Glucose-6-Phosphate Assay[Metabolic]

Muscle Processing

1.      Place 100% ethanol in a sytrofoam container and cool to between 0-5°C by adding dry ice.  Add 400 ml of 1M PCA to Eppendorf tubes for each sample and put them in the cooled ethanol.

2.      Weigh out approximately ~20 mg (record weight) of frozen tissue and pulverize under liquid N₂.  Add pulverized muscle to the PCA and homogenize on ice for 15-20 seconds.

3.      Spin samples in Biofuge at 3,000 rpm for 10 minutes at -5°C.  Transfer 200 ml of supernatant to an Eppendorf tube with 120 ml KIK (2N KOH, 0.4M imidazole, 0.4M KCl) buffer and vortex.

4.      Spin samples in Biofuge at 13,000 rpm for 1 minute at -5°C.  Remove supernatant and store at -80°C until analysis.

Assay

1.      Prepare G-6-P standards in the 25-400 mM range (with a zero concentration being water, 25, 50, 100, 200, 400 mM ).

2.      Add the following to the wells of a 200 ml microplate:

• 1.      100 ml 0.2M Tris, pH 7.5
• 2.      20 ml 0.1M MgCl₂
• 3.      10 ml 1% NADP
• 4.      30 ml ddH₂O
• 5.      40 ml Sample

3.      Read the A₃₄₀ for all samples....PRINT these out or save them.

4.      Add 5 ml of G-6-P dehydrogenase (1 mg/ml) diluted 1:5 in 50 mM Tris, pH 7.5.

5.      Allow samples to sit > 10 min and read A₃₄₀ again. You may want to read the samples every 2-3 minutes to determine when no further change in absorbance occurs.

6.      Determine the DA₃₄₀ for the standards as follows and create a regression equation relating the DA₃₄₀ and G-6-P concentrations:

[DA₃₄₀ final (+ G6PDH) - DA₃₄₀ initial (w/o G6PDH)] - DA₃₄₀ (H₂O)