GLYCOGEN is a branched polysaccharide of glucose units linked by α- 1,4 glycosidic bonds and α-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. BioAssay Systems' 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.

Use as little as 10 μL samples. Linear detection range: 2 to 200 μg/mL glycogen for colorimetric assays and 0.2 to 20 μg/mL for fluorimetric assays.

KIT CONTENTS

Assay Buffer: 12 mL Enzyme A: 120 μL Enzyme B: 120 μL

Dye Reagent: 120 μL Standard: 50 μL 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.

Samples can be prepared according to established methods [1-3].

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 5 μL Standard with 1.25 mL dH2O to give 200 μg/mL standard. Dilute standard in dH2O as follows.

RSAMPLE and RBLANK are the OD570nm or fluorescence intensity values

of the sample and blank (water, or sample blank, see below).

1. If the sample contains glucose, a Sample Blank well should be added: Prepare Sample Blank reagent by mixing 90 μL Assay Buffer, 1 μL Enzyme B, and 1 μL 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, β- mercaptoethanol) may interfere with this assay and should be avoided in sample preparation.

Pipeting devices, centrifuge tubes, clear flat bottom 96-well plates and plate reader.

1. Murat JC, Serfaty A. (1974). Simple enzymatic determination of polysaccharide (glycogen) content of animal tissues. Clin Chem. 20(12):1576-1577.

2. Bueding, E. and Orrell, S.A. (1964). A Mild Procedure for the Isolation of Polydisperse Glycogen from Animal Tissues. J. Biol. Chem. 239: 4018-4020.

3. Dalrymple, R. H. and Hamm, R. (1973) A method for the extraction of glycogen and metabolites from a single muscle sample. Intl. J. Food Sci & Tech 8(4): 439-444.