Rat CYP1A1 (Cytochrome P450, family 1, subfamily A, polypeptide 1) ELISA Kit (RTES01086)

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RTES01086
€649

Description

ELISA Kit Technical ManualMSDS

Rat CYP1A1 (Cytochrome P450, family 1, subfamily A, 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 CYP1A1 . 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 CYP1A1 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 CYP1A1, 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 CYP1A1. The concentration of Rat CYP1A1 in samples can be calculated by comparing the OD of the samples to the standard curve.

Assay typeSandwich
Format96T
Assay time4.5h
ReactivityRat
Detection MethodColormetric
Detection Range0.16-10 ng/mL
Sensitivity0.10 ng/mL
Sample Volume Required Per Well100uL
Sample TypeSerum, plasma and other biological fluids

Specificity

This kit recognizes Rat CYP1A1 in samples. No significant cross-reactivity or interference between Rat CYP1A1 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
102.33
2.366
2.3482.288
51.476
1.534
1.5051.445
2.50.852
0.844
0.8480.788
1.250.384
0.386
0.3850.325
0.630.251
0.225
0.2380.178
0.320.161
0.151
0.1560.096
0.160.101
0.119
0.110.05
00.057
0.063
0.06--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Rat CYP1A1 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Rat CYP1A1 were tested on 3 different plates, 20 replicates in each plate.

Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (ng/mL)0.491.394.540.521.284.64
Standard deviation0.030.060.220.040.060.22
C V (%)6.124.324.857.694.694.74

Recovery

The recovery of Rat CYP1A1 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)93-10599
EDTA plasma (n=5)87-10394
Cell culture media (n=5)91-10597

Linearity

Samples were spiked with high concentrations of Rat CYP1A1 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)
1:2Range (%)86-10085-9785-101
Average (%)929292
1:4Range (%)92-10685-10084-96
Average (%)999189
1:8Range (%)94-10483-9784-96
Average (%)999089
1:16Range (%)86-10187-10182-97
Average (%)929289

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 Standard2 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 Diluent1 vial, 20 mL4'C, 6 months
Biotinylated Detection Ab Diluent1 vial, 14 mL
HRP Conjugate Diluent1 vial, 14 mL
Concentrated Wash Buffer (25X)1 vial, 30 mL
Substrate Reagent1 vial, 10 mL4'C(shading light)
Stop Solution1 vial, 10 mL4'C
Plate Sealer5 pieces
Product Description1 copy
Certificate of Analysis1 copy

Rat CYP1A1 (Cytochrome P450, family 1, subfamily A, polypeptide 1) ELISA Kit (RTES01086) 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 CYP1A1 (Cytochrome P450, family 1, subfamily A, polypeptide 1) ELISA Kit (RTES01086) Protein Information

UniProt Protein Function:CYP1A1: 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
UniProt Protein Details:

Protein type:Oxidoreductase; Xenobiotic Metabolism - metabolism by cytochrome P450; Amino Acid Metabolism - tryptophan; EC 1.14.14.1; Cofactor and Vitamin Metabolism - retinol

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

Molecular Function:catalytic activity; demethylase activity; enzyme binding; flavonoid 3'-monooxygenase activity; heme binding; iron ion binding; monooxygenase activity; oxidoreductase activity; oxidoreductase activity, acting on diphenols and related substances as donors; 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; protein binding; steroid hydroxylase activity

Biological Process: 9-cis-retinoic acid biosynthetic process; aging; amine metabolic process; camera-type eye development; cell proliferation; coumarin metabolic process; developmental process; dibenzo-p-dioxin catabolic process; dibenzo-p-dioxin metabolic process; drug metabolic process; embryonic development ending in birth or egg hatching; flavonoid metabolic process; gut development; heterocycle metabolic process; hydrogen peroxide biosynthetic process; insecticide metabolic process; liver development; maternal process involved in parturition; porphyrin metabolic process; response to antibiotic; response to arsenic; response to drug; response to food; response to herbicide; response to hyperoxia; response to hypoxia; response to iron(III) ion; response to lipopolysaccharide; response to nematode; response to organic cyclic substance; response to organic substance; response to toxin; response to virus; response to vitamin A; response to wounding; toxin metabolic process

NCBI Summary:monooxygenase that plays a role in dioxin metabolism and detoxification which is 3-methylcholanthrene-inducible [RGD, Feb 2006]
UniProt Code:P00185
NCBI GenInfo Identifier:46048641
NCBI Gene ID:24296
NCBI Accession:NP_036672.2
UniProt Related Accession:P00185
Molecular Weight:59,393 Da
NCBI Full Name:cytochrome P450 1A1
NCBI Synonym Full Names:cytochrome P450, family 1, subfamily a, polypeptide 1
NCBI Official Symbol:Cyp1a1  
NCBI Official Synonym Symbols:AHH; AHRR; CP11; CYP1; Cyp45c; P1-450; P450-C; P450DX; Cypc45c; P-450MC  
NCBI Protein Information:cytochrome P450 1A1
UniProt Protein Name:Cytochrome P450 1A1
UniProt Synonym Protein Names:CYPIA1; Cytochrome P450-C; Cytochrome P450MT2
Protein Family:Cytochrome
UniProt Gene Name:Cyp1a1  
UniProt Entry Name:CP1A1_RAT
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Additional Information

Reactivity:
Rat
ELISA Type:
Sandwich
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