EMBL Hamburg Biological
Small Angle Scattering

Sample requirements

There are three main options for SAXS data collection at P12.

Batch measurements with the robotic sample changer

  • Sample concentration: from 0.5 to 10 mg/ml (or more)
  • Sample volume: 40 μl per exposure
  • The amount of buffer: at least twice that of the sample
  • Buffer composition: no aggressive substances (chloroform, toluene etc)
  • No viscose buffers (eg above 30% glycerol or above 10% sucrose)
  • Temperature range: from 8° to 45°C

SEC-SAXS measurements

  • Stock sample concentration from 7 mg/ml
  • Sample volume about 50–100 μl (depends on the column size)
  • The amount of buffer: at least 500 ml
  • All measurements at room temperature only
  • Users are encouraged to bring their own columns
  • MALLS/RI analysis is available

In-air operation

  • Only available during dedicated slots (2-3 per year)
  • Measurements in quartz capillaries ⌀1.5 mm (provided by the user), temperature range from 4° to 100°C
  • Measurements in cuvettes or custom holders, at room temperature only
  • For stopped-flow, at least 2–3 ml of sample is required
  • Please approach the local contact at least 2 weeks in advance for details

Sample purity

Users must verify that samples are both pure and monodisperse as is possible (preferably above 95%) prior to SAXS measurements at P12. Sample purity can be assessed using native gel filtration (size exclusion chromatography, SEC), ultracentrifugation, dynamic light scattering, etc). In particular, sample contaminants with molecular weights higher than a target of interest must be removed from the sample. If the sample is aggregated, the scattering data will be difficult or even impossible to interpret. Please note that a single band on a denaturing/reducing SDS-PAGE gel does not indicate that the sample is pure or monodisperse. It is advised to run, at the very least, both reducing and non-reducing SDS-PAGE as well as native PAGE to assess sample quality.


The chemical composition of the buffer must exactly match that of the sample. The best results are obtained with the last dialysis buffer. Separately prepared buffers may easily lead to mismatch and to severe difficulties during the buffer subtraction procedure. IMPORTANT: samples in viscose buffers (eg those containing above 30% glycerol or above 10% sucrose) cannot be loaded by the robotic sample changer. If your samples must be measured in viscose buffers, "In-air operation" must be selected in the beamtime application!

Sample concentration

Sample concentrations for most experiments at P12 span 0.5–10 mg/ml and typically a concentration series is measured for each sample (e.g. 1, 2, 5, 10 mg/ml). The volume of sample required depends on whether samples are measured using the BioSAXS sample changer or SEC-SAXS (see below). For sample changer measurements, it is advised to bring an excess of an exactly-matched buffer (e.g., 10–20 ml) to set up the concentration series for each sample. If the sample is well behaved, high concentration stocks can be diluted before measurement. If the sample is known to undergo aggregation at high concentration, it is best to bring the low concentration stocks, measure them, and then concentrate the samples onsite immediately prior to SAXS.

Sample concentrations must be determined as accurately as possible as the concentrations are used to normalize the scattering data and to obtain critical molecular weight information. At P12, a Nanodrop spectrophotometer is available that spans and absorbance range of 220–750 nm. For most proteins, concentrations can be determined at 280 nm. Bradford assays, unless very-well standardised for a specific protein, are usually not sufficiently precise for determining protein concentration. A refractometer is also available at P12 for estimating concentration that is particularly useful for proteins lacking aromatic amino acids or those with poor Abs 280 nm extinction coefficients.

Additional advice on preparing samples for biological solution small-angle scattering is described in Jeffries et al., 2016 Nature Protocols 11, 2122-2153 (EuropePMC Open Access: PMC5402874).

Potentially toxic samples

We do not collect SAXS data from samples classified as biohazard S2, S3, or S4, e.g. viruses, prions and toxins. Please send us a detailed description of you samples in advance if you have any safety concerns.

Getting samples to P12

If you bring your samples and buffers with you to P12 and require additional laboratory resources, e.g., to make up more buffer, set up sample dialyses, perform SEC (with fraction collection), please book the Sample Preparation and Characterisation (SPC) laboratory at least two weeks prior to arrival.

If you decide to post the samples and matched buffers prior to you arrival, please do so well in advance. Even with courier service (FedEx, DHL, TNT, UPS, etc) allow a week for the samples to arrive. Send frozen samples only on Monday or Tuesday to ensure delivery before Saturday. Note that the samples will be stored exactly according to your instructions. Write these instructions clearly and label the samples. Do not randomly scatter individual loose tubes into dry ice or ice packs. Collect the individual tubes into a bag or small box, then place the bag/box into the dry ice container or ice packs container. Make sure to seal the tubes (e.g., with parafilm) to prevent 'lid-popping' during shipment. Do not send samples on water ice.

