低聚糖，蛋白质中二者相互作用检测方案(大分子作用仪)

HORIBAJOBIN YVONSPRi & Life Sciences SPRi & Life SciencesApplication Note SPRi-0707.1-v1-Page 2 SPRi Imaging ofOligosaccharides/Proteins Interactions MERCEY Emilie1， SADIR Rabia2， MAILLART Emmanuel3， ROGET Andre1，LORTAT-JACOB Hugues?， LIVACHE Thierry1 1 CREAB， UMR 5819 (CEA-CNRS-UJF)， DRFMC. CEA Grenoble； 17， rue des Martyrs 38054 GRENOBLE Cedex 9， France. 2 Laboratoire d'Enzymologie Moleculaire， Institut de Biologie Structurale UMR 5075 (CEA-CNRS-UJF)， 41 rue Jules Horowitz， 38027 Grenoble Cedex 1， France. 3 GenOptics SA， Orsay， France Introduction The majority of proteins are glycosylated：they possessoligosaccharide： chains and are hence termedglycoproteins. Oligosaccharides， also called carbohydrateor sugar， are often quite large (as large as some proteindomains for example) and they have many functions inmolecular interactions. In mammals， sulfated glycosaminoglycans (6(GAGs))areamongthe carbohydrates which play an essentialphysiological role. For example， heparin (also known as ananti-coagulant)and heparinsulfate (HS) are linearpolysaccharides expressing a high chemical heterogeneitythat enables a wide range of very diverse functions，essentially through direct interactions with a number ofproteins. The SPR imaging technology developed by GenOptics is alabel-free highly sensitive method of visualization. Thanksto a video CCD camera the whole area of the biochip canbe visualized. This enables the functionalization of biochipsin array format and the collection of data from all thedifferent spots. PUH N (CH2)10 HN-NH2 Pattern and image of SPRi-biochip TM functionalized ·Each sequence is adressed separately · A mixture， diluted in a phosphate buffer， of pyrrole and pyrroles coupledwith oligosaccharide is copolymerized · Spot thickness is controlled by the injected charge via a voltage pulse.Spot diameter： 100 um<0<500 pm · Up to 400 spots sC Abroad monochromatic polarized light (at a specificwavelength) illuminates the whole functionalized area of theSPRi-biochipTM which iss connectedl with adetectionchamber. A CCD video camera gives access to arrayformat (up to 400 spots) by image capture of all localchanges at the surface of the SPRi-biochipTM. Influence of spacer arm length and HP6 probe density onthe interaction kinetics resulting from the injection of 250nM SDF-1a. ·Kinetic results kass(HP6/SDF) 7.1 104±2104 M-1.s-1 kdiss(HP6/SDF) 1.410-2±0.1 10-2.s-1 KD(HP6/SDF) 195± 50 nM ·These results indicate that SDF1a reacts Find us at www.jobinyvon.com/SPRi or telephone： France： +33 (0)1645413 00USA： +1 732 494 86 60 Japan： +81 (0)3 3861 8231Germany：+49 (0)89 46 23 17-0UK：+44 (0)20 8204 8142 Italy： +390257603050China： +86 (0)10 8567 9966Other Countries： +33 (0)1 64 54 13 00 HORIBAExplore the future Combining Polypyrrole with SPRi provides a unique method for fast, easy and reliable studies of oligosaccharide interactions on a biochip. The GenOptics platform, based on SPRi technology, is a powerful device for glycobiology, a rapidly expending area of research.This study demonstrates the process of grafting natural oligosaccharides on SPRi-Biochip™.SPR imaging is ideal to study protein-oligosaccharide interactions without labeling neither the probes nor the targets and to monitor multiple (at least 25) kinetic interactions in real time.This approach enable direct studies of synthetic or natural oligosaccharides properties to be developed even further.