Adding Size Exclusion Chromatography (SEC) and Light Scattering (LS) Devices to Obtain High-Quality Small Angle X-Ray Scattering (SAXS) Data

Graewert M, Da Vela S, Gräwert T, Molodenskiy D, Blanchet C, Svergun D, Jeffries C, Crystals 10(11):975 (2020) DOI

SASDFR8 – Bovine serum albumin, purified dimer - SEC-SAXS coupled to multiangle laser and quasi-elastic light scattering (MALLS and QELS)

Bovine serum albumin
MWexperimental 126 kDa
MWexpected 133 kDa
VPorod 211 nm3
log I(s) 1.23×104 1.23×103 1.23×102 1.23×101
Bovine serum albumin small angle scattering data  s, nm-1
ln I(s)
Bovine serum albumin Guinier plot ln 1.24×104 Rg: 4.0 nm 0 (4.0 nm)-2 s2
Bovine serum albumin Kratky plot 1.104 0 3 sRg
Bovine serum albumin pair distance distribution function Rg: 4.0 nm 0 Dmax: 13.2 nm

Data validation

Fits and models

log I(s)
 s, nm-1
Bovine serum albumin GASBOR model

log I(s)
 s, nm-1
Bovine serum albumin PDB (PROTEIN DATA BANK) model

Synchrotron SAXS data from pre-purified bovine serum albumin dimer in 50 mM HEPES, 150 mM NaCl, 2% v/v glycerol, pH 7 were collected on the EMBL P12 beam line at PETRA III (DESY, Hamburg, Germany) using a Pilatus 6M detector at a sample-detector distance of 3 m and at a wavelength of λ = 0.124 nm (I(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle). In-line size-exclusion chromatography (SEC) SAS was employed. The SEC parameters were as follows: A 100.00 μl sample at 5 mg/ml was injected at a 0.50 ml/min flow rate onto a GE Superdex 200 Increase 10/300 column at 20°C. 25 successive 1 second frames were collected through the major SEC-elution peak and processed using CHROMIXS. The data were normalized to the intensity of the transmitted beam and radially averaged; the scattering of the solvent-blank was subtracted.

BSA dimer underwent pre-purification prior to SEC-SAXS using the following method. All procedures were performed at 4 oC. Protein powder (Sigma) consisting of BSA monomers, dimers, trimers and higher MW species was made to approximately 25 mg/ml in 25 mM HEPES, 50 mM NaCl, 5 mM urea, 1% v/v glycerol, pH 7. Approximately 200 μl of sample were loaded onto a Superdex 200 Increase 10/300 column (GE Healthcare) equilibrated in the same buffer (flow rate = 0.4 ml/min). Fractionated aliquots corresponding to the minor dimer peak (estimated using UV A280 and UV A245 nm) were pooled and concentrated (30 kDa centrifuge spin filter) to a final concentration of 5 mg/ml (the concentration was determined from triplicate UV A280 measurements using an E0.1% of 0.646 (= 1 g/l) calculated from the amino acid sequence (ProtParam)). Approximately 50 μl aliquots were snap-frozen in liquid nitrogen then stored at -80oC prior to the SEC-SAXS analysis that was performed at room temperature in 50 mM HEPES, 150 mM NaCl, 2% v/v glycerol, pH 7. The Rg-correlation through the SEC-SAXS peak, the individual unsubtracted SEC-SAXS frames as well as the results from coupled MALLS and QELS analysis are included in the full entry zip archive. The quoted experimental molecular weight was determined using MALLS in combination with refractive-index (RI) measurements that were recorded from the same sample eluting from the column using a split-flow SEC-SAXS-light scattering configuration (Graewert et al., (2015) Sci. Reports. 5, 10734: doi: 10.1038/srep10734). The average hydrodynamic radius of the protein is 4.6 nm.

Tags: benchmark
Bovine serum albumin (BSA)
Mol. type   Protein
Organism   Bos taurus
Olig. state   Dimer
Mon. MW   66.4 kDa
UniProt   P02769 (25-607)
Sequence   FASTA