UV/RI ratios for the protein and the nucleic acid used for
complex formation.
4. Compute the observed UV/RI ratio for the complex from
SEC/MALS analyses at different concentrations. Check
whether the UV/RI ratios are concentration dependent.
5. Compute the expected UV/RI ratio for the possible complexes
as weight-average UV/RI ratios of protein and nucleic acids
based on the proposed stoichiometries (results of such analysis
for FIR protein complexes are reported in Table 3 and plotted
in Fig. 3).
6. Confirm that both the observed M w and UV/RI ratio are
consistent with the proposed stoichiometry (Fig. 3 and
Table 3; see Note 12).
4 Notes
1. The data for the examples shown were collected using an
SEC/MALS system consisting of a high-performance liquid
chromatography system (HPLC), Waters Alliance 2960, or
Agilent 1200. The elution from SEC was monitored by a
Waters or Agilent photodiode array (PDA) UV/VIS detector,
differential refractometer, and static and dynamic, multiangle
laser light scattering detector. Two software packages were
used for data collection and analysis: the Waters Millennium
software controlled the HPLC operation and data collection
from the multiwavelength UV/VIS detector, while the Wyatt
ASTRA software collected data from the refractive index detector, the light scattering detector, and recorded the UV trace at
280 nm, 295 nm, or 310 nm (see Note 3) sent from the PDA
detector. However, other UV, RI, and LS detectors can be used
(a single-angle LS detector is suitable for analysis of macromolecules with molar masses up to ~500 kDa). The UV detector
should be capable of extracting signals for various UV wavelengths (see Note 3).
2. The 0.1 μm “inline” filter placed between pumps and the
injector retains any particles that are shed from the HPLC
pump’s head. An additional “inline” filter (2 μm PEEK frit)
with small dead volume is installed between the injector and
the SEC column; this filter traps protein aggregates that are
formed during the injection step and that may result from
exposing the protein sample to high pressure; this filter substantially increases the lifetime of the SEC column. These filters
are replaced whenever the system’s operating pressure increases
by more than 5%.
SEC Coupled with Light Scattering
391
complex formation.
4. Compute the observed UV/RI ratio for the complex from
SEC/MALS analyses at different concentrations. Check
whether the UV/RI ratios are concentration dependent.
5. Compute the expected UV/RI ratio for the possible complexes
as weight-average UV/RI ratios of protein and nucleic acids
based on the proposed stoichiometries (results of such analysis
for FIR protein complexes are reported in Table 3 and plotted
in Fig. 3).
6. Confirm that both the observed M w and UV/RI ratio are
consistent with the proposed stoichiometry (Fig. 3 and
Table 3; see Note 12).
4 Notes
1. The data for the examples shown were collected using an
SEC/MALS system consisting of a high-performance liquid
chromatography system (HPLC), Waters Alliance 2960, or
Agilent 1200. The elution from SEC was monitored by a
Waters or Agilent photodiode array (PDA) UV/VIS detector,
differential refractometer, and static and dynamic, multiangle
laser light scattering detector. Two software packages were
used for data collection and analysis: the Waters Millennium
software controlled the HPLC operation and data collection
from the multiwavelength UV/VIS detector, while the Wyatt
ASTRA software collected data from the refractive index detector, the light scattering detector, and recorded the UV trace at
280 nm, 295 nm, or 310 nm (see Note 3) sent from the PDA
detector. However, other UV, RI, and LS detectors can be used
(a single-angle LS detector is suitable for analysis of macromolecules with molar masses up to ~500 kDa). The UV detector
should be capable of extracting signals for various UV wavelengths (see Note 3).
2. The 0.1 μm “inline” filter placed between pumps and the
injector retains any particles that are shed from the HPLC
pump’s head. An additional “inline” filter (2 μm PEEK frit)
with small dead volume is installed between the injector and
the SEC column; this filter traps protein aggregates that are
formed during the injection step and that may result from
exposing the protein sample to high pressure; this filter substantially increases the lifetime of the SEC column. These filters
are replaced whenever the system’s operating pressure increases
by more than 5%.
SEC Coupled with Light Scattering
391