Surface Plasmon Resonance to Study Biomolecular Interactions Using a Sensor Chip
Trascrizione
For the Dual-Channel SPR system, use HBS-EP+ as running buffer, 10 millimolar glycine hydrochloric acid of pH 1.5 to 1.6 as the regeneration buffer, and turn on the instrument degasser, autosampler, and pump. Then, wash the entire system with double-distilled water for 1 hour.
Next, drop immersion oil onto the detector, and mount a glass sensor chip coated with a thin gold film, and on the upper side functionalized with carboxymethyldextran hydrogel directly onto the detector below the three-port flow cell. Then, fix the setting by pulling down the handling.
To immobilize the proteinaceous ligands to sensor chips, open a run table by clicking on Form in the Menu bar, and Run Table Editor in the integrated SPRAutoLink software. Then, choose and click on BASIC_Immobilization from the list of available run tables, and follow the steps of the experimental procedure on the computer screen.
Next, click on Sample Set Editor in the Form section to fill out the reagents list for two racks placed in the autosampler for further analyses. Click on Autosampler Sampler Direct Control as a tool in the Menu bar to bring the racks forward or back home, and choose 4 degrees Celsius as the operating temperature.
Start the pump to infuse double-distilled water by clicking on Tools and "Pump Direct Control," and record data by clicking on "SPR Instrument Direct Control," and each time, start in the newly-appearing window. After adding the coupling reagents in 300-microliter vials, put them into the autosampler racks and start the run table by clicking on Run.
For the simple protein-protein interaction after refilling the pump at 25,000 microliters per minute, perform baseline adjustment for 30 seconds. Allow for the subsequent baseline adjustment with double-distilled water for 1.5 minutes before quenching the activated chip surface with 1 molar ethanolamine hydrochloride, pH 8.5. Then, switch the tubes from the liquid sampler to the degasser from double-distilled water into the bottle with HBS-EP+.
Click on Form, scroll down, and change to "Post-processing" by clicking this operation mode. Then, click on Add to select binding curves generated over time in the data plot form for each flow cell, and export the overlay as a scrubber file. Next, click on File to open the file-saving options and obtain response curves by aligning left and right curves and subtracting signals of the second reference channel from those of the ligand channel.
