foam. Cover plate with aluminum foil to protect from light,
and incubate 30 min at room temperature with gentle shaking
(seeTable2).- Read the plate using an ELISA microplate reader immediately
after incubation at OD570 nm for colorimetric assays or at
Ex535/Em587 for fluorimetric assays (seeNote 16).
3.5 Analysis
and Calculations
- Subtract the background from all the readings. Also subtract
the free glucose background levels from corresponding sam-
ples. Apply the corrected reading to the standard curve to get
the amount of glycogen in the samples (μg).SeeFig. 1 for a
representative standard curve graph. - Apply the following formula to calculate the concentration of
glycogen in your samples:
C¼(μg glycogen in standard curve/samples volume)dilu-
tion factor¼μg/μl(seeNote 17).4 Notes
- Cellular density highly influences glycogen content. Higher
amounts of glycogen are found when cells are confluent. To
have accurate measurements, cells need to be evenly spread in
the dish. - Working on ice is important to slow down the activity of the
enzymes that could break down glycogen. Proceed quickly
during this step to limit glycogen hydrolysis events. - Boiling the samples inactivates the enzymes that could hydro-
lyze glycogen in your samples. - Protein levels could be determined using the Bradford assay.
Other protein assays can also be used if they are compatible
with the used concentration of potassium hydroxide. - Commercially available kits for glycogen determination are
similar in context and content. They are widespread and easily
available at a reasonable price. Because in this assay multiple
enzymes are needed to lead to the end product, we find that
Table 2
Development reagents in colorimetric and fluorimetric assays
Colorimetric assay (volume inμl) Fluorimetric assay (volume inμl)
Development enzyme mix 2 1
Development enzyme buffer 46 48.7
OxiRed 2 0.3Biochemical Titration of Glycogen in Cells and Tissues 63