Assay of Glycogen Synthase Activity

Glycogen synthase assays measure the rate of incorporation of UDP-glucose into glycogen, in the absence and presence of the allosteric activator G6P. The glycogen is precipitated onto glass filters with cold ethanol, free 3H-UDP-glu-cose is washed away, and 3H -incorporation into glycogen is measured by liquid scintillation counting. Reactions are usually performed in duplicate (two — G6P, two + G6P), but each condition can be done in triplicate especially when first learning the assay (see Note 3).

1. Calculate the number of reactions to be run (four to six lysate sample). Multiple reaction number by 50 iL to get the total volume of reaction mix needed. Round up by 200 iL for blanks and to ensure you do not run out. For each milliliter of reaction mix, add 16 mg of glycogen to 1 mL of GSB (without TritonX-100). Warm to 37°C for 5-10 min and vortex to dissolve glycogen.

2. Place GSB + glycogen on ice and add 60 iL/mL of 200 mM UDP-glucose. Add 2iCi/mL of 3H-UDP-glucose and vortex. Split the reaction mix equally into two tubes labeled "—" and "+." Add 62.5 iL/500 iL of double-distilled water to the "—" tube. Add 62.5 iL/500 iL of 200 mM G6P to the "+" tube.

3. Reactions are performed in microfuge tubes in a final volume of 100 iL. Place the tubes on ice in racks that can be transferred to a water bath. Label tubes 1, 2, 3, and so on.

4. Add 50 iL (approx 100 ig) of each lysate to four reaction tubes (see Note 4). Sample 1 goes in tubes 1-4, sample 2 in 5-8, and so forth. Add 50 iL of "—" reaction mix to the odd-numbered tubes and 50 iL of "+" reaction mix to the even-numbered tubes. Label two tubes "blank" and add 50 iL of GSB + 50 iL of reaction mix.

5. Seal tubes and vortex gently. If there are droplets on the sides of the tubes, spin tubes 3 s in a microfuge.

6. Transfer racks to a 37°C water bath for 15 min. Then, place racks in an ice-water bath for 15 min.

7. Label GF/A filters on a piece of aluminum foil using a Schleicher and Schuell marking pen. Do not use regular Sharpies.

8. Pick up the filter with a tweezer and spot 90 iL of reaction onto it. Use aerosol resistant tips and move Pipetman around filter while spotting. Wait 3 s and drop filter into a container on ice with approximately 200 mL of 70% ethanol (4°C).

9. When finished spotting filters, gently wash for 15 min at 4°C. You can either shake in a cold room or keep box on ice and use a room temperature shaker. It is very important to shake the filters gently, as they can disintegrate if agitated too hard. Wash two more times for 15 min in 70% ethanol at room temperature.

10. Remove filters and place on a paper towel in fume hood for 30-60 min to dry. Alternatively, place filters flat in 20-mL scintillation vials and air-dry overnight.

11. Add 5 mL of scintillation fluid to filters in 20-mL scintillation vials and count 1-5 min in a liquid scintillation counter. Average the cpms and divide -G6P counts (active glycogen synthase) by +G6P counts (total glycogen synthase) to get activity ratio.

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