AMPK Methods and Protocols

12. 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) bromophenol blue, and 5% (v/v)β-mercaptoethanol.
    Prepare 1 M Tris–HCl, pH 6.8, by dissolving 121.14 g of
    Tris–HCl in 1 L of distilled water, and adjust the pH with
    HCl. Mix 12.5 mL of Tris–HCl 1 M pH 6.8, 5 g SDS,
    25 mL glycerol, 2.5 mLβ-mercaptoethanol, 25 10 ^3 g bro-
    mophenol blue. Make up with mQ water to 50 mL. Store at
     20 C(seeNote 3).

2.3 Sodium Dodecyl
Sulfate-
Polyacrylamide Gel
Electrophoresis

1. Buffer A, pH 8.8: 750 mM Tris–HCl, pH 8.8, 0.2% (w/v)
   SDS. Weight 90.1 g Tris–HCl, 2 g SDS. Make up to 1 L with
   distilled water. Adjust the pH with HCl.


2. Buffer B, pH 6.8: 250 mM Tris–HCl, pH 6.8, 0.2% (w/v)
   SDS. Weight 30.2 g Tris–HCl, 2 g SDS. Make up to 1 L with
   distilled water. Adjust the pH with HCl.


3. APS: 10% (w/v) ammonium persulfate (APS). Dissolve 5 g
   APS in 50 mL distilled water. Prepare ready-to-use aliquots.
   Store at 4C.


4. 60% (v/v) 2-propanol: Mix 60 mL of 2-propanol in 40 mL of
   distilled water.


5. Mini-PROTEAN vertical electrophoresis system and 1.5 mm
   glass plates.


6. Running buffer 5: 20 mM glycine, 200 mM Tris–HCl, 1%
   (w/v) SDS. Weigh 72 g glycine, 15 g Tris–HCl, and 5 g SDS.
   Make up to 1 L with distilled water. Prepare running buffer 1
   by adding 100 mL of running buffer 5in 400 mL of distilled
   water.


7. Protein ladder.

2.4 Semi-dry
Transfer

1. Transfer buffer 1: 40 mM glycine, 40 mM Tris–HCl, 0.36%
   (w/v) SDS, 20% (v/v) methanol. Dissolve in distilled water.
   Store at 4C.


2. Extra-thick blot paper (filter paper). Cut two pieces of filter
   paper per gel with the necessary size to cover the gel. Submerge
   them in transfer buffer 1before use.


3. Polyvinylidene difluoride (PVDF) membrane. Cut one piece of
   PVDF per gel larger enough to match the gel size.


4. Plastic container (to activate the membranes).


5. Trans-Blot SD semi-dry transfer cell.


6. Tris-buffered saline (TBS) 10: 200 mM Tris–HCl, pH 7.6,
   1.3 mM NaCl. Weight 24.2 g Tris–HCl, 80 g NaCl. Dissolve in
   distilled water. Adjust pH. Make up to 1 L.

436 Patricia Seoane-Collazo and Miguel Lo ́pez