Glycogen Branching Assay to Measure the Degree of Glycogen Branching

протокол

1. Qualitative assessment of glycogen branching

1. Prepare saturated calcium chloride solution by adding 74.5 g of anhydrous calcium chloride to ~40 mL of water and stirring. Add a little more water and continue to stir. Make the volume up to 100 mL with water and continue stirring until the CaCl₂ is fully dissolved.
2. Prepare a working stock of iodine/CaCl₂ color reagent by mixing 50 µL of KI/iodine stock solution with 13 mL of saturated CaCl₂ solution in a 15 mL tube. Mix well and store at 4 °C, protected from the light. The solution is stable for at least 1 week under these conditions.
3. Determination of branching
   1. In a 1.5 mL tube, combine 650 µL of iodine/CaCl₂ color reagent stock with 100 µL of water and mix thoroughly. Transfer the solution to a disposable methacrylate cuvette.
      NOTE: The solution in the cuvette should be clear and pale yellow in color.
   2. Place in the spectrophotometer and, running in a wavelength scanning mode, collect a background spectrum from 330 nm to 800 nm.
   3. In a 1.5 mL tube, combine 650 µL of working iodine/CaCl₂ color reagent with 50 µg of oyster glycogen. Bring the final volume to 750 µL with water and mix thoroughly. Transfer the solution to a disposable methacrylate cuvette.
      NOTE: The solution in the cuvette should be clear and a deep orange/brown color.
4. Place in the spectrophotometer and collect an absorption spectrum from 330 nm to 800 nm.
5. Repeat steps 1.3.3 through 1.4 with 50 µg of amylopectin and 30 µg of amylose.
   NOTE: The amylopectin sample should be yellow/green and the amylose sample should be green/blue. Both samples should be clear. The colored complexes formed are stable, with no change in the absorption spectrum, for at least 1 h at room temperature.
6. To obtain an indication of the branched structure of an uncharacterized glycogen sample, combine 25 µg to 50 µg of glycogen with 650 µL of working iodine/CaCl₂ color reagent. Proceed as above, bringing the volume to 750 µL with water, mixing thoroughly, and transferring to a methacrylate cuvette.
   NOTE: The glycogen sample should yield a yellow/orange to orange/brown color depending upon the degree of branching (length of outer chains) of the glycogen present. Again, the sample should be clear.
7. Collect the absorption spectrum.

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Материалы

Name	Company	Catalog Number	Comments
Amylopectin (amylose free) from waxy corn	Fisher Scientific	A0456
Amylose	Biosynth Carbosynth	YA10257
Glycogen, Type II from oyster	MilliporeSigma	G8751
Methacrylate cuvettes, 1.5 mL	Fisher Scientific	14-955-128	Methacrylate is required since some procedures are conducted at 340 nm or below