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Mouse Alcohol Dehydrogenase ELISA Kit (A4731)

1 Citation
Standard Curve - Mouse Alcohol Dehydrogenase ELISA Kit (A4731) - Antibodies.com
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$40
Dispatched from St. Louis, MO.
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Name
Mouse Alcohol Dehydrogenase ELISA Kit
Description
Mouse Alcohol Dehydrogenase ELISA Kit is a sandwich Enzyme-Linked Immunosorbent Assay (sELISA) designed for the in vitro quantitative determination of mouse Alcohol Dehydrogenase in serum, plasma, tissue homogenates or other biological fluids.
Assay Type
Sandwich (quantitative)
Principle of Assay
Mouse Alcohol Dehydrogenase ELISA Kit (A4731) employs the sandwich enzyme immunoassay technique for the quantitative measurement of mouse Alcohol Dehydrogenase in serum, plasma, tissue homogenates or other biological fluids. An antibody specific for Alcohol Dehydrogenase has been pre-coated onto a 96-well microtiter plate. The standards and test samples are added into the wells and the Alcohol Dehydrogenase present in each sample is bound to the wells by the immobilized antibody. Following incubation, the wells are washed and then incubated with Biotinylated Anti-Alcohol Dehydrogenase Antibody, which binds the captured Alcohol Dehydrogenase present in each well. Following incubation, unbound biotinylated detection antibody is removed by washing, and an HRP-Avidin conjugate is added to the wells and the microtiter plate is incubated. Following incubation and washing, TMB substrate solution is then used to visualize the HRP enzymatic reaction by catalysis to produce a blue-coloured product that changes to yellow after addition of acidic stop solution. The density of yellow is proportional to the amount of Alcohol Dehydrogenase captured in each well. The concentration of Alcohol Dehydrogenase can then be calculated by reading the O.D. absorbance at 450nm in a microplate reader and referring to the standard curve.
Platform
Pre-coated Microplate (12 x 8 well strips)
Detection Type
Colourmetric
Instrument
Colorimetric Microplate Reader
Sample Type
Serum, plasma, tissue homogenates or other biological fluids.
Sensitivity
0.09 ng/ml
Product Range
0.312-20 ng/ml
Assay Time
4h 30m
Reactivity
Mouse
Recovery
Sample Type n Range Average
Serum 5 80% - 102% 91%
EDTA Plasma 5 81% - 100% 90%
Heparin Plasma 5 80% - 89% 84%
Linearity
Sample Type 1:2 1:4 1:8 1:16
Serum (n=5) 87-91% 87-107% 74-101% 92-97%
EDTA Plasma (n=5) 90-105% 84-101% 90-101% 79-108%
Heparin Plasma (n=5) 84-95% 92-105% 82-105% 89-91%
Precision
Intra-assay: CV < 10%, Inter-assay: CV < 12%
General Notes
Information online is representative. Always refer to the Product Manual inside the kit.
Storage
Store kit components at +4°C or -20°C as instructed. Do not use past expiration date!
Synonyms
ADH1, ADH1A, Alcohol dehydrogenase 1A, Alcohol dehydrogenase subunit alpha
Disclaimer
This product is for research use only. It is not intended for diagnostic or therapeutic use.
Kit Components
Item Quantity Storage
Pre-Coated 96 Well Microplate 12 x 8 Well Strips -20°C
Lyopholized Standard 2 Vials -20°C
Detection Solution A 120μl -20°C
Detection Solution B 120µl -20°C
Wash Buffer (30X) 20ml +4°C
Sample Dilution Buffer 45ml -20°C
TMB Substrate 9ml +4°C
Stop Solution 6ml +4°C
Plate Sealers 5 Adhesive Strips -
Required Materials
The following materials are not included in the kit, but are required to run this assay:
  • Microplate reader capable of measuring absorbance at 450nm ± 10nm.
  • Squirt bottle, multichannel pipette reservoir, or automated microplate washer.
  • Graph paper or computer software capable of generating logarithmic functions.
  • Test tubes and Eppendorf tubes to prepare standards and samples.
  • Multi- and single-channel pipettes and sterile pipette tips.
  • Deionized or distilled water.
  • Absorbent paper towels.
  • 37°C incubator.
Important Notes
  • Sample concentrations should be estimated before being used in the assay and a proper dilution factor should be selected to make the diluted target protein concentration fall in the optimal detection range of this kit.
  • Hemolysis can greatly impact the validity of test results. Take care to minimize hemolysis.
  • Samples should be brought to room temperature before starting the assay.
Bring all reagents and samples to room temperature before use.
We recommend that all standards and samples be assayed in duplicate.
Step 1
Prepare all reagents, samples, and working standards as directed in the previous sections.
Step 2
Determine the number of microplate strips required for the assay and insert them into the plate frame. Unused strips should be stored in a foil pouch at -20°C.
Step 3
Add 100μl of standard solutions to the standard wells. Note: A plate layout is provided to record standards and samples assayed.
Step 4
Add 100μl of samples to the sample wells.
Step 5
Cover with an adhesive plate sealer provided and incubate at 37°C for 2 hours.
Step 6
Remove the plate sealer and aspirate the liquid from the plate.
Step 7
Add 100μl of working Detection Solution A to each well.
Step 8
Cover with a new adhesive plate sealer and incubate at 37°C for 60 minutes.
Step 9
Remove the plate sealer, aspirate the liquid from the plate, and wash the plate three times with Wash Buffer. Soak wells with 300μl of Wash Buffer for 1-2 minutes each time. Note: Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Blot the plate onto paper towels or other absorbent material. Do not let the wells dry completely at any time!
Step 10
Add 100μl of working Detection Solution B to each well.
Step 11
Cover with a new adhesive plate sealer and incubate at 37°C for 60 minutes.
Step 12
Remove the plate sealer, aspirate the liquid from the plate, and wash the plate five times with Wash Buffer. Soak wells with 300μl of Wash Buffer for 1-2 minutes each time.
Step 13
Add 90μl of TMB Substrate to each well. Cover with a new plate sealer and incubate at 37°C in the dark for 15-25 minutes (do not exceed 30 minutes). Note: This incubation time is for reference only, the optimal reaction time should be determined by the end-user and can be shortened or extended according to the actual color change. The reaction may be terminated when a gradient is apparent in the standard wells.
Step 14
Add 50μl of Stop Solution to each well. The color in the wells should change from blue to yellow immediately. Note: If the color in the wells is green or the color change does not appear uniform, gently tap the plate to ensure thorough mixing.
Step 15
Determine the optical density (O.D. value) of each well immediately using a microplate reader set to 450 nm.

