Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

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equipped with a pulse dampener, in-line 0.2-µm and 0.1-µm membrane filters,
and a Rheodyne Model 7125 sample injector complete with a 1.0-mL sample
loop; Pharmacia Superose 12 FPLC Gel Filtration column; Wyatt Technology
Corporation Dawn F Multi-Angle Laser Light Scattering Photometer equipped
with a 10-mW 488-nm argon-ion laser and temperature-controlled flow cell and
read head; Wyatt Technology Corporation Optilab Model 903 Differential Refractometer
equipped with temperature-controlled 1-mm path length flow cell;
and finally, a Lauda model K-2/R circulating water bath with insulated hoses
connected to both the Dawn F and Optilab 903.
2. Periodically, the 90° detector in the Dawn F is calibrated with HPLC grade Toluene,
which has a known scattering intensity, or Rayleigh Ratio, R Θ, following the
procedures outlined in the Dawn F instrument manual (1). The Optilab 903 is
calibrated with a solution prepared from ultrapure, anhydrous, NaCl following
the procedures outlined in the Optilab 903 instrument manual (2).
3. The desired run temperature (usually 20°C.) is set for both the Dawn F and
Optilab 903 using the temperature control setting on the Dawn F and on the Lauda
water bath at least 24 h prior to calibrating or running samples to allow the Optilab
signal (DRI) to stabilize, because it is very sensitive to temperature changes. As
well, the Optilab lamp is powered up for a minimum of 24 h before data collection
to reduce signal drift over time.
4. Prior to pumping through the instrument setup, the buffer solution is filtered
through 0.1-µm membrane filters. A minimum volume of 100–200 mL is then
pumped through the set-up at a maximum of 1.0 mL/min to allow the column to
equilibrate and the Optilab signal to stabilize.
2.2. Sample Preparation
1. The protein solution (typically 400–500 µL of 1–5 mg/mL solution) is dialyzed
against the buffer solution for 24 h and then filtered through a 0.22-µm syringe
filter prior to injecting into the instrument set-up. After filtering, the sample solution
is spun for 3–5 min in a microfuge to remove minute air bubbles introduced
in the filtering process.
2.3. Data Collection
1. Prior to running a sample, the intensity of the scattering signal at different angles
is normalized to the intensity of the signal at 90° in the Dawn F using a solution
of BSA (Sigma 98% monomer), prepared in the same manner as the sample solution.
With the HPLC pump operating at a flow rate of 0.8 mL/min, which provides
reasonable separation of peaks and relatively quiet MALLS signals in
reasonably short run times, a 40-µL aliquot of BSA solution (approx 5 mg/mL) is
injected into the system using the Rheodyene injector. Data collection is performed
using Wyatt Technology’s Astra software.
2. Once the MALLS and DRI signals have stabilized after elution of the BSA peaks,
an aliquot of the sample solution is injected (with the Superose 12 column and a
sensitivity setting of 100, an injected mass of approx 0.25 mg will produce a full-