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 0.65 ml/min using your favorite buffer with a maximum of pH 7.5. The eluent from SEC is measured in line with 1) MALS and UV, 2) SAXS, 3) Differential refractivity.

You can use this template for a detailed methods section in your publications.

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. We highly recommend adding 10-20% glycerol in your 10X buffer, so when diluted with dd H₂O your 1x buffer will have 1-2% glycerol.

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 with a short report that includes SAXS parameters and a P(r) function to assess the quality of your data. We will also provide an SEC-MALS report for further quality control. Your results will be deposited to SIMPLE SCATTERING.

Please use the program RAW for viewing your merged SAXS profiles (.dat), derived P(r) functions (.out ), SAXS parameters, or SEC-SAXS chromatograms (.hdf5 ).

For more information please visit our tutorials. You can also choose 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 help you with your analysis.

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

• Rosenberg DJ, Hura GL, Hammel M (2022) "Size exclusion chromatography coupled small angle X-ray scattering with tandem multiangle light scattering at the SIBYLS beamline" Meth. enzymol. 677, 191-219. (SEC-SAXS)
• 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)
• Putnam CD, 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