How to Prepare and Perform Solution SAXS Experiments
We typically do Small Angle X-ray Scattering (SAXS) experiments at an x-ray photon energy of 12 keV.
The Pilatus 1M detector will be used for SAXS. In the case of simultaneously
SAXS and WAXS measurements a second Pilatus 100 K detector will be used
at a short distance from the sample.We have a dedicated SAXS camera with
a sample-to-detector distance of 3.5 m yielding a Q range 0.004--0.33 Å-1
which is suitable for protein molecules ranging from several kDa to a few MDa.
For our WAXS instrument, please contact the beamline scientist to discuss your needs.
To minimize radiation damage, typically 10 - 20 frames of short exposures
(0.1 - 1s) are taken on a flowing sample in a capillary cell. The data are
averaged over the multiple frames to improve the signal/noise ratio after
confirming the absence of radiation damage. A minimum sample volume of
120 μl is recomended for Flow-Thru mode during the X-ray exposure.
However, in special cases even a sample volume of 20 μl may also be able
to yield reasonable SAXS data.
In the case of online chromatography SAXS, a sample volume of 150-500 μl
of a few mg/mL is desirable to load the sample to GE Akta pure. Concerning
the sample quantity and requirements for time-resolved experiments using
the stopped-flow or microfluidic mixers, please contact the beamline scientist
For proteins of a size comparable to lysozyme (14.3 kDa)
or cytochrome c (~ 12 kDa), a concentration of
2 mg/ml can give reasonably good data quality. If the
protein has twice the size of lysozyme or cytochrome c,
the concentration can be reduced by a factor of two.
Higher concentrations can be used to give better data
quality if the protein does not suffer from aggregation.
We can measure samples with a concentration of
0.5 mg/ml at long exposures and have good data
quality in the measured q region.
DNA and RNA scatter x-rays more strongly than proteins, so the required
concentration can be about 5 fold lower than proteins.
Scattering data taken on a protein solution contains
signals from both the protein and the buffer. Scattering
measurements will be done on both the protein solution and
the buffer and then the buffer scattering is then
subtracted from the solution scattering. The
buffer-subtracted data corresponds to the scattering
signal of the protein alone. Therefore, the buffer
measurement is as essential as the protein solution
measurement. The exact chemical composition in the buffer
and in the solution must be matched in order to obtain a
proper buffer subtraction.
One way to get matched buffers is to dialyze the
protein solution in a buffer and then bring both the
protein solution and the buffer for SAXS measurement.
Since the buffer may also be used to flush the capillary
flow-cell before measurement, a volume of 10 ml of
buffer is required for each protein solution
measurement. It is recommended to bring 50-100 ml
of buffer with an additional 10 ml for each protein
sample. SEC SAXS requires more buffer to equilibrate the
size-exclusion column which consumes 50 mL for each
buffer condition. So a rule of thumb is always bring
excess buffer solution. It is also possible to make
buffers in our wetlab onsite. In such cases, please
contact the beamline scientist in advance.
Performing Size Exclusion Chromatography SAXS
The default data collection strategy at BioCAT is
Size-Exclusion Chromatography SAXS (SEC-SAXS), which
involves having a liquid chromatography unit in line
with the SAXS camera which facilitates purification of
samples using an appropriate gel-filtration column
immediately before exposure to the x-ray beam
This ensures optimal sample quality and separates the molecule of interest
from potential aggregates, oligomers, or breakdown products which are often
seen in samples that are purified days to weeks before data acquisition and
can adversely affect data quality and interpretability. The continuously
flowing sample very effectively counters the risk of radiation damage. This
strategy is also particularly elegant because buffer matching is relatively
simple and there is a built-in concentration series which samples a much more
extensive number of concentrations with minimal effort compared to the traditional
data collection strategies.
The FPLC unit being used for SEC-SAXS is called the AKTA-pure
(GE HealthSciences), and it comes equipped with a UV-monitor, capable
of measuring UV absorbance at 3 separate wavelengths (200nm to 700nm)
simultaneously, a loop valve and a column valve which facilitate optimal
We are able to pre-load up to 5 samples at a time and also keep the system
connected with up to 5 columns simultaneously which could be pre-equilibrated
and therefore reduce the time consumed in transitioning to multiple samples
and between different columns.
While we recommend that users bring their own
columns for the sake of consistency and to avoid cross-contamination issues,
we do provide a variety of columns in two different sizes, viz., superdex-200,
superdex-75, and superpose-6 columns in 10/300 and 5/150 sizes (column volumes
of 24mls and 3mls respectively). The default flow rate used for SEC-SAXS
experiments is 0.7mls/min and the typical experiment takes ~40mins. For more
detailed technical information on the AKTA-pure, please refer to the
and for more specific queries about our setup contact the beamline scientists.
