Glycogen Assay Kit, BioAssay™

Glycogen is a branched polysaccharide of glucose units linked by a- 1,4 glycosidic bonds and a-1,6 glycosidic bonds. It is stored primarily in the liver and muscle, and forms an energy reserve that can be quickly mobilized to meet a sudden need for glucose. The most common glycogen metabolism disorder is found in diabetes, in which, due to abnormal amounts of insulin, liver glycogen can be abnormally accumulated or depleted. Genetic glycogen storage diseases have been associated with various inborn errors of metabolism caused by deficiencies of enzymes necessary for glycogen synthesis or breakdown.||Simple, direct and automation-ready procedures for measuring glycogen concentrations find wide applications in research and drug discovery. Glycogen assay uses a single Working Reagent that combines the enzymatic break down of glycogen and the detection of glucose in one step. The color intensity of the reaction product at 570nm or fluorescence intensity at lem/ex=585/530nm is directly proportional to the glycogen concentration in the sample. This simple convenient assay is carried out at room temperature and takes only 30 min.||Key Features:|Use as little as 10ul samples. Linear detection range: 2 to 200 ug/mL glycogen for Colorimetric Assay:s and 0.2 to 20 ug/mL for Fluorometric Assay:s.||Kit Contents:|Assay Buffer: 12ml Enzyme A: 120ul Enzyme B: 120ul|Dye Reagent: 120ul Standard: 50ul 50 mg/mL|Storage conditions. The kit is shipped on ice. Store all reagents at -20°C. Shelf life of six 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.||Sample Preparation:|Samples can be prepared according to established methods [1-3].||Colorimetric Procedure:|1. Equilibrate all components to room temperature. During experiment, keep thawed enzymes in a refrigerator or on ice.|2. Standards and samples: Dilute standard by mixing 5ul Standard with 1.245ml dH2O to give 200 μg/mL standard. Dilute standard in dH2O as follows.|No 200 ug/mL STD+H2O Vol (ul) Glycogen (ug/ml)|1 200ul+0ul 200 200|2 150ul+50ul 200 150|3 100ul+100ul 200 100|4 50ul+150ul 200 50|5 0ul+200ul 200 0|Transfer 10ul standard and samples into separate wells of a clear flat-bottom microplate.|3. Working Reagent. For each reaction well, mix 90ul Assay Buffer, 1ul Enzyme A, 1ul Enzyme B and 1ul Dye Reagent in a clean tube. Transfer 90ul Working Reagent into each reaction well. Tap plate to mix.|4. Incubate 30 min at room temperature. Read optical density at 570nm (550-585nm).||Fluorometric Procedure:|For Fluorometric Assays, the linear detection range is 0.2 to 20 ug/mL glycogen. Follow steps 1-3 of the Colorimetric Procedure:, but prepare 0, 5, 10, 15 and 20 ug/mL Standard and use a black flat-bottom microplate. Incubate 30 min at room temperature and read fluorescence at lex=530nm and lem=585nm.||Calculation:|Subtract Blank reading (OD570nm or fluorescence intensity) from the standard reading values and plot the DOD or DF against standard concentrations. Determine the slope and calculate the glycogen concentration of the sample.|Glycogen =|RSAMPLE-RBLANK|Slope|ug/mL|RSAMPLE and RBLANK are the OD570nm or fluorescence intensity values of the sample and blank (water, or sample blank, see below).||General Considerations:|1. If the sample contains glucose, a Sample Blank well should be added: Prepare Sample Blank reagent by mixing 90ul Assay Buffer, 1ul Enzyme B, and 1ul Dye Reagent (No Enzyme A). Add this reagent only to the Sample Blank wells. Subtract the OD or fluorescence of the Sample Blank from the sample readings to calculate glycogen concentration.|2. This assay is based on a kinetic reaction, the use of a multichannel pipettor for adding the working reagent is recommended.|3. Interference. SH-group containing reagents (e.g., DTT, b-mercaptoethanol) may interfere with this assay and should be avoided in sample preparation.||Materials Required, But Not Provided:|Pipeting devices, centrifuge tubes, clear flat bottom 96-well plates and plate reader.