AMPK Methods and Protocols

ready-made kits where conditions are already optimized are

important. However, based on the content of this chapter,

where we describe the content of every component, it is possi-

ble to extrapolate an easy protocol that serves the same pur-

pose. Such kits are sold in BioVision, Sigma, Abcam, Cayman

Chemical, Bio-labs, and many other companies. Figure1 of

this chapter has been generated using BioVision.

6. This procedure can be slightly modified to extract glycogen
   from tissues (livers and muscles). Briefly, use 50–100 mg of
   tissues. Wash twice with cold 1
   PBS and incubate with 300μl
   of 30% KOH at 70C for 2 h. For a faster procedure, homoge-
   nize the tissues in 300μl of 30% KOH with a homogenizer and
   boil for 10 min. Centrifuge at 12,000
   gat 4C for 10 min to
   remove cellular debris. Transfer the supernatant to prechilled
   conical tubes, and add four volumes of 95% cold ethanol to
   precipitate the glycogen. Wash pellets with 70% ethanol twice

D

Resc. EV

+NaCl

-NaCl

EV

Resc.

300

0

400

500

100

200

ng glucose/μg proteins

Glycogen

600

NaCl - +

700

*

*

*

*

3000

0

4000

5000

1000

2000

Relative fluorescence

6000

7000

0 0.05 0.1 0.15 0.2

Glycogen (mg)

A

Glycogen extraction

Hydrolysis reaction

Development reaction

OD570 nm /

Fluorescence reading

(Ex/Em 535/587 nm)

y = 31107x - 16.033

R2 = 0.9976

B

C

Fig. 1Protocol demonstration using UOK257 EV and FLCN rescued cells. (a) Representative scheme of the
protocol summary. (b) Glycogen standard curve example. (c) Glycogen determination using this protocol in
UOK 257 cells lacking or rescued for folliculin expression at basal level or after exposure to 250 mM NaCl for
24 h. (d) Periodic acid-Schiff staining in UOK 257 cells lacking or rescued for folliculin expression at basal level
or after exposure to 250 mM NaCl for 24 h

64 Elite Possik and Arnim Pause