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.
- 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
The capture antibody provided in this kit recognizes human, mouse and rat Visfatin.
Target Protein Information
Nicotinamide phosphoribosyltransferase (NAmPRTase) (Nampt) (EC 220.127.116.11) (Pre-B-cell colony-enhancing factor 1) (Pre-B cell-enhancing factor) (Visfatin)
NAMPT PBEF PBEF1
- Pre-Coated 96-well Strip Microplate
- Wash Buffer
- Standard Peptide
- Assay Diluent(s)
- Biotinylated Peptide
- 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 OutlinePrepare 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.
Standard, Biotinylated Visfatin 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.