Yaniv Oren, Jindou Sadanari, Frolow Felix, Lamed Raphael, Bayer Edward A
Department of Molecular Microbiology and Biotechnology, The Daniella Rich Institute for Structural Biology, Tel Aviv University, Ramat Aviv, Israel.
Methods Mol Biol. 2012;908:101-7. doi: 10.1007/978-1-61779-956-3_10.
Experimental identification of carbohydrate-binding modules (CBM) and determination of ligand specificity of each CBM are complementary and compulsory steps for their characterization. Some CBMs are very specific for their primary substrate (e.g., cellulose), whereas others are relatively promiscuous or nonspecific in their substrate preference. Here we describe a simple procedure based on in-tube adsorption of a CBM to various insoluble polysaccharides, followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) for determining the distribution of the CBM between the bound and unbound fractions. This technique enables qualitative assessment of the binding strength and ligand specificity for each CBM.
对碳水化合物结合模块（CBM）进行实验鉴定并确定每个CBM的配体特异性是对其进行表征的互补且必要的步骤。一些CBM对其主要底物（如纤维素）具有高度特异性，而其他CBM在底物偏好方面则相对宽泛或非特异性。在此，我们描述了一种基于管内CBM吸附到各种不溶性多糖上，随后进行十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）的简单程序，用于确定CBM在结合和未结合部分之间的分布。该技术能够对每个CBM的结合强度和配体特异性进行定性评估。
