Size-exclusion chromatography–small-angle neutron scattering system optimized for an instrument with medium neutron flux

Morishima K, Inoue R, Nakagawa T, Shimizu M, Sakamoto R, Oda T, Mayumi K, Sugiyama M, Journal of Applied Crystallography 58(2) (2025) DOI

SASDWB3 – Bovine serum albumin, monomer from SEC-SANS

Albumin (natural variant A214T)
MWI(0) 70 kDa
MWexpected 66 kDa
log I(s) 1.32×10-1 1.32×10-2 1.32×10-3 1.32×10-4
Albumin (natural variant A214T) small angle scattering data  s, nm-1
ln I(s)
Albumin (natural variant A214T) Guinier plot ln 1.33×10-1 Rg: 2.6 nm 0 (2.6 nm)-2 s2
(sRg)2I(s)/I(0)
Albumin (natural variant A214T) Kratky plot 1.104 0 √3 sRg
p(r)
Albumin (natural variant A214T) pair distance distribution function Rg: 2.7 nm 0 Dmax: 8.7 nm

Data validation

Fits and models

log I(s)
 s, nm-1
Albumin (natural variant A214T) PDB (PROTEIN DATA BANK) model
SANS data from solutions of bovine serum albumin in 100 mM Tris-HCl, 100 mM NaCl in 100% v/v D₂O, pH 7.5 were collected on the SANS-U beam line at JRR-3 (Japan Atomic Energy Agency; Ibaraki, Japan) using a ORDELA, model 2660N detector at a sample-detector distance of 4 and 1.03 m at a wavelength of λ = 0.6 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 500.00 μl sample at 7.7 mg/ml was injected onto a Cytiva Superdex 200 Increase 10/300 column at 25°C. 24 successive 600 second frames were collected. The data were normalized to the intensity of the transmitted beam and radially averaged; the scattering of the solvent-blank was subtracted.

SEC elution paused during SANS measurements.

Albumin (natural variant A214T) (BSA)
Mol. type   Protein
Organism   Bos taurus
Olig. state   Monomer
Mon. MW   66.5 kDa
 
UniProt   P02769
Sequence   FASTA
 
PDB ID   4F5S