Gradient polyacrylamide gel electrophoresis in presence of sodium dodecyl sulfate: a practical approach to muscle contractile and regulatory proteins.
Two gradient systems for polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS) are described, with emphasis on improvements accumulated over two decades of studies on contractile proteins and regulatory enzymes from smooth muscle. The first "big slab" system utilizes 18 x 20 x 0.1 cm3 gels and a 10-18% acrylamide gradient, optimized for a high resolution of 10 to 500 kDa polypeptides. Eight (or more) gels are cast simultaneously with a gradient formation from "bottom to top" and 20% glycerol is added to the 18% acrylamide solution. The second "minislab" system represents an improved version of the system of Matsudaira and Burgess (Anal. Biochem. 1978, 87, 386-396), with 8 x 10 x 0.05 cm3 gels and 5-15% or 9-18% acrylamide gradient ranges. They are cast from "top to bottom" in 28-piece batches also with the addition of glycerol for improved gradient formation. Both types of gels can also be cast individually using a specially designed pestle-type gradient maker. For gel destaining, a convenient continuous hydrodynamic destainer is also described.
Institute of Molecular Biology, Austrian Academy of Sciences, Salzburg.
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Sobieszek
D000205:Actomyosin / Q000302:isolation & purification; D000818:Animals; D003285:Contractile Proteins / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel / Q000295:instrumentation* / Q000379:methods*; D007202:Indicators and Reagents; D009124:Muscle Proteins / Q000302:isolation & purification*; D018482:Muscle, Skeletal / Q000737:chemistry; D009130:Muscle, Smooth / Q000737:chemistry; D009218:Myosins / Q000302:isolation & purification; D012967:Sodium Dodecyl Sulfate
D003285:Contractile Proteins; D007202:Indicators and Reagents; D009124:Muscle Proteins; D012967:Sodium Dodecyl Sulfate; D000205:Actomyosin; D009218:Myosins