Address the samples to:
User Office L108, Attn. [name of your local contact]
Notkestr. 85, Geb. 48e
22607 Hamburg
Tel: +49 40 8990 2111

We also offer a post-in SAXS service – either coupled with remote beam line access or, in exceptional circumstances, for the measurements performed by the beamline staff – for users who cannot travel to P12. For the post-in service, samples should be supplied in either specific PCR tube strips or 96 well plates. The catalogue numbers are provided below.
Note: Users travelling to P12 do not need to supply their own tubes or plates, they are provided at the beam line.

Standard SAXS Measurements with the sample changer

Standard 'batch mode' SAXS measurements at P12 are performed using the automated sample changer (Round et al., 2015 Acta Cryst. D 71, 67-75). Using a standard set-up, samples and buffers undergo continuous sample flow during SAXS measurements, generally consisting of 20 × 50 ms exposures, for a total exposure time of 1 s. A complete buffer + sample, wash-and-dry cycle takes approximately 2 min.

Samples and matched buffers are stored under 8–40°C in the sample changer using:

  • 8 × 200 μl PCR tube strips;
  • 1.5 ml Eppendorf tubes (usually reserved for buffers);
  • 96 well plates.

PCR strips and 1.5 ml Eppendorf tubes

Lidless 8 × 200 μl PCR tube strips and lidless 1.5 ml Eppendorf tubes are provided for users at the beam line. Usually, samples are pipetted from stock solutions into each PCR tube, while buffers (1 ml) are placed into 1.5 ml Eppendorf tubes prior to loading the sample changer. The sample changer in this configuration has a ~10 μl dead volume, therefore the recommended sample volumes are:

  • For standard operation continuous flow measurements: 40 μl (for 30 μl SAXS sample analysis, one exposure). This setup covers ~80% of user applications.
  • For non-standard very low-volume static (i.e., no-flow) measurements: 20–25 μl (10–15 μl analysis).*

*Note 1: Very low sample volume analyses are performed without sample flow enabled during data acquisition. This increases the chances of X-ray induced aggregation to the sample.

Note 2: It is possible to use the PCR strips for the buffers instead of using 1.5 ml tubes. The collection strategy at P12 usually employs two buffer measurements for every sample measurement, e.g., buffer-sample-buffer. Therefore, it is necessary to pipette ~70–100 μl of buffer into the PCR tubes for each sample.

Note 3: For users who opt for our post-in service and wish to pre-prepare their samples in PCR tubes the supplier and catalogue number of the strips and lids are:

  • Sarstedt. cat# 72.985.992 (Multiply® microstrip PCR Tubes (no lid));
  • Sarstedt. cat# 65.989.002 (Multiply® strip of 8 lids).
Make sure the tubes are labelled and well sealed to prevent 'lid-popping' during transport (especially when using airfreight.)

96 well plates

The BioSAXS sample changer can also use 96 well plates for sample and buffer loading. The dead volume for 96 well plates is larger (~20 μl) compared to the PCR tube option. Therefore it is necessary to load more sample and buffer into each well. For standard measurements, 50 μl of sample (at a minimum) and 100–120 μl of buffer should be loaded per well for a continuous flow SAXS measurement. Again, 96 well plates are provided at the P12 beam line at no cost to users. For those wishing to pre-prepare their 96 well plates prior to arrival, or to use our post-in service, the catalogue numbers for the plates and sealing material are:

  • Greiner Bio-One, cat#. 651201 (plates);
  • Greiner Bio-One, cat#. 676070 (Platesealer, ViewSeal, Transparent Sealing Film).

SEC-SAXS Measurements

We offer three options for size exclusion chromatography SEC-SAXS for the in-line separation of sample components directly coupled to the SAXS measurements.

  1. Simple SEC-SAXS, 'from column to beam line': The sample is injected onto the SEC column, the sample components are separated and elute through the P12 beam line. Users measure SAXS data only.
  2. UV-SEC-SAXS: The sample is injected onto the SEC column, the sample components are separated and elute through a single-wavelength UV-Vis spectrophotometer (wavelength selection 190-400 nm) then immediately to the P12 beam line. Users measure SAXS data plus UV (or Vis) absorption spectra.
  3. UV-MALLS-DLS-RI-SAXS: Parallel SAXS with additional molecular weight validation from multi-angle laser light scattering (MALLS) coupled to hydrodynamic radius measurements from dynamic light scattering (DLS). The sample is injected onto the SEC column, the sample components are separated. The mobile phase is split equally between the P12 beam line and UV-Vis spectrophotometer coupled to a Wyatt continuous-flow miniDAWN TREOS/QELS (DLS) instrument and a Wyatt T-rEX refractive index (RI) instrument. Users measure SAXS, UV (or Vis) absorption data in addition to MALLS/RI molecular weight and hydrodynamic radius correlations through the elution peaks (Due to technical requirements and additional data analysis and interpretation necessary for Wyatt-MALLS-DLS-SAXS experiments, these projects are always performed on a collaborative basis).

