13 Surface Plasmon Resonance - general info..pdf - Surface...

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Surface plasmon resonance P. Anton van der Merwe 1. INTRODUCTION 3 2. PRINCIPLES AND APPLICATIONS OF SURFACE PLASMON RESONANCE. 4 2.1. Principles 4 2.2. Applications 5 2.2.1. What SPR is good for 5 2.2.2. What SPR is notgood for. 7 3. GENERAL PRINCIPLES OF BIACORE EXPERIMENTS 8 3.1. A typical experiment 8 3.2. Preparation of materials and buffers 9 3.3. Monitoring the Dips 9 4. LIGAND 10 4.1. Direct versus indirect immobilisation 10 4.2. Covalent immobilisation 11 4.2.1. A general approach 11 4.2.2. Choice of chemistry 12 4.2.3. Prepare the protein 12 4.2.4. Pre-concentration 12 4.2.5. Amine coupling 14 4.2.6. Regeneration 15 4.2.7. Adjusting the immobilisation conditions. 16 4.3. Non-covalent immobilisation (ligand capture) 16 4.3.1. Using an existing strategy 17 4.3.2. Developing a new strategy 17 4.4. Activity of immobilised ligand 18 4.5. Control surfaces 19 4.6. Re-using sensor chips 19 5. ANALYTE 20 5.1. Purity, activity and concentration 20 - 1 -
5.2. Valency 20 5.3. Refractive index effect and control analytes 21 5.4. Low molecular weight analytes 22 6. QUALITITATIVE ANALYSIS; DO THEY INTERACT? 22 6.1. Positive and negative controls 22 6.2. Qualitative comparisons using a multivalent analyte 23 7. QUANTITATIVE MEASUREMENTS 23 7.1. Affinity 24 7.1.1. Concepts 24 7.1.2. Experimental design 24 7.1.3. Data analysis 26 7.1.4. Controls 27 7.1.5. Non-linear Scatchard Plots 27 7.2. Kinetics 30 7.2.1. Concepts 30 7.2.2. Experimental design 31 7.2.3. Data analysis 33 7.2.4. Controls 34 7.3. Stoichiometry 35 7.4. Thermodynamics 35 7.5. Activation energy 37 8. CONCLUSION 38 9. APPENDIX 38 9.1. Physical basis of SPR 38 9.2. Samples and buffers 39 9.2.1. Guidelines for preparing samples and buffers for use on the BIAcore 39 9.2.2. Standard buffers 40 9.3. Additional information 41 10. REFERENCES 41 11.ACKNOWLEDGEMENTS42 - 2 -
12. GLOSSARY 42 13. FIGURE 1. ANALYSIS OF DIPS. 44 14.TABLES45 14.1.Table 1. BIAcore instruments currently available (January 1999)45 14.2.Table 2. Sensor chips available for use on the BIAcore46 14.3.Table 3. Covalent coupling chemistry47 14.4.Table 4. Techniques for ligand capture.48 14.5.Table 5. Trivial causes of complex binding kinetics49 14.6.Table 6. Distinguishing some non-trivial causes of complex kinetics50 1. IntroductionSince the development almost a decade ago (1,2) of the first biosensor based on surface plasmon resonance (SPR), the use of this technique has increased steadily. Although there are several SPR-based systems (3-5), by far the most widely used one is the BIAcore (1,2), produced by BIAcore AB, which has developed into a range of instruments (Table 1). By December 1998 over 1200 publications had reported results obtained using the BIAcore. It is likely that it would be even more widely used were it not for its high cost and the pitfalls associated with obtaining accurate quantitative data (5-8). The latter has discouraged many investigators and led to the perception that the technique may be flawed. This is unjustified because the pitfalls are common to many binding techniques and, once understood, they are easily avoided (4,8,9). Furthermore, the BIAcore offers particular advantages for analysing weak macromolecular interactions, allowing measurements that

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