Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

MALLS and Sedimentation Equilibrium 131
should not be disassembled, but can be flushed with cleaning solutions if necessary.
However, the need for cleaning can be greatly reduced if the instrument
setup is thoroughly flushed after sample runs with buffer solution and then MilliQ
H 2O/0.01% NaN 3. Once the buffer solution is completely flushed from the setup,
the HPLC pump is left running continuously at a low flow rate, circulating the
MilliQ H 2O/0.01% NaN 3 through the column and flow cells and back to the solvent
reservoir. This also extends the life of the various seals and check valves on
the HPLC pump.
2. Careful filtering of both the buffer solution and sample solution and the use of an
in-line vacuum solvent degasser cannot be emphasized enough, because the
MALLS detectors are extremely sensitive to minute amounts of dust or micro air
bubbles, which will increase the noise levels dramatically.
3. The MALLS detectors are very sensitive to pressure changes and a small peak
invariably appears at the void volume on the MALLS chromatograms due to a
pressure surge from the sample injection. This effect can be reduced by installing
a bypass loop (3 feet of 0.01 in id PEEK tubing) using T-connections on either
side of the Rheodyne injector.
3. Methods: Analytical Ultracentrifugation
3.1. Instrument Setup and Preparation
1. The instrumental setup consists of a Beckman XL-I Analytical Ultracentrifuge,
containing both Interference and Absorbance Optical Systems, an IBM Pentium
II computer for centrifuge control and data analysis, an AN 50 Ti 8-hole rotor,
CFE six-sector sample cells, containing either quartz or sapphire windows, and a
Perkin Elmer Lambda 5 dual beam spectrophotometer. Prior to performing sedimentation
equilibrium runs on the samples at the desired run speeds, the radial
calibration of the optical system being used is performed at low speed, typically
3000 rpm, following the procedures outlined in the XLI Instruction Manual (4).
2. The AN 50 Ti rotor is brought close to the desired run temperature, usually 20°C,
prior to loading in the centrifuge to lessen equilibration time to the set temperature
before the run start.
3.2. Sample Preparation
1. The protein solution, typically 400–500 µL of 1–5 mg/mL solution for use with
the Interference Optical System, and a generally much lower concentration for
use with the Absorbance Optical System, is dialyzed against the buffer solution
for a minimum of 24 h and then both the dialyzate and protein solution are filtered
through 0.22-µm syringe filters.
2. If there are aromatics present in the protein, the absorbance of the protein solution
is measured against the dialyzate and an approximate concentration determined
using a theoretical extinction coefficient calculated from the amino acid
composition using the Sednterp computer program (5).
3. Dilutions are made of the stock solution to provide three loading concentrations,
typically covering an approximate fivefold difference, such as 5.0, 3.0,