in LS signal has increased twofold when compared to the noise
recorded when the SEC/MALS system has been
initially equilibrated.
2.3 Sample
1. 50–500 μg of protein, nucleic acids, and protein–nucleic acid
complex samples (see Table 4 for guidelines regarding optimal
sample amounts) in a volume that corresponds to ~3% of the
total volume of the SEC column (see Note 8).
3 Methods
3.1 System Setup
and Validation
1. The detectors should be connected in the following order: UV,
LS, and RI. The RI detector should be last in this series because
its cell is fragile and it cannot withstand high pressures. Tubing
with an inner diameter of 0.01
00 (0.25 mm) should be used
throughout the system to minimize the delay volumes between
detectors and minimize band-broadening effects (see Note 9).
3.2 System
Equilibration
1. Equilibrate the SEC/MALS system in the buffer of choice;
turn on the RI detector and pump the buffer through the RI
detector in the “purge” mode for at least 12 h at the flow rate
that would be used during data collection.
2. Turn on the LS and UV detectors; the UV detector requires
30 min to warm up the lamp.
3. Stop the purging mode on the RI detector, zero the RI detector, and start monitoring baselines.
4. Check the baseline quality and stability; the noise level in the
LS baseline monitored at 90
should not exceed 50 μV on a
DAWN HELEOS II detector (with the goal being to keep the
noise at less than 20 μV; see Note 10).
3.3 Validation
of SEC/MALS System
in the Buffer of Interest
1. Dissolve 500 μg of protein standard in 500 μl of running buffer
(use at least three different, individually prepared protein standards; see Note 7).
2. Filter the solutions of protein standards through a 0.22 μm
low-protein-binding filter (see Note 6).
3. Individually inject 200 μg of each protein standard and collect
UV, LS, and RI data.
4. Using software for light scattering data analysis, baselinecorrect the signals from all three detectors, determine interdetector delay, apply band-broadening correction (see Note 9),
normalize the LS detector signals, and calculate M w .
5. Check whether the computed M w s are within 5% of expected
molecular weights.
6. Inject the nucleic acid standard (see Note 6) and validate the
value for (dn/dc) nucleic acid (see Note 11).
SEC Coupled with Light Scattering
389
recorded when the SEC/MALS system has been
initially equilibrated.
2.3 Sample
1. 50–500 μg of protein, nucleic acids, and protein–nucleic acid
complex samples (see Table 4 for guidelines regarding optimal
sample amounts) in a volume that corresponds to ~3% of the
total volume of the SEC column (see Note 8).
3 Methods
3.1 System Setup
and Validation
1. The detectors should be connected in the following order: UV,
LS, and RI. The RI detector should be last in this series because
its cell is fragile and it cannot withstand high pressures. Tubing
with an inner diameter of 0.01
00 (0.25 mm) should be used
throughout the system to minimize the delay volumes between
detectors and minimize band-broadening effects (see Note 9).
3.2 System
Equilibration
1. Equilibrate the SEC/MALS system in the buffer of choice;
turn on the RI detector and pump the buffer through the RI
detector in the “purge” mode for at least 12 h at the flow rate
that would be used during data collection.
2. Turn on the LS and UV detectors; the UV detector requires
30 min to warm up the lamp.
3. Stop the purging mode on the RI detector, zero the RI detector, and start monitoring baselines.
4. Check the baseline quality and stability; the noise level in the
LS baseline monitored at 90
should not exceed 50 μV on a
DAWN HELEOS II detector (with the goal being to keep the
noise at less than 20 μV; see Note 10).
3.3 Validation
of SEC/MALS System
in the Buffer of Interest
1. Dissolve 500 μg of protein standard in 500 μl of running buffer
(use at least three different, individually prepared protein standards; see Note 7).
2. Filter the solutions of protein standards through a 0.22 μm
low-protein-binding filter (see Note 6).
3. Individually inject 200 μg of each protein standard and collect
UV, LS, and RI data.
4. Using software for light scattering data analysis, baselinecorrect the signals from all three detectors, determine interdetector delay, apply band-broadening correction (see Note 9),
normalize the LS detector signals, and calculate M w .
5. Check whether the computed M w s are within 5% of expected
molecular weights.
6. Inject the nucleic acid standard (see Note 6) and validate the
value for (dn/dc) nucleic acid (see Note 11).
SEC Coupled with Light Scattering
389