Rat 15-LO (Arachidonate 15-Lipoxygenase) ELISA Kit (RTES00835)

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RTES00835
€649

Description

ELISA Kit Technical ManualMSDS

Rat 15-LO (Arachidonate 15-Lipoxygenase) 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 15-LO . 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 15-LO 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 15-LO, 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 15-LO. The concentration of Rat 15-LO 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 Range31.25-2000 pg/mL
Sensitivity18.75 pg/mL
Sample Volume Required Per Well100uL
Sample TypeSerum, plasma and other biological fluids

Specificity

This kit recognizes Rat 15-LO in samples. No significant cross-reactivity or interference between Rat 15-LO 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
(pg/mL)
O.D Average Corrected
20002.372
2.398
2.3852.319
10001.617
1.633
1.6251.559
5000.994
0.976
0.9850.919
2500.411
0.447
0.4290.363
1250.245
0.241
0.2430.177
62.50.176
0.16
0.1680.102
31.250.116
0.12
0.1180.052
00.065
0.067
0.066--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Rat 15-LO were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Rat 15-LO were tested on 3 different plates, 20 replicates in each plate.

Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (pg/mL)107.65303.91906.35115.58307.86934.63
Standard deviation5.6314.4145.507.8717.6441.50
C V (%)5.234.745.026.815.734.44

Recovery

The recovery of Rat 15-LO 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)87-9994
EDTA plasma (n=5)93-10499
Cell culture media (n=5)87-10094

Linearity

Samples were spiked with high concentrations of Rat 15-LO 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 (%)88-9997-10995-111
Average (%)94104101
1:4Range (%)90-10381-9489-100
Average (%)978694
1:8Range (%)94-10886-9882-95
Average (%)1009288
1:16Range (%)87-9981-9584-97
Average (%)948889

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 15-LO (Arachidonate 15-Lipoxygenase) ELISA Kit (RTES00835) 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 15-LO (Arachidonate 15-Lipoxygenase) ELISA Kit (RTES00835) Protein Information

UniProt Protein Function:ALOX15: Converts arachidonic acid to 15S- hydroperoxyeicosatetraenoic acid. Also acts on C-12 of arachidonate as well as on linoleic acid. Belongs to the lipoxygenase family.
UniProt Protein Details:

Protein type:Lipid Metabolism - arachidonic acid; EC 1.13.11.31; Motility/polarity/chemotaxis; Oxidoreductase; Lipid Metabolism - linoleic acid; EC 1.13.11.33

Cellular Component: extrinsic to internal side of plasma membrane; membrane; cytoplasm; plasma membrane; lipid particle; cytosol; sarcolemma

Molecular Function:phosphatidylinositol-4,5-bisphosphate binding; arachidonate 12-lipoxygenase activity; hepoxilin A3 synthase activity; arachidonate 15-lipoxygenase activity; hepoxilin-epoxide hydrolase activity; iron ion binding; potassium channel inhibitor activity

Biological Process: icosanoid biosynthetic process; ossification; positive regulation of heterotypic cell-cell adhesion; wound healing; phosphatidylethanolamine biosynthetic process; positive regulation of cell growth; bone mineralization; regulation of membrane potential; positive regulation of actin filament polymerization; lipoxygenase pathway; negative regulation of adaptive immune response; positive regulation of cell proliferation; arachidonic acid metabolic process; fatty acid oxidation; hepoxilin biosynthetic process; negative regulation of cell volume; apoptotic cell clearance; negative regulation of apoptosis

NCBI Summary:demonstrates 12-lipoxygenase and some 15-lipoxygenase enzyme activity [RGD, Feb 2006]
UniProt Code:Q02759
NCBI GenInfo Identifier:31542125
NCBI Gene ID:81639
NCBI Accession:NP_112272.2
UniProt Secondary Accession:Q02759,F1MA51,
UniProt Related Accession:Q02759
Molecular Weight:75,378 Da
NCBI Full Name:arachidonate 15-lipoxygenase
NCBI Synonym Full Names:arachidonate 15-lipoxygenase
NCBI Official Symbol:Alox15  
NCBI Official Synonym Symbols:12-LOX; 15-LOX; Alox12; Alox12l  
NCBI Protein Information:arachidonate 15-lipoxygenase
UniProt Protein Name:Arachidonate 15-lipoxygenase
UniProt Synonym Protein Names:12/15-lipoxygenase; Arachidonate 12-lipoxygenase, leukocyte-type (EC:1.13.11.31); 12-LOX; Arachidonate omega-6 lipoxygenase
UniProt Gene Name:Alox15  
UniProt Entry Name:LOX15_RAT
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Additional Information

Product type:
ELISA
Reactivity:
Rat
ELISA Type:
Sandwich
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