Difference between revisions of "Glycogen Determination from Cells"
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==Protocol== | ==Protocol== | ||
# Treat cells as desired and wash 2x with ice cold PBS. | # Treat cells as desired and wash 2x with ice cold PBS. | ||
| − | # Scrape cells into 0.5 mL of Sodium Acetate (for 12 well). | + | # Scrape cells into 0.5 mL of Sodium Acetate (for 12 well). Lyse by either brief sonication (~10s) or 3x freeze thaw cycles. |
# Measure protein content by bradford assay for normalization | # Measure protein content by bradford assay for normalization | ||
# Quantify glucose using kit: | # Quantify glucose using kit: | ||
## Add 1-5 uL glucose standard for standard curve | ## Add 1-5 uL glucose standard for standard curve | ||
| − | ## Add 10 uL glycogen. If signal is too low increase the volume. Also make a blank with the same volume of Sodium acetate. | + | ## Add 10 uL of the glycogen containing sample. If signal is too low increase the volume. Also make a blank with the same volume of Sodium acetate. |
## Mix and incubate at 37C for 5 min | ## Mix and incubate at 37C for 5 min | ||
## Measure absorbance at 505 nm | ## Measure absorbance at 505 nm | ||
Latest revision as of 18:23, 30 June 2022
Materials and Buffers
- 50 mM Sodium Acetate, pH 4.8
- Amyloglucosidease
- Glucose quantification kit (Wako Autokit Glucose Buffer Solution cat # 439-9091)
- Glucose standard solution (500 mg/dL; Wako)
Protocol
- Treat cells as desired and wash 2x with ice cold PBS.
- Scrape cells into 0.5 mL of Sodium Acetate (for 12 well). Lyse by either brief sonication (~10s) or 3x freeze thaw cycles.
- Measure protein content by bradford assay for normalization
- Quantify glucose using kit:
- Add 1-5 uL glucose standard for standard curve
- Add 10 uL of the glycogen containing sample. If signal is too low increase the volume. Also make a blank with the same volume of Sodium acetate.
- Mix and incubate at 37C for 5 min
- Measure absorbance at 505 nm
- Calculate glycogen levels as umoles glucose released per g tissue (should be ~10)
- Add amyloglucosidase (Add 0.3 mg/mL) and place at 37C for 3h-O/N
- Re-measure glucose levels
- Glycogen levels are calculated by the difference in glucose levels before and after glycosidase treatment and are presented in equivalent glucose units/mg protein.
Reference:
