AF4-to-SAXS: Expanded Characterization of Nanoparticles and Proteins at the P12 Biosaxs Beamline

Melissa Graewert.

SASDW94 – Horse apoferritin from AF4-SAXS measurements

Ferritin light chain
MWexperimental 434 kDa
MWexpected 480 kDa
VPorod 632 nm3
log I(s) 4.91×102 4.91×101 4.91×100 4.91×10-1
Ferritin light chain small angle scattering data  s, nm-1
ln I(s)
Ferritin light chain Guinier plot ln 4.92×102 Rg: 5.4 nm 0 (5.4 nm)-2 s2
(sRg)2I(s)/I(0)
Ferritin light chain Kratky plot 1.104 0 3 sRg
p(r)
Ferritin light chain pair distance distribution function Rg: 5.3 nm 0 Dmax: 13.3 nm

Data validation


Fits and models


log I(s)
 s, nm-1
Ferritin light chain PDB (PROTEIN DATA BANK) model

Synchrotron SAXS data from solutions of apoferritin separated using asymmetric flow field flow fractionation (AF4) in phosphate buffered saline, pH 7.2 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 6 m and at a wavelength of λ = 0.12 nm (I(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle). AF4 separation was performed at 20°C using a flow rate of 0.5 mL/min with an applied constant cross flow rate of 3 mL/min. 45 µL of apoferritin at a concentration of 12 mg/mL were injected. 2700 successive 1 second SAXS data frames were collected through the entire AF4 elution (45 min), where 10 sample data frames were selected through the 24mer-separated elution peak. The data were normalized to the intensity of the transmitted beam and radially averaged; the scattering of the solvent-blank was subtracted.

Tags: AFFFF
Ferritin light chain (apoferritin)
Mol. type   Protein
Organism   Equus caballus
Olig. state   24-mer
Mon. MW   20 kDa
 
UniProt   P02791 (1-175)
Sequence   FASTA
 
PDB ID   1IER