- After boiling, take a 100μl aliquot for protein measurement
(seeNote 4). - Transfer the rest to a prechilled 15 ml conical tubes and add
three volumes of 95% ethanol to precipitate the glycogen.
Vortex and keep the tubes on ice for 10 min. - Centrifuge the lysate for 10 min at 12,000gat 4C.
- Discard supernatant and wash twice with cold 70% ethanol.
- Discard the supernatant and remove all traces of ethanol. Dry
the pellet and resuspend with 50μl of Milli-Q H 2 O. - Determine glycogen content using commercially available gly-
cogen determination kit (seeNotes 5and 6 ). - Make sure to dilute the samples to meet the detection range
(seeNote 7).
3.2 Glycogen
Hydrolysis Enzyme
Mix, Development
Enzyme Mix,
and Probe
Preparations
- Dissolve the hydrolysis enzyme mix with 220μl of the glycogen
hydrolysis buffer by gentle pipetting up and down (seeNotes
8 and 9 ). This mix encompasses the amyloglucosidase enzyme
responsible for glycogen degradation (seeNote 10). Bring to
room temperature before use and avoid light exposure. - Dissolve the development enzyme mix with 220μl of Milli-Q
H 2 O by gentle pipetting up and down. Bring to room temper-
ature before use and avoid light exposure (seeNote 11). - Dissolve the probe powder with 220μl of Milli-Q H 2 Oby
gentle pipetting up and down. Bring to room temperature
before use and avoid exposure to light.
3.3 Samples
and Standard Curve
Hydrolysis Reaction
- To start the reactions, use a clear flat bottom 96-well plate for
colorimetric assays and black flat bottom plates for fluorimetric
assays (seeNote 12). For every experiment, include a back-
ground control, a standard curve, and your samples to measure
both glycogen and glucose levels (seeNote 13and Table1). - Use the glycogen standard (2 mg/ml) to prepare the standard
curve. Start by preparing a 0.2 mg/ml of glycogen standard
solution by diluting 10μl of the provided standard with 90μl
of Milli-Q H 2 O(seeNote 14). For the fluorimetric assay, dilute
the 10μl of the glycogen standard in 990μl of Milli-Q H 2 O. - Add a series of 0, 2, 4, 6, 8, and 10μl of the diluted glycogen
standard (0.2 mg/ml for colorimetric and 0.02 mg/ml for
fluorimetric) in the 96-well plates, and complement with
50, 48, 46, 44, 42, and 40μl of glycogen hydrolysis buffer,
respectively (seeTable1). - In other wells, add 1–50μl samples prepared in Subheading3.1
(in triplicates) and complete the volume to 50μl with the
glycogen hydrolysis buffer (seeNote 14).
Biochemical Titration of Glycogen in Cells and Tissues 61