permit the processing of a large number of samples in plate format,
making buffer condition screening easier. An automated setup can
simplify the measurement of the homogeneity of the sample and
the detection of aggregates (and high-order, physiologically irrelevant oligomers) as described above (Subheading 1.2.2). Moreover,
measurements can be performed over time to predict the stability
under the condition of a downstream experiment, as a “good”
buffer at the start of an experiment may appear “not so good”
after a few hours at 20
C.
1.4.3 Buffer Optimization
The physical stability of a protein strongly depends not only on the
protein itself but also on the buffer composition, with pH, salinity,
the presence of additives, cofactors, or ligands all having an impact.
There is presently no effective way to accurately predict the effect of
solution conditions on a protein’s thermostability or solubility
from its intrinsic properties (amino acid composition, pI, secondary
structure elements, etc.). Consequently, researchers must systematically screen a range of different buffer compositions. It is highly
recommended to combine methods such as DLS and DSF that will
separately give information on solubility/aggregation and thermal
stability. This combined approach allows the user to define the best
buffer by seeking conditions that improve both properties. The
buffer matrices for multiparametric screening of pH, salinity, buffer
nature, additives, and cofactors can be generated by hand or using
simple robotics that dilutes the protein in all the different conditions prior to measurement [28].
1.4.4 Storage Issues
It is recommended to work with freshly prepared proteins. However, this is not always practicable, and proteins may have to be
stored for weeks or months before being used. One method that is
commonly used to store proteins is flash-freezing of small aliquots
(1 mL) and subsequent storage of the samples at À80
C. Thinwalled plastic tubes should be used to hold the sample and plunged
into liquid nitrogen so that freezing is as fast as possible to avoid the
formation of ice crystals that may damage the sample. If retaining
catalytic activity is paramount, proteins are usually stored in a buffer
containing 50% (v/v) glycerol at À20
C. An alternative is lyophilization, which has the advantage that long-term storage can be at
4
C or even room temperature. Ideally, the protein is in pure water
before lyophilization. In this case, when the protein is needed, an
aliquot of lyophilisate is weighed and resuspended in the buffer of
choice. Often proteins are not stable in pure water, so more typically a defined sample volume of the protein in its target buffer is
used for lyophilization. In this case, the lyophilisate must be resuspended in the same volume of water. Not all proteins are suitable
for lyophilization as they may undergo irreversible aggregation
when concentrated and not be resuspended properly.
Protein Quality Control
19
making buffer condition screening easier. An automated setup can
simplify the measurement of the homogeneity of the sample and
the detection of aggregates (and high-order, physiologically irrelevant oligomers) as described above (Subheading 1.2.2). Moreover,
measurements can be performed over time to predict the stability
under the condition of a downstream experiment, as a “good”
buffer at the start of an experiment may appear “not so good”
after a few hours at 20
C.
1.4.3 Buffer Optimization
The physical stability of a protein strongly depends not only on the
protein itself but also on the buffer composition, with pH, salinity,
the presence of additives, cofactors, or ligands all having an impact.
There is presently no effective way to accurately predict the effect of
solution conditions on a protein’s thermostability or solubility
from its intrinsic properties (amino acid composition, pI, secondary
structure elements, etc.). Consequently, researchers must systematically screen a range of different buffer compositions. It is highly
recommended to combine methods such as DLS and DSF that will
separately give information on solubility/aggregation and thermal
stability. This combined approach allows the user to define the best
buffer by seeking conditions that improve both properties. The
buffer matrices for multiparametric screening of pH, salinity, buffer
nature, additives, and cofactors can be generated by hand or using
simple robotics that dilutes the protein in all the different conditions prior to measurement [28].
1.4.4 Storage Issues
It is recommended to work with freshly prepared proteins. However, this is not always practicable, and proteins may have to be
stored for weeks or months before being used. One method that is
commonly used to store proteins is flash-freezing of small aliquots
(1 mL) and subsequent storage of the samples at À80
C. Thinwalled plastic tubes should be used to hold the sample and plunged
into liquid nitrogen so that freezing is as fast as possible to avoid the
formation of ice crystals that may damage the sample. If retaining
catalytic activity is paramount, proteins are usually stored in a buffer
containing 50% (v/v) glycerol at À20
C. An alternative is lyophilization, which has the advantage that long-term storage can be at
4
C or even room temperature. Ideally, the protein is in pure water
before lyophilization. In this case, when the protein is needed, an
aliquot of lyophilisate is weighed and resuspended in the buffer of
choice. Often proteins are not stable in pure water, so more typically a defined sample volume of the protein in its target buffer is
used for lyophilization. In this case, the lyophilisate must be resuspended in the same volume of water. Not all proteins are suitable
for lyophilization as they may undergo irreversible aggregation
when concentrated and not be resuspended properly.
Protein Quality Control
19