11. Set the instrument to repeatedly cycle.
12. Start the buffer:buffer run. Preferably, cycle multiple buffer:
buffer runs overnight.
13. Just as a buffer:buffer run is complete, carefully remove buffer
from the sample cell (do not dry the cell and do not stop the
program) and load the protein sample.
14. Start the sample:buffer run.
15. When carrying out a series of experiments, rinse the sample cell
with buffer twice between runs.
After completion of the experiment, data can be analyzed to
obtain the transition temperature (T m ), the calorimetric enthalpy
(ΔH), and the van’t Hoff enthalpy (ΔH VH ) for the protein under
study (more detail can be found in Ref. 32).
When comparisons are to be made between runs, use the same
instrument settings (temperature range, scan rate, etc.) in each case.
In a buffer-screening context, because the denaturation of most
proteins is not reversible or not reversible in all conditions, data
from the increasing temperature ramp only may be used for comparison (but not thermodynamic analysis).
2.4 Buffer
Optimization
Optimizing the solution conditions of a protein sample is important but can be a tedious task. It is much simplified where there is
access to instruments that can measure multiwell plates. Additionally, most (crystallography) core facilities have liquid handling and
pipetting robots that can prepare small amounts of buffers with
high precision directly into such plates. Alternatively, commercial
buffer screens are available in preprepared plates. In order to
exchange the buffer of the protein sample by the screening buffers,
the protein sample is usually simply diluted ~10-fold with the
screening buffer.
Systematic screening to maximize protein solubility would
involve varying the pH from about 4 to about 9 using different
buffers and testing each pH with concentrations of different salts
(NaCl, KCl) in the range from 0 to 1000 mM. Other additives may
be added, such as reducing agents (for proteins containing free
cysteine residues), divalent ions or cofactors for those proteins
dependent on them, and solubilizing agents such as detergents,
sugars, and amino acids. In selecting buffers and salt concentrations, the downstream application must also be borne in mind; e.g.,
NMR studies benefit greatly from lowering conductivity of the
buffer by lowering salt concentration and possibly compensating
with increased buffer or additives such as amino acids, and many
enzymes only function as catalysts in particular pH ranges. Typically, two rounds of screening will be required: the first to identify a
broad range of pH, salt, and additives that are beneficial and the
second with finer increments of the concentration of components
Protein Quality Control
39
12. Start the buffer:buffer run. Preferably, cycle multiple buffer:
buffer runs overnight.
13. Just as a buffer:buffer run is complete, carefully remove buffer
from the sample cell (do not dry the cell and do not stop the
program) and load the protein sample.
14. Start the sample:buffer run.
15. When carrying out a series of experiments, rinse the sample cell
with buffer twice between runs.
After completion of the experiment, data can be analyzed to
obtain the transition temperature (T m ), the calorimetric enthalpy
(ΔH), and the van’t Hoff enthalpy (ΔH VH ) for the protein under
study (more detail can be found in Ref. 32).
When comparisons are to be made between runs, use the same
instrument settings (temperature range, scan rate, etc.) in each case.
In a buffer-screening context, because the denaturation of most
proteins is not reversible or not reversible in all conditions, data
from the increasing temperature ramp only may be used for comparison (but not thermodynamic analysis).
2.4 Buffer
Optimization
Optimizing the solution conditions of a protein sample is important but can be a tedious task. It is much simplified where there is
access to instruments that can measure multiwell plates. Additionally, most (crystallography) core facilities have liquid handling and
pipetting robots that can prepare small amounts of buffers with
high precision directly into such plates. Alternatively, commercial
buffer screens are available in preprepared plates. In order to
exchange the buffer of the protein sample by the screening buffers,
the protein sample is usually simply diluted ~10-fold with the
screening buffer.
Systematic screening to maximize protein solubility would
involve varying the pH from about 4 to about 9 using different
buffers and testing each pH with concentrations of different salts
(NaCl, KCl) in the range from 0 to 1000 mM. Other additives may
be added, such as reducing agents (for proteins containing free
cysteine residues), divalent ions or cofactors for those proteins
dependent on them, and solubilizing agents such as detergents,
sugars, and amino acids. In selecting buffers and salt concentrations, the downstream application must also be borne in mind; e.g.,
NMR studies benefit greatly from lowering conductivity of the
buffer by lowering salt concentration and possibly compensating
with increased buffer or additives such as amino acids, and many
enzymes only function as catalysts in particular pH ranges. Typically, two rounds of screening will be required: the first to identify a
broad range of pH, salt, and additives that are beneficial and the
second with finer increments of the concentration of components
Protein Quality Control
39