- For every sample include a sample background control. Add
the same amount of samples used instep 3and complete the
volume up to 50μl with the glycogen hydrolysis buffer. To
these samples, doNOTadd the hydrolysis enzyme mix. Read-
ings of these wells will reveal the amount of glucose contami-
nation in your samples prior to glycogen hydrolysis (see
Table1). - Add 2μl of the hydrolysis enzyme mix to the standard curve
and sample wells for the colorimetric assay. For the fluorimetric
assay, add only 1μl of the hydrolysis enzyme mix. The “0” well
is the background control. Readings of these wells will measure
the amount of glucose resultant from the hydrolysis of glyco-
gen in the standard glycogen solution and in your samples (see
Note 15). - Wrap your plate with aluminum foil to protect from light and
incubate with gentle shaking for 30 min.
3.4 Development
and Output
Measurement
- During the 30 min incubation, prepare a master mix of the
development mix solution. Depending on the number of reac-
tions used, prepare enough reagents to cover all samples and
controls. For every reaction, consider 46μl of development
buffer, 2μl of Oxiprobe, and 2μl of development enzyme mix
for the colorimetric assay. For the fluorimetric assay, for every
reaction, add 48.7μl of the development buffer, 1μl of the
development enzyme mix, and 0.3μl of OxiRed. - Add 50μl of the development enzyme mix to every well and
mix well by gentle up and down pipetting. Avoid bubbles and
Table 1
Glycogen standard curve and sample preparation
Glycogen standard
volume (μl)Hydrolysis enzyme
buffer (μl)Hydrolysis enzyme
mix (μl)Development
buffer (μl)
050250
248250
446250
644250
842250
10 40 2 50
Sample volume (μl) Hydrolysis enzyme
buffer (μl)Hydrolysis enzyme
mix (μl)Development
buffer (μl)
1–50 Up to 50 2 50
1–50 Up to 50 0 5062 Elite Possik and Arnim Pause