measurements, columns with small diameter are preferred as they
have greater resolution. In order to select the best column and
ensure correct handling, one should read the manufacturer’s
instruction, which gives the molecular weight limit, pressure
limit, the volume of the column, the void volume, and the recommended flow rate. Test runs with small injection volumes of the
protein of interest may be performed to choose a good column
setup.
The running buffer for the sample should be selected such that
it is stable over time, as any change, such as oxidation of a reducing
agent, will lead to a drift in the baseline of the refractometer. The
buffer should contain at least 50 mM of salt to avoid interactions
with the column matrix and 0.1% (w/v) NaN 3 to prevent microbial
growth. All solutions must be made with high purity water (preferably >18 MΩ), freshly filtered (0.1-μm filter), and degassed to
avoid background scattering and an unstable baseline.
A simple protocol for a SEC-SLS experiment is given below:
1. Switch on the instrument.
2. Purge the system with high purity water.
3. Connect the column to the SEC-LALS/SEC-MALS system so
that eluting proteins are analyzed immediately inline.
4. Choose the flow speed and pressure limit according to the
column manufacturer’s instruction.
5. Equilibrate the system with at least 2 column volumes of water.
Columns are usually stored in 20% (v/v) ethanol. Therefore,
the column must first be washed extensively with water (at least
2 column volumes) as ethanol may otherwise precipitate salt in
the buffer when it is introduced.
6. Purge the system with a buffer of interest.
7. Equilibrate the system with at least 2 column volumes of the
buffer. If necessary, equilibration should be continued until the
baseline is stable.
8. Purge the refractometer extensively until the baseline remains
stable for one column run.
9. Prepare your sample in the same buffer at a concentration of at
least 1 mg/mL.
10. Centrifuge the sample for 5 min in a benchtop centrifuge at full
speed.
11. Load the sample into the injection loop (avoiding creating any
bubbles). The volume that will be injected onto the column
should be lower than 1% of the total column volume to ensure
good separation (usually 100μL for a column volume of
24 mL). For accurate quantification, it is necessary to flush
26
Bertrand Raynal et al.
have greater resolution. In order to select the best column and
ensure correct handling, one should read the manufacturer’s
instruction, which gives the molecular weight limit, pressure
limit, the volume of the column, the void volume, and the recommended flow rate. Test runs with small injection volumes of the
protein of interest may be performed to choose a good column
setup.
The running buffer for the sample should be selected such that
it is stable over time, as any change, such as oxidation of a reducing
agent, will lead to a drift in the baseline of the refractometer. The
buffer should contain at least 50 mM of salt to avoid interactions
with the column matrix and 0.1% (w/v) NaN 3 to prevent microbial
growth. All solutions must be made with high purity water (preferably >18 MΩ), freshly filtered (0.1-μm filter), and degassed to
avoid background scattering and an unstable baseline.
A simple protocol for a SEC-SLS experiment is given below:
1. Switch on the instrument.
2. Purge the system with high purity water.
3. Connect the column to the SEC-LALS/SEC-MALS system so
that eluting proteins are analyzed immediately inline.
4. Choose the flow speed and pressure limit according to the
column manufacturer’s instruction.
5. Equilibrate the system with at least 2 column volumes of water.
Columns are usually stored in 20% (v/v) ethanol. Therefore,
the column must first be washed extensively with water (at least
2 column volumes) as ethanol may otherwise precipitate salt in
the buffer when it is introduced.
6. Purge the system with a buffer of interest.
7. Equilibrate the system with at least 2 column volumes of the
buffer. If necessary, equilibration should be continued until the
baseline is stable.
8. Purge the refractometer extensively until the baseline remains
stable for one column run.
9. Prepare your sample in the same buffer at a concentration of at
least 1 mg/mL.
10. Centrifuge the sample for 5 min in a benchtop centrifuge at full
speed.
11. Load the sample into the injection loop (avoiding creating any
bubbles). The volume that will be injected onto the column
should be lower than 1% of the total column volume to ensure
good separation (usually 100μL for a column volume of
24 mL). For accurate quantification, it is necessary to flush
26
Bertrand Raynal et al.