bioSAXS experiments

B21 operates in a fixed camera length configuration (4.014 meters) at 12.4 keV. The camera length range is suitable for particles with an Rg < 200 Angstroms.

We offer two standard data collection methods:

batch mode data collection using the EMBL Arinax sample handling robot
size-exclusion chromatography coupled SAXS (SEC-SAXS) using an Agilent HPLC system.

Batch mode

The bioSAXS robot automates data collection from a user created spreadsheet of samples in either a 96-well plate or a set of PCR strips. Each sample is 30 µL and is stored between 4 and 15 degrees. The samples are loaded into a temperature controlled quartz cell capillary (10 or 100 µm thick) for X-ray scattering measurements.

Standard bioSAXS experiments using the robot require buffer matching to be performed by the client. For buffer matching, we recommend:

dialysis
SEC (be sure to collect buffer after 1.2 column volumes have passed)
spin concentrators

Please speak to us in advance of your experiment if your buffer contains any of the following components as these may strongly affect either compatibility of the bioSAXS robot, or the quality of the data obtainable.

Detergent
High salt and/or high or low pH.
High glyercol content
Organic solvents

For each individual sample, 30 µl of sample is needed at ~5 mg/ml diluted in a concentration series with 1.5 ml of matched sample buffer.

Measurements are performed in a series: buffer; wash with detergent; rinse; dry; protein sample; wash with detergent; rinse; dry; buffer; wash with detergent; rinse; dry. The entire cycle takes 5 mins to complete. Very small particles (< 10 kDa) will require extended exposure times.

SEC-SAXS mode

Inline SEC-SAXS is provided by an Agilent 1200 HPLC system. We offer several columns:

column	vol. (ml)	mass range (kDa)	max pressure (bar)	recommended flow rate (ml/min)
superdex 200 (GE Healthcare)	2.4	10 to 600	35	0.075
superose 6 (GE Healthcare)	2.4	5 to 5000	35	0.075
KW-402.5 (Shodex)	4.6	5 to 100	85	0.160
KW-403 (Shodex)	4.6	10 to 700	85	0.160
KW-404 (Shodex)	4.6	30 to 4000	85	0.160
KW-405 (Shodex)	4.6	40 to 20000	85	0.160

Superose columns operate at comparatively lower pressures, largely composed of conjugated sugars whereas the Shodex columns are higher pressure columns composed of silica. Samples are measured in a specialised cell with ~500x lower background than the bioSAXS robot cell.

For each SEC-SAXS run, 50 µl of sample is needed at 5-10 mg/ml. We recommend allowing 250 ml of buffer which should ideally contain a radiation protective agent such as 1-2% sucrose. One SEC-SAXS run takes approximately 40 mins to complete.

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Batch mode

SEC-SAXS mode