Thin-layer affinity chromatography in analysis of protein-ligand affinity.
Thin-layer affinity chromatography in analysis of protein-ligand affinity.
复制标题
蛋白质-配体亲和力分析中的薄层亲和层析。
DOI：
10.1006/abio.1996.0518
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发表时间：
1996
影响因子：
2.9
通讯作者：
M. Vuento
中科院分区：
文献类型：
--
作者：
M. Laitinen;K. Sojakka;M. Vuento
NOTES & TIPS 279首先（图2，通道10和11），然后是角蛋白检测其配体的蛋白质。因此，能够监测所研究蛋白的亲和力状态是很重要的（图2，通道12和13），与之前获得的结果相同。定量分析克尔相对于原始的原生状态。评估亲和参数的定量方法包括设置受CaCl2处理影响的atin，可以使用肌动蛋白信号作为蛋白质和配体的内部平衡混合物，并通过标准分析同一膜上结合和自由物种的浓度。一旦有一套合适的抗体通过各种物理化学方法生成(1)。从这些测量中，选择结合不同物种的解离常数，通常可以推导出对同一转移膜作用的高灵敏度双免疫检测。不幸的是，这些方法是乏味的，不允许快速检查方法有以下好处。(1)对蛋白的亲和状态进行正确的条带位置（特别是2 -be上的蛋白点）比较。亲和层析法（亲和层析法以前用于估计电泳）是可以制作的。(2)电泳次数可以减少，解离常数可以计算（2,3）。亲和色谱法的最新发展介绍了可被保存的样品。(3)在抗原与抗体结合的快速分析中，这种方法应该比其他双免疫薄层技术更具定量（4-7），类似于蛋白质-配体无检测方法，在第二次检测之前使用SDS“清洁”印迹，因此可能会消除相互作用。特别是，Lou等人(5)使用了固定在膜上的几个离散抗体区来固定一些抗原(8)。在我们的方法中不需要这样的SDS步骤。分析血清脂蛋白浓度。该试验是基于样品中存在的脂蛋白与用胶体硒标记的脂蛋白的竞争
NOTES & TIPS 279 first (Fig. 2, lanes 10 and 11) followed by keratin detec-ing of protein to its ligand. Hence, it is important to be able to monitor the affinity state of the protein studied tion (Fig. 2, lanes 12 and 13), the same results as previously were obtained. Quantitative analysis of the ker-relative to the original native state. Quantitative methods for assessing affinity parameters involve setting up atin as influenced by the CaCl2 treatment could be accomplished using the actin signal as an internal stan-equilibrium mixtures of protein and ligand and analyzing the concentrations of bound and free species by dard on the same membrane. Once an appropriate set of antibodies generated from various physicochemical methods (1). From such measurements, the dissociation constant of the binding redifferent species was chosen, this highly sensitive double immunodetection on the same transfer membrane action can usually be derived. Unfortunately, these methods are tedious and do not allow a rapid check to method has the following benefits.(1) A correct comparison of band positions (especially protein spots on two-be made on the affinity state of the protein. Affinity chromatography has been previously used for the estidimensional electrophoresis) can be made.(2) The number of electrophoresis runs can be reduced and val-mation of dissociation constants (2, 3). Recent developments in affinity chromatography have introduced uable samples can thus be conserved.(3) This method should be more quantitative than other double immu-thin-layer techniques in the rapid analysis of antigen binding to antibody (4–7), analogous to protein–ligand nodetection methods which use SDS to ‘‘clean’’the blot before the second detection is likely therefore to remove interaction. In particular, Lou et al.(5) used several discrete antibody zones immobilized on membrane to some of the antigens (8). Such an SDS step was not required in our method. analyze lipoprotein concentration in serum. That assay was based on a competition of lipoprotein present in a sample with lipoprotein labeled with colloidal selenium