Introduction
Melanin-concentrating hormone (MCH) is a cyclic 19-amino acid neuropeptide. It binds to two G-coupled protein receptors (GCPRs), MCHR1 and MCHR2. MCH is involved in the regulation of the sleep cycle, maternal behavior, eating behaviors, energy conservation, reproduction and skin pigmentation.
The RayBio® MCH Enzyme Immunoassay (EIA) Kit is an in vitro quantitative assay for detecting MCH peptide based on the competitive enzyme immunoassay principle. In this assay, a biotinylated MCH peptide is spiked into the samples and standards. The samples and standards are then added to the plate, where the biotinylated MCH peptide competes with endogenous (unlabeled) MCH for binding to the anti-MCH antibody. After a wash step, any bound biotinylated MCH then interacts with horseradish peroxidase (HRP)-streptavidin, which catalyzes a color development reaction. The intensity of the colorimetric signal is directly proportional to the amount of captured biotinylated MCH peptide and inversely proportional to the amount of endogenous MCH in the standard or samples. A standard curve of known concentration of MCH peptide can be established and the concentration of MCH peptide in the samples can be calculated accordingly.
Product Features
- Strip plates and additional reagents allow for use in multiple experiments
- Quantitative protein detection
- Establishes normal range
- The best products for confirmation of antibody array data
Application Notes
Kit Components
- Pre-Coated 96-well Strip Microplate
- Wash Buffer
- Standard Peptide
- Assay Diluent(s)
- Biotinylated Peptide
- HRP-Streptavidin
- TMB One-Step Substrate
- Stop Solution
- Assay Diagram
- Positive Control Sample
- Capture Antibody
- User Manual
Other Materials Required
- Distilled or deionized water
- Precision pipettes to deliver 2 µl to 1 ml volumes
- Adjustable 1-25 ml pipettes for reagent preparation
- 100 ml and 1 liter graduated cylinders
- Tubes to prepare standard and sample dilutions
- Orbital shaker
- Aluminum foil
- Saran Wrap
- Absorbent paper
- Microplate reader capable of measuring absorbance at 450nm
- SigmaPlot software (or other software that can perform four-parameter logistic regression models)
Protocol Outline
- Prepare all reagents, samples and standards as instructed.
- Add 100 µl detection antibody to each well.
- Incubate 1.5 h at RT or O/N at 4°C.
- Add 100 µl standard or sample to each well.
- Incubate 2.5 h at RT.
- Add 100 µl prepared streptavidin solution.
- Incubate 45 min at RT.
- Add 100 µl TMB One-Step Substrate Reagent to each well.
- Incubate 30 min at RT.
- Add 50 µl Stop Solution to each well.
- Read plate at 450 nm immediately.
Storage/Stability
Standard, Biotinylated MCH peptide, and Positive Control should be stored at -20°C after arrival. Avoid multiple freeze-thaws. The remaining kit components may be stored at 4°C. Opened Microplate Wells and antibody (Item N) may be stored for up to 1 month at 2° to 8°C. Return unused wells to the pouch containing desiccant pack and reseal along entire edge.