Note 1: All SEC-SAXS operations are performed at room temperature.

Note 2: The SEC system is driven by an Agilent 1260 Infinity Bio-Inert HPLC/FPLC machine with an in-built temperature controlled auto-sampler (compatible with both dextran-based FPLC and silicon dioxide HPLC columns).

Note 3: The maximum injection volume is 100 μl. The standard injection volume is 75 μl. The standard sample concentration is 7-15 mg/ml.

Note 4: Users are advised to use their own SEC columns. However, if P12 users cannot bring a column to the beam line, P12 offers a number of in-house FPLC and HPLC columns:

24 ml analytical FPLC columns (recommended)

  • GE Superdex 200 Increase 10/300 - general purpose separation across a wide MW range (10-600 kDa). Two columns available.
  • GE Superdex 200 Increase 5/150
  • GE Superdex 75 Increase 10/300 (for small macromolecules less than 70 kDa).
  • GE Superdex 75 Increase 5/150
  • GE Superose 6 Increase 10/300 (for very-large macromolecules and complexes up to 5 MDa).
  • GE Superose 6 Increase 5/150 (for very-large macromolecules and complexes up to 5 MDa).
  • GE Superose 6 10/300.
Increase columns sample volume, flow rate, pressure

HPLC columns

  • Wyatt WTC-015S5 5μm 150+, 7.8x300 mm.
  • Wyatt WTC-030S5 5μm 300+, 7.8x300 mm.

Note 5: It is advised to bring, or make up onsite, at least 500-1000 ml of SEC buffer. Buffers can be made at the SPC facility next to the beam line (the SPC requires advanced booking).

Note 6: It is recommended that 3% v/v glycerol is added to the SEC column buffer to reduce the effects of radiation damage (see below), assuming that the addition of glycerol does not affect the sample.

Note 8: For additional MALLS/DLS analysis, buffers must be 0.1 mm filtered. It may take 1-2 hrs (even more) to appropriately equilibrate a SEC column to a stable baseline prior to a SEC-SAXS measurement. A stable UV absorption trace is not an indication of a stable scattering base line. Base line equilibration is best performed overnight, the day prior to SEC-SAXS measurements (if possible).

Note 7: It is recommended that a small sample is measured prior to SEC-SAXS using the default sample changer setup (see above) to assess radiation sensitivity.

For more information, please refer to the Wyatt-MALLS-DLS-SAXS page and to Graewert et al., 2015 Scientific Reports 5:10734.

Decreasing the effects of radiation damage

Samples can be damaged by X-rays that induce aggregation during the course of exposure which will ruin data interpretation. Radiation damage can be reduced via the addition of:

  • 3–5% v/v (maximum) glycerol.
  • 1–2 mM dithiothreitol (DTT, note will affect Abs280 nm readings).
  • 1–2 mM ascorbic acid (vitamin C).
  • 2–5 mM Tris(2-carboxyethyl)phosphine (TCEP; a more stable reducing agent).
  • The use of Tris or HEPES buffers.
  • The addition of other polyols at low concentration, e.g., mono-ethylene glycol.

Note 1. It is very important to know prior to SAXS that the addition of these small molecules does not destabilise the solute (e.g., cause the protein to aggregate).

Note 2. The addition of polyols, e.g., glycerol, mono-ethylene glycol, sucrose, etc, should not generally exceed 5% v/v as the contrast of the system will be lowered. For SEC-SAXS using dextran FPLC columns, 3% v/v is the recommended maximum due to column pressure limits.

Note 3. The exact same quantities of additive must be added to both the sample and to the matched buffer.

Note 4. It is also possible to attenuate the X-ray beam to reduce the X-ray flux and thus decrease X-ray damage. However, beam attenuation results in a significant reduction in data quality. Please consult with your local contact.

For more details on limiting radiation damage, please refer to Jeffries et al., 2015 J. Synchrotron Rad. 22, 273-279.

General reference for the EMBL P12 bioSAXS beam line


  Last modified: October 5, 2021

© BioSAXS group 2021