  • Scientific Validation DataValidation Data (1)

Standard Curve - Mouse Alcohol Dehydrogenase ELISA Kit (A4731) - Antibodies.comEnlarge Image

Standard Curve - Mouse Alcohol Dehydrogenase ELISA Kit (A4731)

Representative standard curve for Mouse Alcohol Dehydrogenase ELISA Kit (A4731).

  • Product Citations (1)

PubMed LogoView Publication
Interaction between dual specificity phosphatase-1 and cullin-1 attenuates alcohol-related liver disease by restoring p62-mediated mitophagy
This publication cites Mouse Alcohol Dehydrogenase ELISA Kit (A4731).
Article Abstract

Besides abstinence, no effective treatment exists for alcohol-related liver disease (ALD), a dreaded consequence of alcohol abuse. In this study, we assessed the roles on ALD of dual specificity phosphatase-1 (DUSP1), an hepatoprotective enzyme, and Cullin-1 (CUL1), a member of the E3 ubiquitin ligase complex that exerts also transcriptional suppression of mitochondrial genes. Alcohol treatment downregulated hepatic DUSP1 expression in wild-type mice. Notably, DUSP1 transgenic (Dusp1Tg ) mice showed resistance to alcohol-mediated hepatic dysfunction, as evidenced by decreased AST/ALT activity, improved alcohol metabolism, and suppressed liver fibrosis, inflammation, and oxidative stress. Functional experiments demonstrated that DUSP1 overexpression prevents alcohol-mediated mitochondrial damage in hepatocytes through restoring mitophagy. Accordingly, pharmacological blockade of mitophagy abolished the hepatoprotective actions of DUSP1. Molecular assays showed that DUSP1 binds cytosolic CUL1 and prevents its translocation to the nucleus. Importantly, CUL1 silencing restored the transcription of p62 and Parkin, resulting in mitophagy activation, and sustained mitochondrial integrity and hepatocyte function upon alcohol stress. These results indicate that alcohol-mediated DUSP1 downregulation interrupts DUSP1/CUL1 interaction, leading to CUL1 nuclear translocation and mitophagy inhibition via transcriptional repression of p62 and Parkin. Thus, targeting the DUSP1/CUL1/p62 axis will be a key approach to restore hepatic mitophagy as well as alleviate symptoms of ALD.

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