Experiments
The design of your experiment is very important for the end-result. Carefully planned experiments with the proper controls will give the best results with a minimum of experimental time. This part wil help you to make the right decisions for your situation. Take some time to plan your experiment and you will gain a lot of time when you analyse the results
Please read the Best results part about preparing your instrument for the best measurements. In addition, read the Sensor chips section and Immobilization section for choosing a suitable sensor chip and immobilization strategy.
This section will discuss
- The injection strategy
- Replicates
- Runnen an experiment
- Recognising problems
- Reporting results
At the end of this section, there is a quiz to test your knowledge.
Flow buffer
De flow buffer is very important in the experiments. It is the carrier of the analyte to the ligand and can greatly affect the interaction between the ligand and analyte by its osmolality, composition and pH (1). Start with one of the common used flow buffers listed in the table below.
| buffer | components |
|---|---|
| HBS-PE | 10 mM HEPES pH 7.4, 150 mM NaCl, 3.4 mM EDTA, 0.01% P20 |
| TBS-P | 50 mM TRIS-HCl pH 7.4, 150 mM NaCl, 0.01% P20 |
| PBS-P | 10.1 mM Na2PO4, 1.8 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, pH 7.4, 0.01% P20 |
When necessary, add compounds to the buffer to enhance the interaction or to suppress non-specific binding. For instance, adding 0.1% BSA to the running buffer can minimize analyte adsorption to the vials and tubing in the system. Raising the detergent (up to 0.1%) or salt concentration (up to 250 mM NaCl) can suppress non-specific binding. In case of dextran sensor chips, adding CM-dextran (0.1–10 mg/ml) can help to reduce non-specific binding. Some interactions benefit from additions of ions like Ca2+ or Zn2+(2),(3). When DMSO is used to dissolve the analyte, take special care to match ligand and reference surface. In addition, a calibration of the system may be necessary to compensate for surface behaviour differences.
The flow buffer must be free of particles and proper degassed to minimize the forming of micro bubbles in the flow system. The interaction between ligand and analyte is influenced by the temperature of the system. Normally the machine is maintained at a pre-set temperature and the flow buffer arriving at the interaction point will have the correct temperature. However, cooling the buffer after degassing will result in the uptake of new gas which can form small air bubbles in the fluidic system.
References
| (1) | Alexander-Brett, J. M. and Fremont, D. H. Dual GPCR and GAG mimicry by the M3 chemokine decoy receptor. J.Exp.Med. 204: 3157-3172; (2007). |
| (2) | Sen, Prosenjit et al Zinc Modulates the Interaction of Protein C and Activated Protein C with Endothelial Cell Protein C Receptor. Journal of Biological Chemistry 285: 20410-20420; (2010). Goto reference |
| (3) | Vales-Gomez, M. et al The role of zinc in the binding of killer cell Ig-like receptors to class I MHC proteins. Proc.Natl.Acad.Sci.U.S.A 98: 1734-1739; (2001). |