Stochaj Wayne R, Berkelman Tom, Laird Nancy
CSH Protoc. 2006 Oct 1;2006(5):pdb.prot4584. doi: 10.1101/pdb.prot4584.
INTRODUCTIONThe equilibration step serves to saturate the IPG strip with the SDS buffer system required for the second-dimension separation. The equilibration solution consists of buffer, urea, glycerol, reductant, SDS, and dye. The buffer (50 mM Tris-HCl, pH 8.8) maintains the appropriate pH range for electrophoresis. Urea and glycerol are added to reduce the effects of electroendosmosis, thus helping improve protein transfer from the IPG strip to the second dimension. The reductant (dithiothreitol) ensures that disulfide bridges are broken. SDS ensures that the proteins are denatured and also provides a net negative charge to all proteins. Iodoacetamide, introduced during a second equilibration step, alkylates thiol groups on the proteins, preventing their reoxidation during electrophoresis, and thus reducing streaking and other artifacts in the second-dimension separation. Iodoacetamide also alkylates residual dithiothreitol, preventing point streaking and other silver staining artifacts. Finally, a tracing dye (bromophenol blue) is added to allow the electrophoresis to be monitored during the run.
引言
平衡步骤用于使IPG胶条用二维分离所需的SDS缓冲系统饱和。平衡溶液由缓冲液、尿素、甘油、还原剂、SDS和染料组成。缓冲液（50 mM Tris-HCl，pH 8.8）维持电泳所需的合适pH范围。添加尿素和甘油以减少电渗作用的影响，从而有助于改善蛋白质从IPG胶条转移到二维分离中。还原剂（二硫苏糖醇）确保二硫键断裂。SDS确保蛋白质变性，并为所有蛋白质提供净负电荷。在第二个平衡步骤中引入的碘乙酰胺使蛋白质上的巯基烷基化，防止其在电泳过程中重新氧化，从而减少二维分离中的条纹和其他假象。碘乙酰胺还使残留的二硫苏糖醇烷基化，防止斑点条纹和其他银染假象。最后，添加一种示踪染料（溴酚蓝）以便在电泳过程中进行监测。
