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SEC-SAXS: Introduction

In order to carry out a successful solution x-ray scattering (SAXS) data collection a highly monodispersed sample is vital. Even small amounts of higher oligomers can interfere with the data analysis and make interpretation of the resulting scattering curves difficult and potentially misleading. Many of the interesting problematic proteins we would like to examine using SAXS can be initially obtained in monodispersed forms by size-exclusion chromatography or similar methods, but tend to quickly aggregate shortly afterwards or form aggregates at re-concentrating after purification. Today SEC-SAXS is a powerful tool to overcome and/or mitigate those problems. The SSRL BioSAXS beamline BL4-2 has been accommodating the SEC-SAXS technique to users for several years. We recently had the first large upgrade on BL4-2 SEC-SAXS experimental setup and it is now open for all BL4-2 users.

Figure 1: UHPLC Setup - Thermo Fisher Scientific UltiMate 3000 system can be easily moved in and out of place via a mobile cart. The UHPLC system can be directly connected to our autosampler (static) system allowing for rapid comparison of SEC-SAXS and autosampler data. Figure 2 (Right): BSA regular and autosampler data comparison- BSA in acetate buffer actually consists of a mixture of monomer and a small amount of dimer and high-order oligomers. This is almost undetectable using our regular static setup; however by using our SEC-SAXS system we can separate the two peaks and collect data on both forms.

Advantages of BL4-2 SEC-SAXS (2nd Gen)
Thermo Fisher Scientific UltiMate 3000 system has been recently installed for Bl4-2 SEC-SAXS setup. This is the first large upgrade on BL4-2 SEC-SAXS setup. This UHPLC system (Ultra-High Performance Liquid Chromatography) having smaller internal volume with automated small volume injection system provides more precise separation with the small size analytical SEC column. Dual columns setup (tandem operation) contributes to high-throughput SEC-SAXS data collection. Thanks to the small bet volume of the column, the user could save sample consumption and minimize sample dilution during SEC, leading to better scattering signal. The user easily performs automated, continuous and strategic SEC-SAXS data collections and analyses from newly updated Blu-Ice beamline control software. Some options for strategic SEC-SAXS data collection against undesired radiation damages are also available on the Blu-Ice. Real-time automatic SEC-SAXS analysis pipeline program, SECPipe, is also available by default and the user can perform SEC-SAXS data collection with watching real-time Rg/I(0) plots calculated by Guinier analysis as well as UV absorbance. Individual features are described below.
The new system is still easily switchable to regular static measurement using our SAXS autosampler as the need arises. New Pilatus3 X 1M detector providing less signal-to-noise ratio of the scattering data also supports the best data possible from those problematic samples.

High resolution mode is now available with specific SEC columns. Sample dilution between the SEC column and X-ray position is virtually negligible. The degree of sample separation on SEC-SAXS results from the column quality and the chromatographic condition. This mode is currently incompatible with Autosampler mode. Please contact beamline staff for more details.

SEC-SAXS High Selectivity Mode
Figure. SEC-SAXS profile of BSA performed by the high resolution mode. The UV signal was recorded at UHPLC UV detector located right after the SEC column.


Available SEC columns at BL4-2
Currently the following SEC columns are available for BL4-2 users on Thermo Fisher Scientific UltiMate 3000 system. Two same columns are used for high-throughput SEC-SAXS data collections by default (tandem operation). This allows the user to wash the used column and then re-equilibrate it with buffer in the background.

UV profile of BSA using Superdex 200 Increase column
[Example] UV profile (A280) of BSA using
Superdex 200 Increase PC 3.2/300 column on
Thermo Fisher Scientific UltiMate 3000 UHPLC
(5ul of 20mg/ml BSA injection at 0.05ml/min flow rate)

Superdex 200 Increase PC 3.2/300 (Product#: 28990946)
     Recommended Mw for SEC-SAXS at BL4-2: 30-250 kDa
     Recommended flow rate: 0.05-0.075 ml/min (Default: 0.05ml/min)
     Approximate column bed volume: 2.4ml
Superdex 75 PC Increase 3.2/300 (Product#: 29148723)
     Recommended Mw for SEC-SAXS at BL4-2: 10-50 kDa
     Recommended flow rate: 0.05-0.075 ml/min (Default: 0.05ml/min)
     Approximate column bed volume: 2.4ml
Superose 6 Increase PC 3.2/30 (Product#: 29091598)
     Recommended Mw for SEC-SAXS at BL4-2: >200 kDa
     Recommended flow rate: 0.04-0.075 ml/min (Default: 0.04ml/min)
     Approximate column bed volume: 2.4ml

If the user uses BL4-2 SEC column, all sample should be centrifuged at BL4-2 right before sample setup to the UHPLC (see instruction at BL4-2). Also, the use of VWR 0.5mm in-line filter is mandatory with BL4-2 SEC column. Please order and bring the in-line filter (VWR Cat #: 49650-005) for your beamtime.
We encourage the user to bring own column(s) for the best SEC performance, especially if the sample is very sensitive to separation, contact with residual sample, etc. It is possible to mount larger size columns but please note that the maximum injection volume is currently 100ml for automatic SEC-SAXS data collection. Since the large SEC column(s) requires large alterations on the instruments, if the user plans to bring own column(s), please contact BL4-2 staff very well in advance of your beamtime (ideally, please contact BL4-2 staff before the user submit beamtime request).