Adipolysis Assay Kit, BioAssay(TM)

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. 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.\n\nKey Features:\nSensitive and accurate. Use as little as 10ul samples. Linear detection range in 96-well plate: 0.92 to 100 ug/mL (10 to 1000uM) glycerol for Colorimetric Assay:s and 0.2 to 5 ug/mL for Fluorimetric Assay:s.\nRapid and convenient. The procedure involves addition of a single working reagent and incubation for 20 min at room temperature.\nRobust 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.\n\nApplications:\nDirect Assays: adipolysis (glycerol in cell culture media).\nDrug Discovery/Pharmacology: effects of testing drugs on adipolysis.\n\nKit Contents: (bulk reagents available)\nAssay Buffer: 24ml Enzyme Mix: 500ul ATP: 250ul\nDye Reagent: 220ul Standard: 100ul 100mM Glycerol\nStorage conditions. The kit is shipped on ice. Store Assay Buffer at 4 degrees C and other reagents at -20 degrees C. Shelf life of 6 months after receipt. Precautions: reagents are for research use only. Normal precautions for laboratory reagents should be exercised while using the reagents. Please refer to Material Safety Data Sheet for detailed information.\n\nColorimetric Procedure:\nSH-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.\n1. 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.\n2. Standards and Samples. Prepare a 100ug/mL standard by mixing 10ul 100mM glycerol standard with 910ul in the same medium used for cell culture. Dilute standard in the medium as follows. Transfer 10ul standards into wells of a clear 96-well assay plate (5ul for 384-well assay plate).\nNo 100 ug/mL STD+Medium Vol (ul) Glycerol (ug/mL)\n1 400ul+0ul 400 100\n2 300ul+200ul 500 60\n3 150ul+350ul 500 30\n4 0ul+500ul 500 0\nCollect cell culture supernatants from culture wells. Such samples should be assayed immediately or stored at -20 degrees C. Transfer 10ul samples (5ul for 384-well assay plate) into separate wells of the assay plate.\n3. Enzyme Reaction. For each assay well, mix 100ul Assay Buffer, 2ul Enzyme Mix, 1ul ATP and 1ul Dye Reagent in a clean tube. Transfer 100ul Working Reagent into each assay well. Tap plate to mix. For assays in a 384-well plate, use 50ul Working Reagent per well.\n4. Incubate 20 min at room temperature. Read optical density at 570nm (550-585nm).\nNote: if the Sample OD is higher than the Standard OD at 100 ug/mL, dilute sample in water and repeat the assay. Multiply result by the dilution factor.\n\nCalculation:\nSubtract blank OD (#4) from the standard OD values and plot the OD against standard concentrations. Determine the slope using linear regression fitting. The glycerol concentration of Sample is calculated\n[Glycerol] =\nODSAMPLE

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SPECIFICATIONS

Catalog Number

A0885-68A

Size

1Kit

References

1. Duncan RE, et al. (2007). Regulation of lipolysis in adipocytes. Annu Rev Nutr. 27: 79-101.\n2. Moller F, Roomi MW. (1974). An enzymatic, spectrophotometric glycerol assay with increased basic sensitivity. Anal Biochem. 59(1): 248-58.\n3. MacRae AR. (1977). A semi-automated enzymatic assay for free glycerol and triglycerides in serum or plasma. Clin Biochem. 10(1): 16-9.