Application

This kit can be used to measure the glycogen content in animal liver and muscle samples. 

Detection significance

Glycogen is mainly produced by liver and muscle. Glycogen content is mainly regulated by glycogen synthase and glycogen phosphorylase. In the liver, glycogen acts as a glucose store for other tissues and maintains blood glucose levels. In muscle, glycogen is mainly used as energy for the supply of adenosine triphosphate (ATP) during muscle contraction. Due to the lack of glucose-6-phosphatase, muscle glycogen cannot maintain the level blood glucose.

Detection principle

Under the presence of concentrated sulfuric acid, glycogen can be dehydrated to furfural derivatives. Furfural derivatives can form blue compound with anthracenone. The concentration of the compound can be measured by colorimetric quantification at 620 nm with glucose standard buffer of same treatment. Glycogen is quite stable in concentrated alkali solution. Heating the tissue sample in concentrated alkali solution before color development will remove other components and keep the glycogen. 

Experiment instruments

Spectrophotometer (620 nm), Vortex mixer, Micropipettor,Centrifuge, Water bath, Micropipettor.

Sample pretreatment

It is recommended to take 2~3 samples which expected large difference to do pre-experiment before formal experiment.

1. Sampling: Wash the fresh liver or muscle tissue sample with saline and dry with filter paper. Weigh the sample. It is recommended that the weight of the sample should be no more than 100 mg.

2. Hydrolysis: Add the Reagent 1 into a tube with a ratio of Weight (mg): Volume (μL) =1:3. Heat the tube in boiling water bath for 20 min. Cool the tube with running water.

Examples: according to the ratio of w: v=1:3, 75 mg of liver tissue sample should be mixed with 225 μL alkali solution. 85 mg of liver tissue sample should be mixed with 255 μL alkali solution.

*Notes: Seal the tube with preservative film to avoid water evaporation. Make a small hole on the film to allow vapour expanding and contracting.

3. Prepare the hydrolyzed glycogen testing solution for measurement

Concentration of the liver glycogen testing solution is 1%, the volume of the double-distilled water added should be: liver weight × 100 - liver weight×4* = liver weight × 96.

Concentration of the muscle glycogen testing solution is 5%, the volume of the double-distilled water added should be: muscle weight × 20 - muscle weight × 4*= muscle weight × 16.

Notes: 4* is the volume of the dehydrated sample and Reagent 1 mixture.

※  For example: weigh 80 mg liver tissue, add 240 μL reagent 1 to hydrolyze, it should add 7680 μL double-distilled water to prepare 1% liver glycogen testing solution

Operation steps

1. Blank tube: Take 1 mL of double-distilled water into a 10 mL glass tube..

Standard tube: Take 1 mL of 0.01 mg/mL Glucose standard solution into a 10 mL glass tube.

Sample tube: Take 0.1 mL of Liver/ muscle glycogen testing solution into a 10 mL glass tube and add 0.9 mL of double-distilled water.

Note: this experiment must be carried out with glass tubes.

2. Add 2.0 mL of Reagent 3 and oscillate fully with vortex mixer (The Reagent 3 should be added slowly after addition of Standard or Sample).

3. Fasten the tubes with plastic film and make a small hole, and incubate the tubes in 100℃ water bath for 5 min, then cool the tubes with running water immediately.

Notes: the mixture must be mix fully after adding the Reagent 3, then heat the tubes in boiling water bath. Otherwise floccule will be formed during heating.

4. Set the spectrophotometer to zero with double-distilled water and measure the OD value of each tube at 620 nm with 1 cm cuvette.

Note: It can be refer to the following operating table.

Technical parameters

1. The sensitivity of the kit is 1.80 mg/g (Liver samples) and 0.36 mg/g (Muscle samples).

2. The intra-assay CV is 3.7 % and the inter-assay CV is 7.3 %.

3. The recovery of the kit is 101 %.

4. The linear range of the kit is 1.80-180 mg/g (Liver samples) and 0.36-36 mg/g (Muscle samples).

Notes

1. This kit is for research use only.

2. Instructions should be followed strictly, changes of operation may result in unreliable results.

3. The validity of kit is 6 months.

4. Do not use components from different batches of kit.