Menguy T, Chenevois S, Guillain F, le Maire M, Falson P, Champeil P
Unité de Recherche Associée 2096 Centre National de la Recherche Scientifique, CEA Saclay, Gif-sur-Yvette, France.
Anal Biochem. 1998 Nov 15;264(2):141-8. doi: 10.1006/abio.1998.2854.
We describe a method for estimating ligand binding to a macromolecular sample under conditions where this binding is of low affinity and must be measured under equilibrium conditions, without removal of the unbound ligand. The method is based on centrifugal ultrafiltration through a membrane with a molecular mass cut-off intermediate between that of the ligand and that of the target, and the amount of bound ligand is calculated from the difference between the (total) ligand in the concentrated sample and the (free) ligand in the ultrafiltrate. Centrifugal ultrafiltration makes it possible to separate free ligand from bound ligand (without changing its concentration) and to simultaneously concentrate the target (such that the proportion of bound ligand becomes significant, even under low-affinity binding conditions). We applied this technique, using Centricon 10 (Amicon) devices, to several cases (soluble proteins, intact membranes, detergent-solubilized proteins, and pure detergent micelles) and assessed its value with respect to the common artifacts that occur in other protocols involving protein retention on nitrocellulose filters (nonspecific ligand adsorption and protein denaturation).
我们描述了一种在配体与大分子样品的结合亲和力较低且必须在平衡条件下测量且不除去未结合配体的情况下，估算配体与大分子样品结合的方法。该方法基于通过一种分子量截留值介于配体和靶标之间的膜进行离心超滤，结合配体的量由浓缩样品中的（总）配体与超滤液中的（游离）配体之间的差值计算得出。离心超滤使得能够将游离配体与结合配体分离（而不改变其浓度），并同时浓缩靶标（从而即使在低亲和力结合条件下，结合配体的比例也变得显著）。我们使用Centricon 10（Amicon）装置将该技术应用于几种情况（可溶性蛋白质、完整膜、去污剂增溶的蛋白质和纯去污剂胶束），并针对其他涉及蛋白质保留在硝酸纤维素滤膜上的方案中出现的常见假象（非特异性配体吸附和蛋白质变性）评估了其价值。
