Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

106 Julenius
3. Mix 50 µL of NHS solution with 50 µL of EDC solution. Inject 40 µL of the
surface activation mixture into one of the flow cells on the sensor chip (see Note 6).
With an automated instrument, both mixing and injecting can be done automatically.
4. Note the SPR signal after the surface activation is finished. This is our second
reference point.
5. Make 100 µL of an immobilization mixture using the protein to be immobilized
(see Note 7). Use one of the immobilization buffers, 10–100 µg/mL of protein
and 1 mM CaCl 2 if the protein binds calcium. Inject 45 µL of the immobilization
mixture.
6. Note the SPR signal after the immobilization is finished. Compare it to the second
reference point. The difference is an estimate of the protein now immobilized
to the surface. 1000 response units (RU) given by a BIAcore instrument
equal a protein surface concentration of about 1 ng/mm 2 . An absolute minimum
is that it should be significantly larger than the instrument noise. For a very small
peptide, this may be enough, but for a medium-sized protein it should be larger.
Typical responses for surface binding of proteins are of the order of 100–20,000
RU (see Note 8). If it seems like the immobilization has not worked properly, it is
possible to try again, as long as the surface has not been deactivated. Go back to
step 4 and change one or more of the conditions (change the pH, the protein
concentration, the injected volume, the flow rate, replace CaCl 2 by EDTA, use
neither CaCl 2 nor EDTA, and so on).
3. Deactivate the surface by injecting 20 µL ethanolamine hydrochloride.
4. Free the surface of any noncovalently attached protein molecules (regeneration)
by injecting 8 µL of a regenerator. For a calcium-dependent association (see Subheading
3.3.), one may use 10 mM EDTA. Otherwise, 0.1 M HCl is the standard
regenerator. Other examples of regenerators are found in the manual of the
instrument.
5. Note the SPR signal after the regeneration. This should be compared to the first
reference point and the difference is the true amount of immobilized protein.
3.3. Qualitative Experiment: Is the Interaction Calcium-Dependent?
1. Mix the analyte stock with calcium buffer to obtain three different concentrations
between 1 nM and 1000 nM. Use lower concentrations for large proteins and
higher for small proteins. Each sample should be 150 µL. Make the same concentrations
of samples dissolved in EDTA buffer.
2. See to that the pump inlet tubing is in the calcium-buffer flask and do not forget
to initiate the flow system if the buffer has been changed. Set the flow rate to
5 µL/min.
3. Start recording the SPR signal (start a sensorgram).
4. Inject 75 µL of one of the calcium samples.
5. After the association phase is over, keep on recording the signal for approx 30 min.
6. Inject 8 µL regenerator.
7. Stop recording the signal. A typical sensorgram is shown in Fig. 2.