Myosin Heavy Chain Gels

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

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

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

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

3. Upper Running Buffer (COLD) Component Grams Tris (base) 12.114 Glycine 11.260 SDS 1.000

H2O 1.000 Liter

4. Lower Running Buffer (COLD) Combine 350 mls of upper buffer with 350 mls of H2O.

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

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

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

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

3. Upper Running Buffer (COLD) Component Grams Tris (base) 12.114 Glycine 11.260 SDS 1.000

H2O 1.000 Liter

4. Lower Running Buffer (COLD) Combine 350 mls of upper buffer with 350 mls of H2O.

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

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

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

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

3. Upper Running Buffer (COLD) Component Grams Tris (base) 24.228 Glycine 22.520 SDS 2.000

H2O 1.000 Liter

4. Lower Running Buffer (COLD) Combine 500 mls of upper buffer with 1500 mls of H2O.

5. Running Conditions: 5.0 mA constant current for 24-36 hours with cooling pump at 4oC.