Sobieszek A
Institute of Molecular Biology, Austrian Academy of Sciences, Salzburg.
Electrophoresis. 1994 Aug-Sep;15(8-9):1014-20. doi: 10.1002/elps.11501501151.
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.
本文描述了两种在十二烷基硫酸钠（SDS）存在下用于聚丙烯酰胺凝胶电泳（PAGE）的梯度系统，重点介绍了在对平滑肌收缩蛋白和调节酶长达二十年的研究中积累的改进方法。第一种“大板”系统使用18×20×0.1 cm³的凝胶和10 - 18%的丙烯酰胺梯度，针对10至500 kDa多肽的高分辨率进行了优化。八块（或更多）凝胶同时灌制，梯度从“下到上”形成，并向18%的丙烯酰胺溶液中添加20%的甘油。第二种“小板”系统是Matsudaira和Burgess系统（《分析生物化学》，1978年，87卷，386 - 396页）的改进版本，凝胶尺寸为8×10×0.05 cm³，丙烯酰胺梯度范围为5 - 15%或9 - 18%。它们以28块一批的方式从“上到下”灌制，同样添加甘油以改善梯度形成。这两种类型的凝胶也可以使用专门设计的杵式梯度制备仪单独灌制。对于凝胶脱色，还介绍了一种方便的连续流体动力学脱色器。
