the injection loop with twofold the injection volume of buffer
to ensure complete loading onto the column.
12. At the end of the run (1 column volume), analyze all peaks of
the chromatogram to assess sample recovery and for molecular
mass determination according to the manufacturer’s manual.
The baseline for the chromatograms should be set up in a
region where no proteins elute, such as the region prior to
the void volume of the column (first third of the column
volume). The baseline should remain constant if the sample is
in the same buffer as the elution buffer and if the temperature is
controlled. If the column type and the buffer conditions are
chosen correctly to avoid nonspecific binding of the protein,
you should recover nearly 100% of your sample.
13. If you intend to run another sample in the same buffer, rinse
the injection syringe and injection loop with buffer and you
may then start another run at (7).
14. When not in use, the instrument should not be left with buffer
for more than an hour. Rinse the injection syringe and injection
loop and purge the pump refractometer and LALS/MALS
detector with water and then fill with 20% (v/v) ethanol.
Columns may be left in buffer for short periods but should
be rinsed with least 2 column volumes of water and then filled
with 20% (v/v) ethanol for longer-term storage.
If the sample is pure and homogeneous, SEC will give one
sharp and symmetric peak in the elution profile/chromatogram
associated with the molecular mass of the target protein. Polydispersity due to oligomers or complex formation will be immediately
obvious if there is more than one peak. The amounts of each species
may be estimated by integrating the area under each peak. Protein
aggregates will usually elute in the void volume. However, one
should keep in mind that SEC is not an equilibrium method; not
only will it apply shear forces on the sample that may disturb
protein oligomers/complexes, but the sample will also be diluted
during the SEC run and will consequently change species populations as the run proceeds.
2.2.3 DLS Measurement
of Homogeneity
DLS is a quick method to test the homogeneity of a sample and the
presence of aggregates. The detected signal is proportional to the
sixth power of the particle radius, meaning that even small numbers
of large aggregates can be easily detected. However, this strong size
dependence also means that the even small fractions of a large
species (such as an oligomer) can lead to inaccurate results or
entirely hide the presence of a smaller one. For example, in a
solution containing 99.9% of homogeneous protein with a 3 nm
R h and 0.1% by mass of an aggregate with a 30 nm R h , the peaks
corresponding to the two populations will have approximately the
Protein Quality Control
27
to ensure complete loading onto the column.
12. At the end of the run (1 column volume), analyze all peaks of
the chromatogram to assess sample recovery and for molecular
mass determination according to the manufacturer’s manual.
The baseline for the chromatograms should be set up in a
region where no proteins elute, such as the region prior to
the void volume of the column (first third of the column
volume). The baseline should remain constant if the sample is
in the same buffer as the elution buffer and if the temperature is
controlled. If the column type and the buffer conditions are
chosen correctly to avoid nonspecific binding of the protein,
you should recover nearly 100% of your sample.
13. If you intend to run another sample in the same buffer, rinse
the injection syringe and injection loop with buffer and you
may then start another run at (7).
14. When not in use, the instrument should not be left with buffer
for more than an hour. Rinse the injection syringe and injection
loop and purge the pump refractometer and LALS/MALS
detector with water and then fill with 20% (v/v) ethanol.
Columns may be left in buffer for short periods but should
be rinsed with least 2 column volumes of water and then filled
with 20% (v/v) ethanol for longer-term storage.
If the sample is pure and homogeneous, SEC will give one
sharp and symmetric peak in the elution profile/chromatogram
associated with the molecular mass of the target protein. Polydispersity due to oligomers or complex formation will be immediately
obvious if there is more than one peak. The amounts of each species
may be estimated by integrating the area under each peak. Protein
aggregates will usually elute in the void volume. However, one
should keep in mind that SEC is not an equilibrium method; not
only will it apply shear forces on the sample that may disturb
protein oligomers/complexes, but the sample will also be diluted
during the SEC run and will consequently change species populations as the run proceeds.
2.2.3 DLS Measurement
of Homogeneity
DLS is a quick method to test the homogeneity of a sample and the
presence of aggregates. The detected signal is proportional to the
sixth power of the particle radius, meaning that even small numbers
of large aggregates can be easily detected. However, this strong size
dependence also means that the even small fractions of a large
species (such as an oligomer) can lead to inaccurate results or
entirely hide the presence of a smaller one. For example, in a
solution containing 99.9% of homogeneous protein with a 3 nm
R h and 0.1% by mass of an aggregate with a 30 nm R h , the peaks
corresponding to the two populations will have approximately the
Protein Quality Control
27