Rat CYP2E1 (Cytochrome P450, family 2, subfamily E, polypeptide 1) CLIA Kit (RTES00170)
Rat CYP2E1 (Cytochrome P450, family 2, subfamily E, polypeptide 1) CLIA Kit
This kit uses Sandwich-CLIA as the method. The micro CLIA plate provided in this kit has been pre-coated with an antibody specific to Rat CYP2E1 . Standards or samples are added to the appropriate micro CLIA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Rat CYP2E1 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 CYP2E1, biotinylated detection antibody and Avidin-HRP conjugate will appear fluorescence. The Relative light unit (RLU) value is measured spectrophotometrically by the Chemiluminescence immunoassay analyzer. The RLU value is positively associated with the concentration of Rat CYP2E1. The concentration of Rat CYP2E1 in the samples can be calculated by comparing the RLU of the samples to the standard curve.
|Detection range||0.63-40 ng/mL|
|Sample type||Serum, plasma and other biological fluids|
|Repeatability||CV < 15%|
This kit recognizes Rat CYP2E1 in samples. No significant cross-reactivity or interference between Rat CYP2E1 and analogues was observed.
As the RLU 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.
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Rat CYP2E1 were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Rat CYP2E1 were tested on 3 different plates, 20 replicates in each plate.
|Intra-assay Precision||Inter-assay Precision|
|C V (%)||12.09||8.09||8.20||8.91||7.57||8.60|
The recovery of Rat CYP2E1 spiked at three different levels in samples throughout the range of the assay was evaluated in various matrices.
|Sample Type||Range (%)||Average Recovery (%)|
|EDTA plasma (n=5)||100-116||106|
|Cell culture media (n=5)||102-114||108|
Samples were spiked with high concentrations of Rat CYP2E1 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)|
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.
|Micro CLIA 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 A||1 vial, 5 mL||4'C (shading light)|
|Substrate Reagent B||1 vial, 5 mL||4'C (shading light)|
|Plate Sealer||5 pieces|
|Product Description||1 copy|
|Certificate of Analysis||1 copy|
Rat CYP2E1 (Cytochrome P450, family 2, subfamily E, polypeptide 1) CLIA Kit (RTES00170) 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 100µL of Substrate mixture solution to each well. Cover with a new plate seal andincubate for no more than 5 min at 37°C. Protect the plate from light.
- 11. Determine the RLU value of each well immediately.
Rat CYP2E1 (Cytochrome P450, family 2, subfamily E, polypeptide 1) CLIA Kit (RTES00170) Protein Information
|UniProt Protein Function:||CYP2E1: Metabolizes several precarcinogens, drugs, and solvents to reactive metabolites. Inactivates a number of drugs and xenobiotics and also bioactivates many xenobiotic substrates to their hepatotoxic or carcinogenic forms. Belongs to the cytochrome P450 family.|
|UniProt Protein Details:|
Protein type:Xenobiotic Metabolism - metabolism by cytochrome P450; Oxidoreductase; Xenobiotic Metabolism - drug metabolism - cytochrome P450; Mitochondrial; EC 1.14.13.n7; Lipid Metabolism - linoleic acid; Lipid Metabolism - arachidonic acid; Endoplasmic reticulum
Cellular Component: endoplasmic reticulum; Golgi membrane; intracellular membrane-bound organelle; intrinsic to endoplasmic reticulum membrane; mitochondrion
Molecular Function:arachidonic acid epoxygenase activity; enzyme binding; FMN binding; heme binding; iron ion binding; monooxygenase activity; oxidoreductase activity; oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen, NADH or NADPH as one donor, and incorporation of one atom of 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; steroid hydroxylase activity
Biological Process: drug metabolic process; epoxygenase P450 pathway; heterocycle metabolic process; monoterpenoid metabolic process; response to drug; response to ethanol; response to organic nitrogen; response to ozone; steroid metabolic process; triacylglycerol metabolic process; xenobiotic metabolic process
|NCBI Summary:||facilitates hepatocyte sensitization to tumor necrosis factor-alpha (TNF-alpha)-mediated cell death; may play a role in xenobiotic metabolism [RGD, Feb 2006]|
|NCBI GenInfo Identifier:||1352193|
|NCBI Gene ID:||25086|
|UniProt Related Accession:||P05182|
|Molecular Weight:||56,627 Da|
|NCBI Full Name:||Cytochrome P450 2E1|
|NCBI Synonym Full Names:||cytochrome P450, family 2, subfamily e, polypeptide 1|
|NCBI Official Symbol:||Cyp2e1|
|NCBI Official Synonym Symbols:||Cyp2e|
|NCBI Protein Information:||cytochrome P450 2E1|
|UniProt Protein Name:||Cytochrome P450 2E1|
|UniProt Synonym Protein Names:||4-nitrophenol 2-hydroxylase (EC:1.14.13.n7); CYPIIE1; Cytochrome P450-J; Cytochrome P450RLM6|
|UniProt Gene Name:||Cyp2e1|
|UniProt Entry Name:||CP2E1_RAT|