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实验方法> 生物信息学技术> 数据库>Surface Plasmon Resonance for Measurements of Biological Interest

Surface Plasmon Resonance for Measurements of Biological Interest

关键词： surface plasmon resonance来源： 互联网
  • Abstract
  • Table of Contents
  • Materials
  • Figures
  • Literature Cited

Abstract

 

Genetic manipulations, including gene knock?outs and mutant screens, provide an initial hint as to the function of a gene product and indicate possible associated factors. To unravel complicated biological processes, which control the development of organisms, one must identify the interacting components. An in vitro technique based on an optical phenomenon, called surface plasmon resonance (SPR), can simultaneously detect interactions between unmodified proteins and directly measure kinetic parameters of the interaction. This technique is gaining popularity, due to the increased availabilty oif user?friendly machines, and an overview of the technology is presented in this unit.

     
 
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Table of Contents

  • Basic Protocol 1: SPR Using BIAcore Chips
  • Basic Protocol 2: SPR Using NTA‐SAM Chips
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: SPR Using BIAcore Chips

  Materials
  • CM‐5 dextran chips (BIAcore)
  • PBS ( appendix 22 )
  • Ligand protein
  • Target protein
  • Amine‐coupling kit (BIAcore), containing:
  • N ‐ethyl‐N′‐[(dimethylamino) propyl] carbodiimide hydrochloride (EDC)
  • N ‐hydroxysuccinimide (NHS)
  • Sodium acetate buffer, low pH
  • Ethanolamine
  • BIAcore SPR equipment
  • BIA evaluation point‐and‐click software

Basic Protocol 2: SPR Using NTA‐SAM Chips

  Materials
  • NTA‐SAM chips (3% to 5% NTA relative to an inert ethylene glycol–terminated thiol)
  • PBS or HeBS ( appendix 22 )
  • 1 mM NaOH
  • 1% (w/v) Ni(II)SO 4
  • Histidine‐tagged ligand protein
  • Target protein
  • BIAcore SPR equipment
  • BIA evaluation point‐and‐click software
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Figures

  •   Figure 20.4.1 The physical configuration of modern SPR devices. Light passing through a dense medium (n1 ) incident at an angle (Θ1) greater than the critical angle, to a second medium of lower refractive index (n2 ) is totally reflected off the interface, except for an evanescent field that extends into the second medium for a distance of one wavelength. At a certain angle (Θ2 ) exquisitely dependent on the refractive index of the second medium, the incident light wave can couple to surface plasmon waves (electron scillations) propagating through a thin metal film (n3 ) sandwiched between the two media. The incident light energy is fed into the surface plasmon wave and a corresponding minimum in reflected light is observed. Proteins immobilized at (or recruited to) the metal‐medium interface alter the dielectric of the region, which causes a shift in the angle of minimum reflectivity (Θ3 ).
    View Image
  •   Figure 20.4.2 BIAcore sensorgram showing that binding of hTBPc in solution to peptide motif surfaces is dependent upon density of ligand immobilization. The BIAcore SPR instrument plots changes in the angle of minimum reflectance in resonance units (RUs) as a function of time. The “square waves” represent injections of protein “plugs” that interrupt the constant buffer flow. The net change in measured RUs following a protein plug injection (arrows) indicates binding and can be correlated to a mass of protein recruited to the surface. An association constant can be derived from analysis of the initial phase of the injection, and a dissociation rate can be extracted from analysis of the system as it returns to buffer flow. Histidine‐tagged fusion proteins terminated with the desired recognition motif were separately immobilized on NTA‐SAMs presenting 3.8% (dashed line) or 5.7% NTA (solid line). hTBPc in solution was injected over the surfaces in two separate experiments. An overlay of the two resultant SPR sensorgrams shows that hTBPc cannot bind (Δ5 RUs; arrowhead) to the repeats when they are immobilized at low density (3.8% NTA; average distance = 29 Å), but bind very tightly (Δ550 RUs; arrowhead) when the peptides are positioned closer together at a higher density (5.7% NTA; average distance = 23 Å).
    View Image

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Literature Cited

Literature Cited
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