and let it dry. Resuspend with Milli-Q H 2 O. Dilute the samples
to be within the detection range.
- Dilution of the samples to meet the detection range is essential.
When the samples contain high amounts of glycogen, the
hydrolysis reaction leads to high amounts of glucose, which
could interfere with the OxiRed probe. With concentrated
samples, it is very common to see the pink and brown colors
develop and then disappear. Dilute the samples to have accurate
measurements.
- Avoid foaming or bubbles while reconstituting this mix since
they could interfere with the readings later on.
- The reconstituted hydrolysis enzyme mix is stable for 2 months
at 20 C. To avoid repetitive freeze/thaw cycles, aliquot the
reconstituted hydrolysis enzyme mix and store in the dark at
20 C. When using frozen aliquots, make sure to equilibrate
it to room temperature before use because cold temperatures
lower the enzyme activity, which would slow down the glyco-
gen hydrolysis process.
- In several organisms and tissues, the presence of acid-insoluble
pools of glycogen has been reported. These pools differ struc-
turally from the acid-soluble glycogen. In this case, the degra-
dation efficiency of glycogen by glucoamylase enzyme is
different between samples, and the results obtained with this
kit may not reflect the “real” glycogen concentration in the
samples. In this situation, the determination of glycogen levels
using other techniques such as periodic acid-Schiff or iodine
staining of tissues/organisms is recommended.
- Similar toseeNotes 8and 9 , mix gently by pipetting up and
down the development enzyme mix. This mix could be stored
at 20 C in the dark for 2 months. Frozen aliquots are
recommended to avoid repetitive freeze and thaw cycles.
Bring to room temperature frozen aliquots before
experimental use.
- These kits could be used as fluorimetric kits. In this case, the
volume of hydrolysis enzyme mix used is lower. Consult
Table1 for information. Black flat bottom 96-well plates
should be used for the fluorimetric assay, and readings should
be taken at Ex¼535 nm/Em¼587 nm.
- The sample volume depends on the glycogen concentration in
these samples. It is recommended to test several dilutions of
samples to make sure that the readings are within the standard
curve range. For highly glycogen-rich samples, dilute your
samples with the glycogen hydrolysis buffer.
- Accurate pipetting is important to get good results. Bubbles
could lead to pipetting errors and interfere with the reading.
Biochemical Titration of Glycogen in Cells and Tissues 65
