<p> Myosin Heavy Chain Gels</p><p>A. Biorad Mini Gels (0.75 mm thick) 1. Separating Gel Component 8% 7% 6% Glycerol 3.00 (3.78g) 3.00 (3.78g) 3.00 (3.78g) 40% Acrylamide 2.00 1.75 1.50 1.5 M Tris (pH 8.8) 1.35 1.35 1.35 1M Glycine 1.00 1.00 1.00 10% SDS 0.40 0.40 0.40</p><p>H2O 2.15 2.40 2.65 Mix well and place under vacuum for 20 min. To initiate polymerization add: Temed------0.005 ml 10% APS---0.100 ml</p><p>2. Stacking Gel (1.0 cm high) Component 4% Glycerol 3.00 (3.78g) 40% Acrylamide 1.00 0.5 M Tris (pH 6.8) 1.40 100 mM EDTA (pH 7) 0.40 10% SDS 0.40</p><p>H2O 3.70 Mix well and place under vacuum for 20 min. To initiate polymerization add: Temed------0.005 ml 10% APS---0.100 ml</p><p>3. Upper Running Buffer (COLD) Component Grams Tris (base) 12.114 Glycine 11.260 SDS 1.000</p><p>H2O 1.000 Liter</p><p>4. Lower Running Buffer (COLD) Combine 350 mls of upper buffer with 350 mls of H2O.</p><p>5. Running Conditions: 80 volts constant voltage for 18 to 24 hours in cold (styrofoam) box with ice. B. Large CBS Gels (0.75 mm thick) 1. Separating Gel Component 8% 7% 6% Glycerol 6.00 (7.56g) 6.00 (7.56g) 6.00 (7.56g) 40% Acrylamide 4.00 3.50 3.00 1.5 M Tris (pH 8.8) 2.70 2.70 2.70 1M Glycine 2.00 2.00 2.00 10% SDS 0.80 0.80 0.80</p><p>H2O 4.30 4.80 5.30 Mix well and place under vacuum for 20 min. To initiate polymerization add: Temed------0.010 ml 10% APS---0.200 ml</p><p>2. Stacking Gel (2.0 – 2.5 cm high) Component 4% Glycerol 3.00 (3.78g) 40% Acrylamide 1.00 0.5 M Tris (pH 6.8) 1.40 100 mM EDTA (pH 7) 0.40 10% SDS 0.40</p><p>H2O 3.70 Mix well and place under vacuum for 20 min. To initiate polymerization add: Temed------0.005 ml 10% APS---0.100 ml</p><p>3. Upper Running Buffer (COLD) Component Grams Tris (base) 12.114 Glycine 11.260 SDS 1.000</p><p>H2O 1.000 Liter</p><p>4. Lower Running Buffer (COLD) Combine 350 mls of upper buffer with 350 mls of H2O.</p><p>5. Running Conditions: New: 4.0 mA constant current for 36 hours in cold box with ice packs. Old: 285 volts constant voltage (current should start at ~12 - 14 mA) for 18 hours. C. Large Biorad Gels (1 side) (0.75 mm thick) 1. Separating Gel Component 8% 7% 6% Glycerol 6.00 (7.56g) 6.00 (7.56g) 6.00 (7.56g) 40% Acrylamide 4.00 3.50 3.00 1.5 M Tris (pH 8.8) 2.70 2.70 2.70 1M Glycine 2.00 2.00 2.00 10% SDS 0.80 0.80 0.80</p><p>H2O 4.30 4.80 5.30 Mix well and place under vacuum for 20 min. To initiate polymerization add: Temed------0.010 ml 10% APS---0.200 ml</p><p>2. Stacking Gel (2.0 – 2.5 cm high) Component 4% Glycerol 3.00 (3.78g) 40% Acrylamide 1.00 0.5 M Tris (pH 6.8) 1.40 100 mM EDTA (pH 7) 0.40 10% SDS 0.40</p><p>H2O 3.70 Mix well and place under vacuum for 20 min. To initiate polymerization add: Temed------0.005 ml 10% APS---0.100 ml</p><p>3. Upper Running Buffer (COLD) Component Grams Tris (base) 24.228 Glycine 22.520 SDS 2.000</p><p>H2O 1.000 Liter</p><p>4. Lower Running Buffer (COLD) Combine 500 mls of upper buffer with 1500 mls of H2O.</p><p>5. Running Conditions: 5.0 mA constant current for 24-36 hours with cooling pump at 4oC.</p>