AMPK Methods and Protocols

Prepare PBS at required glucose concentration (i.e., 3, 11, 17 mM). Add 1 ml of PBS in each well of a nontreated 24-well plate. ...

Incubate at 37C and spot 10μl (or 5μl if using 25μl) of reaction mix on labeled p81 papers at 10, 20, and 30 min (a different ...

Boil the samples for 5 min at 95C and place them on ice ready to be loaded onto an 8% polyacrylamide gel. We generally use a M ...

Add 2.2 volumes of ethanol. Vortex. Incubate the tubes at RT for 10 min or for a longer (20 min–d- ays) period of time on ice o ...

Add to islet pellet 1 ml of lysis buffer. Sonicate the islets in lysis buffer for 10 s at 10 W, then place on ice. Proceed to i ...

The islets can be first placed in a fresh tube and washed by adding islet media into the tube, spinning during 2 min at 1000 rp ...

little as ten islets (< 100 μm) per lane when immunoblot was performed using a WES system (http://www.proteinsimple. com/wes. ...

The amount of protein loaded per lane is low (we estimate ~1–2μg of total protein/100< 100 μm islets), and therefore signals ...

Ferrer J, Thorens B, Reimann F, Gribble F, McGinty JA, Chen L, French PM, Birzele F, Hildebrandt T, Uphues I, Rutter GA (2014) L ...

Hunter Roger W, Foretz M, Bultot L, Full- erton Morgan D, Deak M, Ross Fiona A, Haw- ley Simon A, Shpiro N, Viollet B, Barron D ...

Chapter 27 Analyzing AMPK Function in the Hypothalamus Patricia Seoane-Collazo and Miguel Lo ́pez Abstract Hypothalamic AMPK pla ...

In the hypothalamus, AMPK integrates peripheral hormonal signals to modulate both sides of the energy balance equation, namely, ...

Surgical drill. Tungsten carbide drill (KOMPH141010 for stereotaxic surgery and KOMPH141014 for intracerebroven- tricular (ICV) ...

Loading buffer 5: 250 mM Tris–HCl, pH 6.8, 10% (w/v) sodium dodecyl sulfate (SDS), 50% (v/v) glycerol, 0.0005% (w/v) bromophen ...

TBST: 1% (v/v) Tween-20 in TBS. Mix 100 mL TBS 10and 1 mL Tween-20. Make up to 1 L with distilled water. Blocking solution (BS ...

with a surgical blade and identify the bregma. Target the spe- cific nucleus bilaterally using a 25 gauge needle connected to a ...

Activators of Hypothalamic AMPK 1.Ghrelin. ICV administration of ghrelin to fed satiated rats induced a time-dependent increase ...

the rats receive 1μL of 1 nM 17β-estradiol in each bilateral injection (total 2μL in the ARC/4μL in the VMH). Rats are sacrifice ...

3.2 Dissection of the Hypothalamus and Protein Extraction Carry out all procedures in ice otherwise specified. The whole hypotha ...

curve dilution as described: 0.7 mg/mL¼ 350 μL of 1 mg/ mL standard in 150μL of distilled water; 0.5 mg/mL¼ 250 μL of 1 mg/mL st ...