Human Glucagon EasyTest™ EIA Kit

Product Features

Product Name            Human Glucagon EasyTest™ EIA Kit
Assay Type                   Solid Phase Competitive ELISA
Format                           96-well strip plate
Assay Process             Two wash step, three incubations
Assay Length              2.0 hours
Sample Volume         Serum , Plasma, 50µL
Detection Method    Colorimetric
Sensitivity                   120pg/mL
Assay Range                9.8-40000pg/mL
Reactivity                     Human
Cross-Reactivity       No cross-reactivity observed with available related molecules
Gene Names               GCG; GLP1; GLP2; GRPP
Gene ID                          2641
Swissprot ID               P01275

Standard Curve        Human Glucagon EasyTest™ EIA Kit

Kit Components

• Pre-Coated 96-well Strip Microplate
• Wash Buffer
• Stop Solution
• Assay Diluent
• Anti-Glucagon Antibody
• Standard Glucagon peptide
• Biotinylated Glucagon peptide
• TMB One-Step Substrate

Protocol Outline

1. Prepare all reagents, samples and standards as instructed in the manual.
2. Add 25 µl of anti-Glucagon antibody to all wells.
3. Add 75 µl of 1 x assay diluent to the blank wells and 50 µl to B0 wells.
4. Add 50 µl of standard peptide/samples to the appropriate wells.
5. Add 25 µl of biotinylated peptide into each well except the blank wells.
6. Incubate 1.0 h at RT.
7. Wash and add 100 µl of diluted streptavidin-HRP solution to all wells.
8. Incubate 45 min at RT.
9. Wash and add 100 µl of TMB Substrate Solution into each well.
10. Incubate 15 min at RT.
11. Add 50 µl of Stop Solution to each well.
12. Read at 450 nm/570 nm.

Application

In blood, Glucagon binds with big proteins. Acidification of blood is required to separate and purify Glucagon.

Background

Glucagon is a peptide hormone, produced by alpha cells of the pancreas, which raises the concentration of glucose in the bloodstream. Its effect is opposite that of insulin, which lowers the glucose concentration. The pancreas releases glucagon when the concentration of glucose in the bloodstream falls too low. Glucagon generally elevates the concentration of glucose in the blood by promoting gluconeogenesis and glycogenolysis.

When glucagon binds to the glucagon receptors, the liver cells convert the glycogen into individual glucose molecules and release them into the bloodstream, in a process known as glycogenolysis. As these stores become depleted, glucagon then encourages the liver and kidney to synthesize additional glucose by gluconeogenesis. Glucagon turns off glycolysis in the liver, causing glycolytic intermediates to be shuttled to gluconeogenesis. Glucagon also regulates the rate of glucose production through lipolysis. Glucagon induces lipolysis in humans under conditions of insulin suppression.

Glucagon binds to the glucagon receptor, a G protein-coupled receptor, located in the plasma membrane. The conformation change in the receptor activates G proteins, a heterotrimeric protein with α, β, and γ subunits. The alpha subunit specifically activates adenylate cyclase. Glucagon stimulation of PKA also inactivates the glycolytic enzyme pyruvate kinase.

Storage

The entire kit may be stored at -20°C for up to 1 year from the date of shipment. Avoid repeated freeze-thaw cycles. The kit may be stored at 4°C for up to 6 months. For extended storage, it is recommended to store at -80°C.

Regulatory Status      

Human Glucagon EasyTest™ EIA Kit is for Research Use Only.

• Manual
• AssayProcedure
• MSDS