6. Setup a program containing 5–20 acquisitions with an acquisition time of 2–10 s per sample depending on the presence of
dust or particles that may disturb the measurement. Measurements should be done in triplicates using automatic laser power
and detector attenuation.
7. If the DLS instrument has a camera, check for potential bubble
or precipitate.
8. During data analysis, the buffer quality should be first checked.
9. Check the shape of the autocorrelation curve for the protein
(Fig. 1b, inset). Check the fit and the error of the measurement
and adjust the fitting limit accordingly. The presence of aggregates is indicated if particles with a R h of 10–100 nm are
observed (Fig. 1b).
10. If the protein is not detectable due to the presence of aggregates or artifacts, the sample should be centrifuged using a
benchtop centrifuge (15 min, at full speed). However, it is
important to consider the potential loss of protein during
centrifugation; it is therefore recommended to measure the
concentrations before and after centrifugation.
3.3.2 Size Exclusion
Chromatography (SEC)
with Multiangle Light
Scattering (SEC-MALS)
Test runs with small injection volumes of the protein of interest
may be performed to choose a good column setup (see Note 17). In
line with the column, the refractometer and the light scattering
detectors allow the measurement of the molecular mass of all the
peaks of interest.
A simple protocol for a SEC-MALS experiment is given below:
1. Switch on the instrument.
4. Purge the system with water.
5. Connect the column to the SEC-SLS/SEC-MALS system.
6. Choose the flow speed and pressure limit according to the
column manufacturer’s instruction.
7. Equilibrate the system with at least 2 column volumes of water
(see Note 18).
8. Purge the system with the buffer of interest (see Note 19).
9. Equilibrate the system with at least 2 column volumes of buffer.
10. Purge the refractometer extensively until the baseline remains
stable for one column run (see Note 20).
11. Centrifuge the sample for 5 min in a benchtop centrifuge at full
speed.
12. Load the sample into the injection loop. The volume (usually
100 μl) that will be injected onto the column should be lower
than 1% of the total column volume to ensure a good separation. If the sample concentration needs to be quantified, flush
the injection loop to homogenize the sample injection volume.
PDZ Sample Quality Assessment
113
dust or particles that may disturb the measurement. Measurements should be done in triplicates using automatic laser power
and detector attenuation.
7. If the DLS instrument has a camera, check for potential bubble
or precipitate.
8. During data analysis, the buffer quality should be first checked.
9. Check the shape of the autocorrelation curve for the protein
(Fig. 1b, inset). Check the fit and the error of the measurement
and adjust the fitting limit accordingly. The presence of aggregates is indicated if particles with a R h of 10–100 nm are
observed (Fig. 1b).
10. If the protein is not detectable due to the presence of aggregates or artifacts, the sample should be centrifuged using a
benchtop centrifuge (15 min, at full speed). However, it is
important to consider the potential loss of protein during
centrifugation; it is therefore recommended to measure the
concentrations before and after centrifugation.
3.3.2 Size Exclusion
Chromatography (SEC)
with Multiangle Light
Scattering (SEC-MALS)
Test runs with small injection volumes of the protein of interest
may be performed to choose a good column setup (see Note 17). In
line with the column, the refractometer and the light scattering
detectors allow the measurement of the molecular mass of all the
peaks of interest.
A simple protocol for a SEC-MALS experiment is given below:
1. Switch on the instrument.
4. Purge the system with water.
5. Connect the column to the SEC-SLS/SEC-MALS system.
6. Choose the flow speed and pressure limit according to the
column manufacturer’s instruction.
7. Equilibrate the system with at least 2 column volumes of water
(see Note 18).
8. Purge the system with the buffer of interest (see Note 19).
9. Equilibrate the system with at least 2 column volumes of buffer.
10. Purge the refractometer extensively until the baseline remains
stable for one column run (see Note 20).
11. Centrifuge the sample for 5 min in a benchtop centrifuge at full
speed.
12. Load the sample into the injection loop. The volume (usually
100 μl) that will be injected onto the column should be lower
than 1% of the total column volume to ensure a good separation. If the sample concentration needs to be quantified, flush
the injection loop to homogenize the sample injection volume.
PDZ Sample Quality Assessment
113