Mouse Visfatin PharmaGenie ELISA Kit (SBRS0089)

1. Pre-Coated 96-well Strip Microplate
2. Wash Buffer
3. Standard Peptide
4. Assay Diluent(s)
5. Biotinylated Peptide
6. HRP-Streptavidin
7. TMB One-Step Substrate
8. Stop Solution
9. Assay Diagram
10. Positive Control Sample
11. Capture Antibody
12. Technical Manual

Other materials and equipment required:

The Assay Genie Mouse Visfatin PharmaGenie ELISA Kit (SBRS0089) will require other equipment and materials to carry out the assay. Please see list below for further details.

• Distilled or deionized water
• Precision pipettes to deliver 2 ul 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)

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

Obesity, which is characterized by excessive accumulation of adipose tissue in the body, has become one of the greatest public health challenges. Obesity is not only associated with health problems linked to increased weight-dependent pressure overload on lung, joints and bones, but also a important risk factor for life-threatening diseases such as cardiovascular diseases, type 2 diabetes and certain cancers. Visfatin, an adipokine that is highly enriched in the visceral fat, has increased expression level in plasma during the development of obesity, implying its important role in obesity. Visfatin (also known as PBEF and Nampt), a 52 kDa cytoplasmic protein composed of 473-amino acids, is expressed primarily in bone marrow, muscle and liver tissue but can also be detected in placenta, lung, kidney and heart tissue. In addition to its role in obesity, visfatin also has other biological functions. Visfatin is a nicotinamide phosphoribosyltransferase (Nampt) enzyme that catalyzes first step in the biosynthesis of NAD from nicotinamide. Visfatin has also been reported to be a cytokine (PBEF) that promotes B cell maturation and inhibits neutrophil apoptosis.