General sample handling

For every Prometheus Panta experiment, please carry out the following steps:

• Clean the capillary tray surface
  • 99.8% ethanol
  • Lens paper
• Load capillaries completely and align them centered before placing the lid
• Avoid liquid on the outside of the capillary 

As always, avoid scratches to the surface of the tray.

Sample preparation 

Prometheus Panta performs measurements over a large protein concentration range. We recommend the following concentrations as starting points:

• 1 mg/mL for nanoDSF measurements (increase for membrane proteins)
• 2 mg/mL for DLS measurement (increase for proteins below 50 kDa)

For DLS measurements it is important to avoid dirt and dust as they will directly interfere with the measurement and create artefacts in the data. For this reason, we recommend the following:

• Keep capillary vial closed when not in use
• Use a fresh box of pipet tips and a fresh bag of sample tubes or plates
• Keep boxes for pipet tips or tube lids closed when not in use
•  Filter all buffers (see below)
•  Filter samples or spin down (see below)

1.1. Filtration of water and buffers

To record high-quality DLS data, it is advisable to work with highly pure water (e.g. AnalaR NORMAPUR; VWR #102927G) and buffer components. Filter water and buffer prior to usage with filters having a cutoff of 0.2 μm (e.g. Filter devices: Nalgene 25 mm; Thermo Fisher Scientific #724-2020 or Nalgene 13 mm; Thermo Fisher Scientific #720-1320)

To get the best possible result, filter water or buffer twice through the same filter into fresh and clean 15 mL/ 50 mL tubes.

1.2. Pre-rinsing sample tubes

Similar to filtering water and buffer, keeping sample solutions in clean and dust-free sample containers can improve DLS data quality. When unsure whether the sample or buffer containers are free of any particulate matter or when dust-artifacts are detected  in the data despite having filtered all solutions, try pre-rinsing sample containers:

1. Place sample tubes in sample holder
2. Set a pipet to the matching volume
3. Rinse the pipet tip once with filtered water, discard
4. Use a clean tip to fill filtered water into all tubes
5. Clean tubes by shaking / inverting
6. Discard water by tipping it out (make sure no water remains in the tube, e.g. in the lid)
7. Close tubes again to avoid contamination

 1.3. Filtration of protein solutions

Working with dirt- and dust-free sample solutions is key to determine the correct size of particles in a sample. It is therefore advisable to filter protein solutions directly before starting an experiment.

An alternative method to remove dirt and dust is spinning down the sample directly before the experiment (e.g., 14.000g, 10-15 minutes at 4º C in a tabletop centrifuge).

When filtering protein solutions, we recommend carrying out the following steps:

1. Start with clean tubes (as described under 1.2.)
2. Filter the protein solution:
   • For standard DLS application, filter protein solution through 0.22 μm pore size filters with a sufficiently small dead volume. e.g. Nalgene 13 mm; Thermo Fisher Scientific #720-1320 or Costar Spin-X Centrifuge Tube filter; Corning Inc. # CLS8161-100EA
   •  For optimal DLS results, filter protein solution through 0.02 μm pore size filter e.g. Whatman Anotop 10 Plus; GE Healthcare #6809-3002
3. Take a fresh, clean 1 mL syringe, fill the syringe with the protein solution and filter it into the clean tube (discard first droplet or subsequently filter the sample for a 2nd time)

Please note:

For all subsequent pipetting / dilution steps, always use clean tubes and pipet tips pre-rinsed with filtered water!

Of course, to filter or not to filter depends on your application. If, for example, one is interested in the long-term stability of proteins, one might want to filter the sample at the beginning of the experiment, but then keep it as it is for all subsequent measurements to assess if larger particles form over time.