Pellet was dried under reduced pressure and dissolved in 2D sample buffer containing 9.5M Urea, 4% NP-40, 5% (v/v) 2-mercaptoethanol, 2% v/v ampholytes
of pH 5-8 and pH 3-10.
Acrylamide, bisacrylamide, Biolyte ampholyte
solution, and concentrated Coomassie Brilliant Blue protein reagent were obtained from BioRad Laboratories, Hercules, CA, USA.
We transferred 4 [micro]L of each sample to a hydrated Immobiline dry gel (ampholyte
: Servalyt, pH 5-7; GE Healthcare) and run on a PhastSystem (GE Healthcare) according to the manufacturer's conditions and protocol.
Frozen muscle tissue (100 mg) was incubated for 40 min in 1 ml of 8 M urea, 2 M thio-urea, 65 mM DTT, 2% CHAPS, 1% bio-lyte ampholyte
(3-10, Bio-Rad, Hercules, CA, USA) and protease inhibitor cocktail (Roche Diagnostics GmbH, Mannheim, Germany).
In the first dimensional isoelectric focusing (IEF), 120 [micro]g proteins of each sample were diluted to 450 [micro]L with rehydration buffer containing 8 M urea, 2% (w/v) CHAPS, 50 mM DTT, and 0.5% (v/v) ampholyte
(pH 4-7, Amersham Biosciences), and IPG strips were allowed to rehydrate in the above solution under mineral oil.
The cell homogenate (approx 0.98 mL) and 3 mL of ampholyte
solution (pH range 3-10, Bio-Rad, Hercules, CA) were mixed in 58 mL of distilled water.
A mixture of 4 mL [H.sub.2]O, 1 mL of acrylamide and bisacrylamide stock (4.75 g acrylamide and 0.25 g bisacrylamide in 10 mL [H.sub.2]O), 1 mL of ammonium persulfate stock (0.025 g in 10 mL [H.sub.2]O), 0.5 mL of ampholyte
(pH 4-9), and 0.2 mL of TEMED stock (0.20 mL TEMED in 10 mL [H.sub.2]O) was used to make four slabs.
For isoelectric focusing (IEF), dry acrylamide gels (6% T) were reswollen to their original thickness in 3% ampholyte
solution (Ampholine 3.5-9.5; Amersham Biosciences) with or without the addition of 9 mol/L urea.
Isoelectric focusing of the hemolysate was performed on a polyacrylamide gel plate containing carrier ampholyte
(pH range 6-8 and 3.5-10.0; Pharmacia LKB Biotechnology).
In this study, three sheets of cellulose acetate membranes that had absorbed 100 g/L ampholyte
Sepaline (pH 3.5-10) (Fuji Photo Film) and 100 g/L sucrose were used simultaneously.
solution consisted of a mixture of Pharmalyte pH 6-8 and Pharmalyte pH 7-9 (3:1, by vol), at a final concentration of 20 mL/L in a 4 g/L methylcellulose solution.