Biacore生物大分子互作.

1、 The sensorgram provides quantitative information in real-time on specificity of binding, active concentration of molecule in a sample, kinetics and affinity. Illustration of detector and IFC ligand analyte ligand analyte capturing molecule Ligand: the interactant immobilized on the sensor surface T

2、he ligand can be immobilized directly to the sensor surface, or indirectly via an immobilized capturing molecule Analyte: the interactant in free solution v Quantitative measurements of the binding interaction between one or more molecules are dependent on the immobilization of a target molecule to

3、the sensor chip surface. v Binding partners to the target can be captured from a complex mixture, in most cases, without prior purification (for example, clinical material, cell culture media) as they pass over the chip. Interactions between proteins, nucleic acids, lipids, carbohydrates and even wh

4、ole cells can be studied. v The sensor chip consists of a glass surface, coated with a thin layer of gold. This forms the basis for a range of specialized surfaces designed to optimize the binding of a variety of molecules. v In the most widely used sensor chip, the gold surface is modified with a c

5、arboxymethylated dextran layer. This dextran hydrogel layer forms a hydrophilic environment for attached biomolecules, preserving them in a non- denatured state. A range of other derivatized surfaces is also available to enable various immobilization chemistries v Sensor Chip CM5 for applications fr

6、om basic research to quality control v Sensor Chip SA for capture of biotinylated peptides, proteins and DNA v Sensor Chip NTA for capture of ligands via metal chelation v Sensor Chip HPA for membrane biochemistry and the study of membrane associated receptors in a near native environment v Pioneer

7、Chips as tools for users novel applications in terms of chemistry or binding specificity. vInteractions involving all types of biomolecules such as proteins, lipids, carbohydrates, and nucleic acids can be studied. v Studies can also be made of reagents ranging in size from small organic molecules,

8、e.g. drug candidates, to large molecular assemblies, such as membrane receptors embedded in lipid bilayer vesicles or even whole cells. Amine coupling: most common method of coupling. Surface is activated with a 1:1 mixture of NHS:EDC, to give reactive sucinimide esters. The ligand (in a buffer givi

9、ng a + charge) is passed over the surface and the esters react spontaneously with amino groups. Any free amine group can react with the surface. C OH OC O OC NH O EDC/NHS N OO NH2 ligand ligand Thiol coupling: an active disulfide moiety can be introduced either on the dextran matrix or on the ligand

10、 molecule. 2-(2-pyrdinyldithio) ethaneamine (PDEA) can be used to introduce the actice di-sulfide group. C OH OC O OC NH O EDC/NHS N OO SH ligand ligand PDEA C NH O S S S S N v Sensor Chip SA is designed to bind biotinylated molecules. v Streptavidin-biotin coupling provides an alternative to other

11、ligand coupling methods e.g. covalent amine or thiol coupling, and is particularly valuable for applications in molecular biology where immobilization of nucleic acids through a 5-biotin moiety is often the most practical approach. B-cat/TCF activation of certain promoters requires splicing variants containing an “E-tail” Using the Biacore we showed that TCF1 E-tail binds non-specifically to DNA (Syed et al 2005) Ma c i n t o s h P I CT i ma g e f o r ma t i s n o t s u p p o r t e d Response (RU) Time (s) ka = 1.03e+05/Ms kd = 2.49 e-02/s KA = 4.14 e+06/M KD = 2.42 e-07 M Macintosh P