RBP4 (retinol binding protein-4) is an adipocyte-secreted molecule that is elevated in the serum before the development of frank diabetes. In obesity and type 2 diabetes, expression of the GLUT4 glucose transporter is decreased selectively in adipocytes. Adipose-specific Glut4 knockout mice show insulin resistance secondarily in muscle and liver. Recent studies have shown that serum RBP4 levels are elevated in insulin-resistant mice and humans with obesity and type 2 diabetes. RBP4 levels are normalized by rosiglitazone, an insulin-sensitizing drug. Transgenic overexpression of human RBP4 or injection of recombinant RBP4 in normal mice causes insulin resistance. Conversely, genetic deletion of RBP4 enhances insulin sensitivity. Fenretinide, a synthetic retinoid that increases urinary excretion of RBP4, normalizes serum RBP4 levels and improves insulin resistance and glucose intolerance in mice with obesity induced by a high-fat diet. Increasing serum RBP4 induces hepatic expression of the gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK) and impairs insulin signalling in muscle. Thus, RBP4 is an adipocyte-derived 'signal' that may contribute to the pathogenesis of type 2 diabetes. Lowering RBP4 could be a new strategy for treating type 2 diabetes. Thus, RBP4 appears to identify insulin resistance and associated cardiovascular risk factors in subjects with varied clinical presentations.
- 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 Retinol Binding Protein 4 (RBP4).
Target Protein Information
Retinol-binding protein 4 (Plasma retinol-binding protein) (PRBP) (RBP)
- 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 Retinol Binding Protein 4 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.