reference Standard & Sample Diluent | 1 vial, 20 mL | 4'C, 6 months |
| Biotinylated Detection Ab Diluent | 1 vial, 14 mL | |
| HRP Conjugate Diluent | 1 vial, 14 mL | |
| Concentrated Wash Buffer (25X) | 1 vial, 30 mL | |
| Substrate Reagent | 1 vial, 10 mL | 4'C(shading light) |
| Stop Solution | 1 vial, 10 mL | 4'C |
| Plate Sealer | 5 pieces | |
| Product Description | 1 copy | |
| Certificate of Analysis | 1 copy |
Rat CYP1B1 (Cytochrome P450, family 1, subfamily B, polypeptide 1) ELISA Kit (RTES01088) Assay procedure
- 1. Set standard, test sample and control (zero) wells on the pre-coated plate and record theirpositions. It is recommended to measure each standard and sample in duplicate. Note: addall solutions to the bottom of the plate wells while avoiding contact with the well walls. Ensuresolutions do not foam when adding to the wells.
- 2. Aliquot 100µl of standard solutions into the standard wells.
- 3. Add 100µl of Sample / Standard dilution buffer into the control (zero) well.
- 4. Add 100µl of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids) into test sample wells.
- 5. Cover the plate with the sealer provided in the kit and incubate for 90 min at 37°C.
- 6. Aspirate the liquid from each well, do not wash. Immediately add 100µL of BiotinylatedDetection Ab working solution to each well. Cover the plate with a plate seal and gently mix.Incubate for 1 hour at 37°C.
- 7. Aspirate or decant the solution from the plate and add 350µL of wash buffer to each welland incubate for 1-2 minutes at room temperature. Aspirate the solution from each well andclap the plate on absorbent filter paper to dry. Repeat this process 3 times. Note: a microplatewasher can be used in this step and other wash steps.
- 8. Add 100µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37°C.
- 9. Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
- 10. Add 90µL of Substrate Reagent to each well. Cover with a new plate seal and incubate forapproximately 15 min at 37°C. Protect the plate from light. Note: the reaction time can beshortened or extended according to the actual color change, but not by more than 30min.
- 11. Add 50 µL of Stop Solution to each well. Note: Adding the stop solution should be done inthe same order as the substrate solution.
- 12. Determine the optical density (OD value) of each well immediately with a microplate readerset at 450 nm.
Rat CYP1B1 (Cytochrome P450, family 1, subfamily B, polypeptide 1) ELISA Kit (RTES01088) Protein Information
| UniProt Protein Function: | CYP1B1: Cytochromes P450 are a group of heme-thiolate monooxygenases. In liver microsomes, this enzyme is involved in an NADPH-dependent electron transport pathway. It oxidizes a variety of structurally unrelated compounds, including steroids, fatty acids, and xenobiotics. Defects in CYP1B1 are the cause of primary congenital glaucoma type 3A (GLC3A). GLC3A is an autosomal recessive form of primary congenital glaucoma (PCG). PCG is characterized by marked increase of intraocular pressure at birth or early childhood, large ocular globes (buphthalmos) and corneal edema. It results from developmental defects of the trabecular meshwork and anterior chamber angle of the eye that prevent adequate drainage of aqueous humor. Defects in CYP1B1 are a cause of primary open angle glaucoma (POAG). POAG is a complex and genetically heterogeneous ocular disorder characterized by a specific pattern of optic nerve and visual field defects. The angle of the anterior chamber of the eye is open, and usually the intraocular pressure is increased. The disease is asymptomatic until the late stages, by which time significant and irreversible optic nerve damage has already taken place. In some cases, POAG shows digenic inheritance involving mutations in CYP1B1 and MYOC genes. Defects in CYP1B1 are a cause of Peters anomaly (PAN). Peters anomaly is a congenital defect of the anterior chamber of the eye. Belongs to the cytochrome P450 family. |
| UniProt Protein Details: | Protein type:Motility/polarity/chemotaxis; Oxidoreductase; Membrane protein, peripheral; Endoplasmic reticulum; Amino Acid Metabolism - tryptophan; EC 1.14.14.1; Xenobiotic Metabolism - metabolism by cytochrome P450 Cellular Component: endoplasmic reticulum membrane; integral to membrane; intracellular membrane-bound organelle; mitochondrion; nucleus Molecular Function:heme binding; iron ion binding; monooxygenase activity; oxidoreductase activity; oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen; 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 Biological Process: adrenal gland development; angiogenesis; arachidonic acid metabolic process; aromatic compound metabolic process; benzene and derivative metabolic process; blood vessel morphogenesis; cell adhesion; collagen fibril organization; dibenzo-p-dioxin metabolic process; DNA modification; endothelial cell migration; estrogen metabolic process; induction of apoptosis by oxidative stress; inhibition of NF-kappaB transcription factor; male gonad development; membrane lipid catabolic process; negative regulation of cell adhesion mediated by integrin; negative regulation of cell migration; negative regulation of cell proliferation; nitric oxide biosynthetic process; positive regulation of angiogenesis; positive regulation of apoptosis; positive regulation of JAK-STAT cascade; positive regulation of smooth muscle cell migration; positive regulation of translation; response to arsenic; response to estradiol stimulus; response to follicle-stimulating hormone stimulus; response to nutrient; response to organic cyclic substance; response to organic substance; response to steroid hormone stimulus; response to toxin; retinal metabolic process; retinol metabolic process; steroid metabolic process; toxin metabolic process; xenobiotic metabolic process |
| NCBI Summary: | plays a role in polycyclic aromatic hydrocarbon metabolism in adrenal microsomes [RGD, Feb 2006] |
| UniProt Code: | Q64678 |
| NCBI GenInfo Identifier: | 6978737 |
| NCBI Gene ID: | 25426 |
| NCBI Accession: | NP_037072.1 |
| UniProt Related Accession: | Q64678 |
| Molecular Weight: | 61.9 kDa |
| NCBI Full Name: | cytochrome P450 1B1 |
| NCBI Synonym Full Names: | cytochrome P450, family 1, subfamily b, polypeptide 1 |
| NCBI Official Symbol: | Cyp1b1 |
| NCBI Protein Information: | cytochrome P450 1B1 |
| UniProt Protein Name: | Cytochrome P450 1B1 |
| UniProt Synonym Protein Names: | CYPIB1; Cytochrome P450RAP |
| Protein Family: | Cytochrome |
| UniProt Gene Name: | Cyp1b1 |
| UniProt Entry Name: | CP1B1_RAT |
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