Glycogen Branching Assay to Measure the Degree of Glycogen Branching
Transcrição
To determine the glycogen branching, combine 650 microliters of iodine/calcium chloride color reagent stock with 100 microliters of water in a 1.5-milliliter tube, and mix the solution thoroughly before transferring to a disposable methacrylate cuvette. Place the cuvette in the spectrophotometer to conduct a run in a wavelength-scanning mode for collecting the background spectrum from 330 to 800 nanometers.
In a separate 1.5-milliliter tube, combine 650 microliters of the working iodine/calcium chloride color reagent with 50 micrograms of the oyster glycogen, and make up the final volume to 750 microliters with water. After blending the mixture thoroughly, transfer the solution to a disposable methacrylate cuvette to collect an absorption spectrum from 330 to 800 nanometers.
Similarly, obtain an absorption spectrum with 50 micrograms of amylopectin and 30 micrograms of amylose as described before.
To obtain an indication of the branched structure of an uncharacterized glycogen sample, combine 25 to 50 micrograms of the glycogen with 650 microliters of the working iodine/calcium chloride color reagent, and proceed as explained earlier to acquire the absorption spectrum.
