Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

124 Lopez and Makhatadze
2.5 mL are required to it fill up. Similarly, to fill a 250-µL stirring syringe for
VP-ITC, it is necessary to prepare approx 400 µL of ligand solution.
2. The protein concentration in the cell can be very low, and thus it may be advantageous
to prepare a stock solution of protein. It is crucial that the dilution of the
stock protein solution to the desired concentration is done with the buffer used for
the last dialysis. This will save time when experiments at different concentrations
are required or when different experimental conditions are tested (see Note 2).
3. Remove the protein and ligand solutions from the dialysis bag and centrifuge
them for at least 20 min at 14,000 rpm and 4°C to remove insoluble particles and
dust. Never filter solutions because there might be nonspecific absorption of protein
to the membrane!
4. Measure concentrations of the protein and ligand. Knowledge of exact concentrations
of protein and ligand are very important. Rapid and accurate method for
measuring concentration is optical absorbance in the UV-range. The extinction
coefficient can be calculated from the number of aromatic residues and disulfide
bonds in a protein using an empirical Eq. 2:
ε0. 280 1%,
nm 1cm = (5690 · NTrp + 1280 · NTyr + 120 · NSS) / Mw (2)
where Mw is the molecular mass of the protein in daltons, and NTrp, NTyr, and NSS are number of tryptophan, tyrosine, and disulfide bonds, respectively. A simple
experimental procedure for estimating the extinction coefficient is described (3).
In many cases, the correction for light scattering is required and can be taken into
account according to (4).
5. Wash the cell with the buffer from the last dialysis. Introduce the needle very
carefully in the cell until it touches the bottom. At this time, lift it up about 1 mm
from the bottom and remove the liquid in the cell. The cells should never be
dried! Inject the buffer solution until you see it overflowing at the top (in the reservoir
area) and then remove the buffer from the cell. Repeat this procedure 20–25
times (approx 50 mL of buffer). After last washing, remove all buffer left in the
cell. Remove all remaining buffer from the washing syringe by gently shaking it,
but do not dry.
6. Fill the cell with the protein solution. It is important to avoid any air bubbles
trapped in the cell. For this, after filling up the syringe, pump out all air bubbles.
Insert the needle into the calorimetric cell (so that the tip of the needle is barely
above the bottom of the cell) and slowly lower the plunger until the solution
appears in the overflow reservoir. At this point, start abruptly pumping solution
in and out of the cell. This abrupt pumping will force trapped air bubbles out
from the cell.
7. The ITC reference cell is generally loaded with 0.1% sodium azide solution in
water and needs to be changed only periodically (once a month).
8. Fill up the ITC stirring syringe (the VP-ITC comes with special narrow tubes
necessary to load the stirring syringe). Once the syringe is loaded, make sure
there are no bubbles, wipe the needle of the syringe with a Kimwipe towel. Avoid
touching the wholes in the paddle.