SEC-SAXS Instructions the basics

Below is a schematic diagram of the Size-Exclusion Chromatography Small Angle X-ray Scattering (SEC-SAXS) workflow that you may take advantage of at the SIBYLS beamline 12.3.1 at the Advanced Light Source in Berkeley:

Separation of analytes is typically accomplished by size-exclusion chromatography (SEC) using an Agilent 1260 series HPLC with a Shodex analytical column at a flow rate of 0.5 ml/min using a your favorite buffer with the maximum of pH 7.5. Eluent is split 2 to 1 between X-ray synchrotron radiation and a series of four inline analytical instruments:

1. Aglient 1260 series multiple wavelength detector (MWD)
2. Wyatt Dawn Helos multi-angle light scattering (MALS) detector
3. Wyatt DyanPro Titan quasi-elastic light scattering (QELS) detector
4. Wyatt Optilab rEX refractometer, see the diagram below:

Samples are then examined using small-angle X-ray scattering (SAXS) as they come off of the column with λ=1.03 Å incident light at a sample to detector distances of 1.5 m. This results in scattering vectors, q, ranging from 0.013 Å ^-1 to 0.5 Å^-1, where the scattering vector is defined as q = 4πsinθ/λ and 2θ is the measured scattering angle.

3 s exposures are collected for each frame over the course of 40 min.

Additionally, UV, MALS, QELS, and differential refractive index data is collected and analyzed using Wyatt Astra 6 software.

All SIBYLS MailinSAXS users must first register for a MailinSAXS account

Request Mail-in Account

All users must register with the ALS User Office, and submit one ALS RAPIDD proposal for every three MailinSAXS slots

Submit Proposal

To expedite processing and sending your data, your data will be sent to Simple Scattering. Please register by clicking the button below

Simple Scattering

After completing the previous 3 steps, you may book a mail-in slot for beamtime

Book Mail-in Slot

Each beamtime slot is limited to 7 samples using 1 column and 1 buffer.

Each column run has the following sample and buffer requirements:

1. A sample with a minimum volume of 60 µl
2. 50 ml of 10x SEC buffer

The sample concentration should be from 3-10 mg/ml with smaller molecules requiring higher concentrations. Ideally, the sample should be prepared in a buffer that is identical or very similar to the SEC buffer.

For SEC buffers, the salt concentration must not exceed 500 mM and the pH of the buffer must be less than 7.5 to be accepted. If glycerol is desired, the concentration should not exceed 2%.

Please use small concentrations of other stabilizers such as glucose, ATP, etc. to minimize scattering background and to avoid high column-pressures.

We recommended that samples are shipped in standard Eppendorf PCR tubes together with 10x buffer using 4 °C cold packs (NOT frozen) in a styrofoam shipping container. Tightly pack the styrofoam box by surrounding assembly with more cold packs. While in some cases it is ok if the packs are frozen, do not allow the tubes to touch them. 

DO NOT ship wet ice.

Clearly label the outside of your shipping box with the temperature of your samples. This is extremely important if a shipping delay occurs.

Lastly, please provide us with a shipping tracking number.

Ship samples overnight to arrive at SIBYLS on the first day of your shift:

We will provide a merged SAXS curve for each sample together with a short report that includes SAXS parameters and a P(r) function to assess the quality of your data. We will also provide a SEC-MALS report for further quality control.

After the data has been successfully collected, you have a choice of the amount of SIBYLS staff involvement in processing and analyzing the results. Our team uses a variety of programs and decades of combined experience to assist you in your analysis.

Your data will be copied to your home directory on our "kona" server. To transfer files to your local computer, you may go into your local target directory and either remote sync rsync or secure copy scp the files from kona:

Or you may use sftp or other tools to transfer data to your local computer.

This is helpful in particular if you want to reprocess unsubtracted data.

Link to the ScÅtter IV download page.

We are continuing to improve our systems with the aim of making data of higher quality. Please provide us with constructive feedback. For additional information please feel free to contact:

Please cite all relevant SIBYLS beamline papers below in the 'References' section of your journal article or book chapter

• Dyer KN, Hammel M, Rambo RP, Tsutakawa SE, Rodic I, Classen S, Tainer JA, Hura GL (2014) “High-Throughput SAXS for the Characterization of Biomolecules in Solution: A Practical Approach” Methods Mol. Biol. 1091, 245-58. (Specific Methods paper for HT-SAXS at SIBYLS)
• Classen S, et al. (2013) “Implementation and performance of SIBYLS: a dual endstation small-angle X-ray scattering and macromolecular crystallography beamline at the Advanced Light Source” J. Appl. Crystallogr. 46(Pt 1), 1-13. (SIBYLS Beamline hardware)
• Hura GL, et al. (2009) "Robust, high-throughput solution structural analyses by small angle X-ray scattering (SAXS)" Nature Methods 6, 606–612. (General High-Throughput SAXS)
• Putnam CD1, Hammel M, Hura GL, Tainer JA (2007) "Solution scattering (SAXS) combined with crystallography and computation: defining accurate macromolecular structures, conformations and assemblies in solution” Q. Rev. Biophys. 40, 191-285. (General SAXS)

Please also acknowledge SIBYLS when using data generated at beamline 12.3.1.

These citations and acknowledgments are absolutely essential in keeping the MailinSAXS program funded and the beamline continually repaired and upgraded, as well as to provide access to experienced staff and the development of new technologies, such as SEC-SAXS.

We regularly present online webinars to highlight new SAXS features, and how to address and solve specific problems that occur during SAXS data collection and processing:

Webinars