1. Weigh out muscle (use ~ 10 mg for rat and 5-8 mg for human) in triplicate and place each piece in a 1.5 mL microfuge tube with 500 uL of 2 N HCl. Make sure muscle is completely submerged in the HCl. Puncture a hole in the top of the tubes before weighing to allow for venting during boiling.

Note: Make sure the muscle doesn't thaw until it is put into the acid by keeping it on dry ice. After it is in the tube the tubes can be kept at RT until boiling.

2. Weigh tubes to the nearest mg.

3. Boil tubes in heat block for 2 hrs. with occasional gentle vortexing. Note: While the tubes are boiling start warming up the flourometer.

4. Spin down tubes in microcentrifuge to get all of the liquid to the bottom of the tube.

5. Reweigh the tubes and reconstitute each tube to its original volume with dd H₂O (1uL

H₂O = 1mg).

6. Add 500 uL of 2N NaOH to each tube to neutralize the acid.

7. Cover tubes with parafilm and vortex using multivortexer to break up the muscle. Vortex at maximum speed for 1 min or until the muscle is broken up.

8. Make up 500µl of the following glucose standards in dd H₂O from a 7 mMstock

solution: 700, 350, 175, 87.5, and 43.75 uM.

9. Add (50-200uL) of standard or sample to 10 X 75 mm tubes in duplicate

(Volumes within each assay must be the same for standards and samples))

10. Add 1 mL of Hexokinase Reagent (Sigma) to each tube and vortex tubes.

11. Incubate at RT for 10 min.

12. Read tubes on flourometer or spectrophometer. 340nm