filter units have low dead volume and are thus preferred over
syringe filters.
7. Protein standards that are routinely used for SEC calibration
are good choices for validation of SEC/MALS performance
(the individual standards, not the premixed sets). The list of
16 commercially available proteins, with M w ranging from
6.5 kDa to 475 kDa, that our laboratory analyzed by SEC/MALS system is posted at https://medicine.yale.edu/keck/
biophysics/technologies/lightscatter/results/tables.aspx. All
proteins included in the list are monodisperse, so data collected
for these proteins can be used to normalize the LS detector. For
nucleic acid standard, the exact piece of DNA or RNA used for
complex formation is sufficient.
8. Preferably, the samples subjected to SEC/MALS analysis are
fractions collected from SEC purification that are not concentrated postfractionation to avoid protein aggregation.
9. Band broadening inherent to the SEC/MALS system can be
corrected for using software algorithms during data processing.
These parameters are computed during processing of SEC/MALS data collected at the same flow rate for a standard that is
known to be monodisperse. Since changes in sample concentration influence its diffusion, this correction should be used
with great caution; when sample is analyzed at a broad range of
concentrations, the band-broadening correction should be
validated at a comparable concentration range.
10. Proper equilibration of the SEC/MALS system is critical for
obtaining high precision in the M w determination; the baselines need to be monitored for at least 60 min to ensure that
there is no substantial drift in RI signal, and that the LS
baseline is free of noise and stable. Since the response of the
LS detector determines the lower limit of detection, low noise
is critical when working with a low amount of sample (e.g.,
20 μg of a 40 kDa macromolecule). When not in use, the
SEC/MALS system should be stored with 20% ethanol continuously recirculating throughout it. For long-term storage,
the LS cell should be filled with methanol filtered through a
0.02 μm filter.
11. The error in the M w measurement of the complex results
mostly from the uncertainty in (dn/dc) complex . The value of
(dn/dc) protein can be estimated with high precision during
ASTRA analysis of SEC/MALS data collected for protein
standards; starting with the commonly used value of
0.187 ml/g [5, 6, 17, 21, 22], the iterative process leads to a
single (dn/dc) protein value within the range of 0.18–0.20 ml/g
that provides the best match between the expected and
measured M w for all standards analyzed and taking into
SEC Coupled with Light Scattering
393
syringe filters.
7. Protein standards that are routinely used for SEC calibration
are good choices for validation of SEC/MALS performance
(the individual standards, not the premixed sets). The list of
16 commercially available proteins, with M w ranging from
6.5 kDa to 475 kDa, that our laboratory analyzed by SEC/MALS system is posted at https://medicine.yale.edu/keck/
biophysics/technologies/lightscatter/results/tables.aspx. All
proteins included in the list are monodisperse, so data collected
for these proteins can be used to normalize the LS detector. For
nucleic acid standard, the exact piece of DNA or RNA used for
complex formation is sufficient.
8. Preferably, the samples subjected to SEC/MALS analysis are
fractions collected from SEC purification that are not concentrated postfractionation to avoid protein aggregation.
9. Band broadening inherent to the SEC/MALS system can be
corrected for using software algorithms during data processing.
These parameters are computed during processing of SEC/MALS data collected at the same flow rate for a standard that is
known to be monodisperse. Since changes in sample concentration influence its diffusion, this correction should be used
with great caution; when sample is analyzed at a broad range of
concentrations, the band-broadening correction should be
validated at a comparable concentration range.
10. Proper equilibration of the SEC/MALS system is critical for
obtaining high precision in the M w determination; the baselines need to be monitored for at least 60 min to ensure that
there is no substantial drift in RI signal, and that the LS
baseline is free of noise and stable. Since the response of the
LS detector determines the lower limit of detection, low noise
is critical when working with a low amount of sample (e.g.,
20 μg of a 40 kDa macromolecule). When not in use, the
SEC/MALS system should be stored with 20% ethanol continuously recirculating throughout it. For long-term storage,
the LS cell should be filled with methanol filtered through a
0.02 μm filter.
11. The error in the M w measurement of the complex results
mostly from the uncertainty in (dn/dc) complex . The value of
(dn/dc) protein can be estimated with high precision during
ASTRA analysis of SEC/MALS data collected for protein
standards; starting with the commonly used value of
0.187 ml/g [5, 6, 17, 21, 22], the iterative process leads to a
single (dn/dc) protein value within the range of 0.18–0.20 ml/g
that provides the best match between the expected and
measured M w for all standards analyzed and taking into
SEC Coupled with Light Scattering
393