same intensity. Furthermore, the resolution of DLS permits one to
distinguish between monomeric and oligomeric states of a protein
only if the molecular mass of the two states differs significantly (see
Subheading 1.2.2). Thus, samples that contain a mixture of monomers and dimers of a protein will usually give rise to only one peak.
Modern backscattering DLS instruments can measure scattering even for protein concentrations >50 mg/mL but will tend to
provide progressively more inaccurate results at high concentrations. Each instrument will have an ideal operating concentration
range, which depends on the particle sizes present. Larger particles
scatter more and lower concentrations are better. The manufacturer’s information should be followed, but a typical rule of thumb
would be to aim for a concentration ¼ 10/(particle molecular
weight in kDa) in mg/mL, i.e., about 1 mg/mL for a 10-kDa
particle and 0.1 mg/mL for a 100-kDa species. Experiments
should be repeated at concentrations differing by a factor of 2 to
ensure that there are no substantial concentration-dependent
effects.
DLS experiments can be performed using nearly any buffer.
However, the buffer must be filtered as it is very important to
remove dust or salt crystals that would cause scattering and interfere with measurements. When working with membrane proteins
containing detergent micelles, or other buffers containing colloids,
or crowding agents, it is important to measure all conditions with
and without protein to be able to distinguish the behavior of empty
micelles/buffer from the solubilized protein. In such cases, the
objective is to identify conditions where a monodisperse protein:
detergent or protein:amphipol complex is formed.
A simple protocol for a DLS measurement is given below:
1. Switch on the instrument and select the experimental
temperature.
2. Wait for temperature stabilization.
3. If using reusable cuvettes or plates, clean them with detergent
(e.g., Hellmanex 2% (v/v)) and water, and dry it with ethanol.
4. Filter and degas buffers using a 0.1-μm or, preferably, 0.02-μm
filter.
5. Prepare the protein sample at an appropriate concentration,
diluting with buffer as necessary. If the purpose of the DLS
measurement is to detect aggregation, then the sample should
not be filtered or centrifuged. Otherwise, it is advisable to filter
the sample and centrifuge it for 5 min in a benchtop centrifuge
at full speed prior to loading.
6. Load buffer and samples into the wells or into the cuvettes.
Avoid the formation of bubbles. However, if bubbles are present, centrifuge the plate or cuvette (e.g., for plates: 1 min at
500–1000 Â g).
28
Bertrand Raynal et al.
distinguish between monomeric and oligomeric states of a protein
only if the molecular mass of the two states differs significantly (see
Subheading 1.2.2). Thus, samples that contain a mixture of monomers and dimers of a protein will usually give rise to only one peak.
Modern backscattering DLS instruments can measure scattering even for protein concentrations >50 mg/mL but will tend to
provide progressively more inaccurate results at high concentrations. Each instrument will have an ideal operating concentration
range, which depends on the particle sizes present. Larger particles
scatter more and lower concentrations are better. The manufacturer’s information should be followed, but a typical rule of thumb
would be to aim for a concentration ¼ 10/(particle molecular
weight in kDa) in mg/mL, i.e., about 1 mg/mL for a 10-kDa
particle and 0.1 mg/mL for a 100-kDa species. Experiments
should be repeated at concentrations differing by a factor of 2 to
ensure that there are no substantial concentration-dependent
effects.
DLS experiments can be performed using nearly any buffer.
However, the buffer must be filtered as it is very important to
remove dust or salt crystals that would cause scattering and interfere with measurements. When working with membrane proteins
containing detergent micelles, or other buffers containing colloids,
or crowding agents, it is important to measure all conditions with
and without protein to be able to distinguish the behavior of empty
micelles/buffer from the solubilized protein. In such cases, the
objective is to identify conditions where a monodisperse protein:
detergent or protein:amphipol complex is formed.
A simple protocol for a DLS measurement is given below:
1. Switch on the instrument and select the experimental
temperature.
2. Wait for temperature stabilization.
3. If using reusable cuvettes or plates, clean them with detergent
(e.g., Hellmanex 2% (v/v)) and water, and dry it with ethanol.
4. Filter and degas buffers using a 0.1-μm or, preferably, 0.02-μm
filter.
5. Prepare the protein sample at an appropriate concentration,
diluting with buffer as necessary. If the purpose of the DLS
measurement is to detect aggregation, then the sample should
not be filtered or centrifuged. Otherwise, it is advisable to filter
the sample and centrifuge it for 5 min in a benchtop centrifuge
at full speed prior to loading.
6. Load buffer and samples into the wells or into the cuvettes.
Avoid the formation of bubbles. However, if bubbles are present, centrifuge the plate or cuvette (e.g., for plates: 1 min at
500–1000 Â g).
28
Bertrand Raynal et al.