Alpha-Melanocyte-stimulating hormone (alpha-MSH), a proteolyic cleavage product from ACTH (1-13), is an endogenous peptide hormone and neuropeptide of the melanocortin family. It is a non-selective full agonist of the melanocortin receptors MC1, MC3, MC4, and MC5, but not MC2. Alpha-MSH is important for pigmentation primarily of the hair and skin. It also plays a role in feeding behavior, energy homeostasis, sexual activity, and protection against ischemia and reperfusion injury.

In this assay, a biotinylated Alpha-MSH peptide is spiked into the samples and standards. The samples and standards are then added to the plate, where the biotinylated Alpha-MSH peptide competes with endogenous (unlabeled) Alpha-MSH for binding to the anti-Alpha-MSH antibody. After a wash step, any bound biotinylated Alpha-MSH 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 Alpha-MSH peptide and inversely proportional to the amount of endogenous Alpha- MSH in the standard or samples. A standard curve of known concentration of Alpha-MSH peptide can be established and the concentration of Alpha-MSH peptide in the samples can be calculated accordingly.

• 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

• 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

• 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 450 nm
• SigmaPlot software (or other software that can perform four-parameter logistic regression models)

1. Prepare all reagents, samples and standards as instructed.
2. Add 100 µl detection antibody to each well.
3. Incubate 1.5 h at RT or O/N at 4°C.
4. Add 100 µl standard or sample to each well.
5. Incubate 2.5 h at RT.
6. Add 100 µl prepared streptavidin solution.
7. Incubate 45 min at RT.
8. Add 100 µl TMB One-Step Substrate Reagent to each well.
9. Incubate 30 min at RT.
10. Add 50 µl Stop Solution to each well.
11. Read plate at 450 nm immediately.

Standard, Biotinylated Alpha-MSH 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°C to 8°C. Return unused wells to the pouch containing desiccant pack and reseal along entire edge.