2.2 Hydrolysis
and Development
Reagent Preparation
In this section we provide the components that are utilized by most
commercial glycogen detection kits, with their composition, and
method of action.- Hydrolysis enzyme mix: Lyophilized mixture encompassing
the amyloglucosidase enzyme. - Hydrolysis buffer: This is an acetate buffer. Most kits use
between 25 mM and 50 mM sodium acetate at pH 4.5. - Development enzyme mix: It is a lyophilized mixture that
encompasses the glucose oxidase enzyme. This enzyme oxi-
dizes glucose molecules to produce gluconic acid and hydro-
gen peroxide. - Development buffer: This is a concentrated phosphate buffer at
pH 7.4. Most kits utilize between 0.1 M and 0.5 M K 2 HPO 4. - OxiRed probe also called Amplex Red: It is a lyophilized pow-
der of 10-acetyl-3.7-dihydroxyphenoxazine (ADHP). It is a
highly sensitive and stable substrate for horseradish peroxidase
(HRP) that specifically reacts with hydrogen peroxide to pro-
duce resorufin that could be detected by colorimetric
(lmax¼570 nm) or fluorimetric (Ex/Em¼535/587 nm)
detection methods.
2.3 Glycogen
Determination
in Standard Curve
and Samples
- Black flat bottom 96-well plate (if performing the fluorimetric
assay). - Clear flat bottom 96-well plate (if performing the colorimetric
assay). - Glycogen standard (2 mg/ml, provided in the commercial
glycogen determination kit). - ELISA microplate reader.
3 Methods
All procedures should be carried out at room temperature unless
specified otherwise.3.1 Cell Lysis
and Glycogen
Extraction
- Grow the cells in 100 mm diameter dish (seeNote 1)at37C.
Harvest the cells when they reach ~80% confluence. Place the
plates on ice. - Wash the cells three times with PBS buffer to remove the
remaining glucose molecules from culture medium. - On ice, add 300μl of 30% potassium hydroxide and scrape the
cells (seeNote 2). - Transfer the samples to Eppendorf tubes and boil at 95C for
10 min to inactivate the enzymes (seeNote 3).
60 Elite Possik and Arnim Pause