Adipolysis Assay Kit

Produktnummer ABIN1000290

Kurzübersicht für Adipolysis Assay Kit (ABIN1000290)

Target: Adipolysis

Applikation: Biochemical Assay (BCA)

Proben: Cell Culture Supernatant

Produktdetails

Spezifität: 0.92 μg/mL

Produktmerkmale: Sensitive and accurate. Use as little as 10 µL samples. Linear detection range in 96-well plate: 0.92 to 100 µg/mL (10 to 1000 µM) glycerol for colorimetric assays and 0.2 to 5 µg/mL for fluorimetric assays.
Rapid and convenient. The procedure involves addition of a single working reagent and incubation for 20 min at room temperature.
Robust and amenable to HTS assays. Potential interference by testing drugs is greatly reduced at 570nm.
Compatible with culture media containing phenol red. Assays can be performed in 96 or 384- well plates.

Bestandteile: Assay Buffer: 24 mL. Enzyme Mix: 500 µL. ATP: 250 µL. Dye Reagent: 220 µL. Standard: 100 µL 100 mM Glycerol.

Benötigtes Material: Pipeting devices, centrifuge tubes, appropriate 96- or 384-well plates and plate reader.

Anwendungsinformationen

Applikationshinweise: Direct Assays: adipolysis (glycerol in cell culture media).
Drug Discovery/Pharmacology: effects of testing drugs on adipolysis.

Protokoll: SH-group containing reagents (e.g. mercaptoethanol, DTT) may interfere with this assay and should be avoided in sample preparation. Prior to the assay, equilibrate all components to room temperature. Keep thawed Enzyme Mix in a refrigerator or on ice during assays.
1. Cell Culture. Note: Cells and testing drugs are to be provided by the customer and are not included in this reagent kit. Grow cells (e.g. preadipocytes, adipocytes) in culture plate (24-well, 96-well or 384- well). If desired, treat cells with testing drugs such as insulin, isoproterenol, and incubate for the desired time period.
2. Standards and Samples. Prepare a 100 µg/mL standard by mixing 10 µL 100 mM glycerol standard with 910 µL in the same medium used for cell culture. Dilute standard in the medium. Transfer 10 µL standards into wells of a clear 96-well assay plate (5 µL for 384-well assay plate). Collect cell culture supernatants from culture wells. Such samples should be assayed immediately or stored at -20°C. Transfer 10 µL samples (5 µL for 384-well assay plate) into separate wells of the assay plate.
3. Enzyme Reaction. For each assay well, mix 100 µL Assay Buffer, 2µL Enzyme Mix, 1 µL ATP and 1 µL Dye Reagent in a clean tube. Transfer 100 µL Working Reagent into each assay well. Tap plate to mix. For assays in a 384-well plate, use 50 µL Working Reagent per well.
4. Incubate 20 min at room temperature. Read optical density at 570nm (550-585nm). Note: if the Sample OD is higher than the Standard OD at 100 µg/mL, dilute sample in water and repeat the assay. Multiply result by the dilution factor.

Ergebnisberechnung:

Subtract blank OD (#4) from the standard OD values and plot the OD against standard concentrations. Determine the slope using linear regression fitting.
Conversions: 1 µg/mL glycerol equals 10.9 µM. FLUORIMETRIC

Beschränkungen: Nur für Forschungszwecke einsetzbar

Handhabung

Lagerung: -20 °C

Referenzen

Li, Guan, Yang: "Neuropeptide Y potentiates beta-adrenergic stimulation of lipolysis in 3T3-L1 adipocytes." in: Regulatory peptides, Vol. 178, Issue 1-3, pp. 16-20, (2012) (PubMed).

Antigendetails

Target: Adipolysis

Hintergrund: Quantitative assay of adipolysis by measuring glycerol released in cell culture using colorimetric (570nm) or fluorimetric (530nm/590nm) methods.
Procedure: 20 min.

Obesity is a chronic condition that develops from storage of excessive energy in the form of adipose tissue. The resulting adiposity presents a high risk factor for diseases such as type 2 diabetes, cardiovascular diseases, and cancer. Adipolysis or lipolysis is a highly regulated process in fat metabolism, in which triglycerides are broken down into glycerol and free fatty acids. Rapid, robust and accurate procedures for adipolysis quantification in cell culture are very useful in research and drug discovery. This adipolysis assay kit directly measures glycerol released during adipolysis. This homogeneous assay uses a single Working Reagent that combines glycerol kinase, glycerol phosphate oxidase and color reactions in one step. The color intensity of the reaction product at 570nm is directly proportional to glycerol concentration in the sample.