Shainoff J R, Smejkal G B, Mitkevich O, DiBello P M
Research Institute of The Cleveland Clinic Foundation, OH, USA.
Electrophoresis. 1996 Jan;17(1):179-84. doi: 10.1002/elps.1150170129.
A preparative method for isolating centigram quantities of high molecular weight polypeptide chains with high resolution and recovery uses linear polyacrylamide/agarose composite (LPAC) gels as electrophoretic media from which the polypeptides can be easily extracted. The composites are prepared in a manner yielding linear copolymers of acrylamide and 1-allyloxy-2,3-propanediol within 2% agarose gels. After electrophoresis in sodium dodecyl sulfate (SDS), protein bands were rapidly visualized for excision by briefly immersing the gel in cold 0.1 M KCl which precipitates the protein-associated SDS. The gel slices are then freeze-thawed to disrupt the agarose matrix and promote syneresis of fluid upon centrifugation. The polypeptides are then separated from the polyacrylamide in the supernatant solution by precipitating with either acidic isopropanol, trichloroacetic acid, ammonium sulfate or other general protein precipitants. As determined with polypeptide chains of fibrinogen and its cross-linked derivatives, recoveries were virtually complete (95.4% +/- 2.2%), and were independent of molecular weights over the range tested (10(4) --10(6)).
一种用于分离毫克级高分子量多肽链的制备方法，该方法具有高分辨率和高回收率，它使用线性聚丙烯酰胺/琼脂糖复合物（LPAC）凝胶作为电泳介质，多肽可从该介质中轻松提取。复合物的制备方式是在2%琼脂糖凝胶中生成丙烯酰胺和1-烯丙氧基-2,3-丙二醇的线性共聚物。在十二烷基硫酸钠（SDS）中进行电泳后，通过将凝胶短暂浸入冷的0.1 M KCl中使与蛋白质结合的SDS沉淀，从而快速可视化蛋白质条带以便切除。然后将凝胶切片进行冻融以破坏琼脂糖基质，并在离心时促进液体的脱水收缩。然后通过用酸性异丙醇、三氯乙酸、硫酸铵或其他一般蛋白质沉淀剂沉淀，从上清液中分离出聚丙烯酰胺中的多肽。用纤维蛋白原及其交联衍生物的多肽链测定，回收率几乎是完全的（95.4%±2.2%），并且在所测试的分子量范围（10⁴ - 10⁶）内与分子量无关。