Sample and Buffer Requirements:
Most commonly used buffer systems (e.g. Tris, HEPES, Phosphate buffer, MES,
etc) are compatible with our data-acquisition strategy. Salt concentrations
up to 1M are tolerated but lower salt concentrations are recommended. Use
of reducing agents such as DTT and TCEP, which are also known free radical
scavengers is recommended to counter the possibility of radiation damage in
concentrations of up to 1mM. Glycerol has been successfully used in the past
at concentrations as high as 10%. Higher concentrations of Glycerol are
discouraged as they would preclude the optimal flow-rate of 0.7ml/min on
account of higher back pressures thus increasing the likelihood of radiation
damage. Optimal sample concentrations are dependent on the size of the molecule.
For relatively small molecules in the 10-25kDa range, we recommend preparing
~500Åµl of ~5mg/ml and for medium sized molecules in the 25-75kDa
range, ~300Åµl of ~5mg/ml and for large molecules (75kDa and above),
~200Åµl of ~5mg/ml is sufficient for one SEC-SAXS run.
Samples with nucleic acids (DNA, RNA or protein-nucleic acid complexes) also show
strong scattering and need relatively less sample than protein. We strongly
suggest test-runs with analytical columns before arriving at the beam line,
thus, facilitating a higher degree of predictability and easier assessment
of results on site. Please find more helpful sample preparation guidelines
presentation on sample preparation.
We also provide the more traditional equilibrium SAXS setup without in-line
chromatography for which please find instructions below.
Performing Equilibrium SAXS Experiments
|Sample loading system with a Hamilton syringe pump.
The protein solution and buffer are contained in eppendorf
tubes 1.5 ml or 0.5 ml before being loaded into
the brass flow-cell. The sample is then flowed back and
forth during SAXS/WAXS measurements with a Hamilton
programmable syringe pump. We provide centrifuges for both
eppendorf tube sizes. You will need to bring your own
filters if you want to filter your samples. The protein
solution is measured immediately following the measurement
of its matched buffer. Before switching to the next buffer
and protein solution, the flow cell must be flushed with
the sequence: water, 20% bleach, water, 100% ethanol,
water, acetone, and water to remove any possible protein
deposits left on the flow-cell wall by interactions with
the high-flux x-ray beam.
Data collection for equilibrium SAXS
The User Interface shown below controls the sample loading before the X-ray
exposure and flow through the quartz capillary during the X-ray exposure.
It also automates the washing process between measurements. When 'Wash cell'
is clicked, the capillary flow cell is flushed subsequently by water, bleach,
water, isopropyl alcohol, water and then is air dried. 'Load cell' will pick
up the fluid in the Eppendorf tube and pull it into the capillary and 'Pull'
and 'Push' can be used to fine tune the position of the sample segment inside
the capillary and the tubing. In order to reduce the potential effects of
radiation damage, the sample will flow through the X-ray beam during the X-ray
irradiation. Immediately before starting the detector image collection, one
needs to click 'Flow-thru' to initiate the flow. The 'Speed' and 'Step' can
be set according to the sample volume and image collection protocol. After
the measurements if the user would like to recover the sample and save for
further analysis, it can be done by click 'Return sample'. Otherwise, one can
click 'Wash cell' and it will be flushed to waste.
|The flow control interface for equilibrium SAXS.
mesurement of the incident and transmitted X-ray
The X-ray intensity of the incident beam is measured with a National
Instruments analog I/O card and a LabView program is developed to acquire
the incident and transmitted beam intensity during the X-ray exposure time.
The X-ray scattering is collected with a Pilatus 1M detector mounted at the
end of the vacuum flight tube of a fixed camera length. The exposure time
is set using the following EPICS interface which communicates with the
Dectris camserver. The camserver controls and communicates directly with the
colleciton with Pilatus 1M.
The raw data for solution SAXS consists of the detector images and the
corresponding incident and transimitted intensity values which are used
to normalize the SAXS curves. The 2-D images are converted to SAXS curves
after radial averaging and q-calibration. Automatic data reduction pipeline
is available at the beamline computer. SAXS curves are available to the user
for further processing in real time (only a few minutes after the images are
Online data analysis for quality control
The Linux version of ATSAS is installed on the beamline computer 'dracula'
and the program 'SAS Data Analysis' (primusqt) can be used to plot, average,
and subtract SAXS curves immediately after it is reduced. Guinier analysis,
p(r) calculation and ab initio modeling can also be done using the ATSAS software
in order to check on the data quality.
Data Analysis and Modeling
For detailed data analysis and modeling we rely on the ATSAS software package
developed at EMBL Hamubrg and the programs in CCP-SAS. Please refer to the
respected website for more information. Our beamline scientists are experinced
BioSAXS expert and if you would like to collaborate with us on data analysis
and modeling, please contact them.