Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

Surface Plasmon Resonance of CaBP 105
4. Flow buffers (store at 4°C, stable for 2 wk):
a. 10 mM HEPES, 3.4 mM ethylenediaminetetracetic acid (EDTA), 0.15 M
NaCl, approx 0.005% Tween-20 (a surfactant; see Note 4), approx 0.02%
NaN 3 (toxic), pH 7.4.
b. 10 mM HEPES, 2 mM CaCl 2, 0.15 M NaCl, approx 0.005% Tween-20, approx
0.02% NaN 3 (toxic), pH 7.4.
5. Immobilization buffers (store at 4°C, stability depending on pH):
10 mM sodium acetate, adjust pH with 5 M acetic acid. Make 3–4 with different
pH, slightly under the isoelectic point of the protein to be immobilized (e.g.,
pH 3.5, 4.0, and 4.5 for a protein with isoelectric point approx 4.5).
6. EDTA and CaCl 2 solutions:
a. 10 mM EDTA, pH 8.0.
b. 0.5 M EDTA, pH 7.4.
c. 0.5 M CaCl 2.
7. 0.1 M HCl.
8. Sterile, disposable filters, 0.22 µm (e.g., Corning):
a. Syringe tip filters ∅ approx 25 mm and 5 mL sterile, disposable syringes to
go with them.
b. Bottle top filters (either 33- or 45-mm neck size, depending on what kind of
glass bottles you readily have; see Note 5).
9. Coupling reagents:
a. 0.1 M N-hydroxysuccinimide (NHS).
b. 0.4 MN-ethyl-N'-(dimethylaminopropyl)carbodiimide (EDC).
c. 1 M ethanolamine hydrochloride.
3. Methods
3.1. Preparation
1. Filter all solutions. The amounts needed are such that the flow buffers should be
filtered using the bottle top filters and all other solutions should be filtered using
syringe tip filters. Do not forget to filter the protein stock solutions. Solutions
older than 1 wk should be filtered again before use.
2. Degas the flow buffers under vacuum for approx 5 min.
3. Start the SPR instrument and insert the sensor chip (see the instrument manual
for a detailed description of all instrument-related instructions).
4. Place the pump inlet tubing in the calcium-buffer flask. Place a beaker at the
waste outlet. Initiate the flow system with the new buffer.
5. Set the temperature to the desired value. Wait until it is equilibrated.
6. If the chip is new, it should be normalized using a glycerol solution.
3.2. Immobilization
1. Set the flow rate to 5 µL/min.
2. Wait for equilibrium baseline and note the SPR signal. This is our first reference
point.