Size-exclusion chromatography small-angle X-ray scattering of water soluble proteins on a laboratory instrument.

Bucciarelli S, Midtgaard SR, Nors Pedersen M, Skou S, Arleth L, Vestergaard B, J Appl Crystallogr 51(Pt 6):1623-1632 (2018) Europe PMC

SASDEZ4 – Carbonic Anhydrase from in-house SEC-SAXS

Carbonic anhydrase 2
MWexperimental 31 kDa
MWexpected 29 kDa
VPorod 38 nm3
log I(s) 1.59×10-1 1.59×10-2 1.59×10-3 1.59×10-4
Carbonic anhydrase 2 small angle scattering data  s, nm-1
ln I(s)
Carbonic anhydrase 2 Guinier plot ln 1.59×10-1 Rg: 1.9 nm 0 (1.9 nm)-2 s2
(sRg)2I(s)/I(0)
Carbonic anhydrase 2 Kratky plot 1.104 0 3 sRg
p(r)
Carbonic anhydrase 2 pair distance distribution function Rg: 1.8 nm 0 Dmax: 5.3 nm

Data validation


Fits and models


log I(s)
 s, nm-1
Carbonic anhydrase 2 PDB model

log I(s)
 s, nm-1
Carbonic anhydrase 2 DAMFILT model

SAXS data from solutions of Carbonic Anhydrase from in-house SEC-SAXS in PBS, pH 7.4 were collected on a Xenocs BioXolver L instrument at the Copenhagen University, Department of Drug Design and Pharmacology (Copenhagen, Denmark) using a 20Hz Pilatus 300K detector at a sample-detector distance of 0.7 m and at a wavelength of λ = 0.134 nm (l(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle).

The CAH sample (500 microlitres at 5.4 mg/ml) was injected onto a GE Healthcare S200 Increase 10/300 (24 ml) column at a flow rate of 0.1 ml/min at 23°C. A total of 165 x 30 second SAXS data frames were recorded throughout the CAH elution. After background solvent corrections, SEC-SAXS data frames 96-105, corresponding to monomeric CAH, were scaled, averaged and binned logarithmically to produce the data displayed in this entry. Additional files, including plots of the selected frames and of I(0), HPLC and exposure cell UV traces and Rg vs. time (determined by AUTORG) are included in the additional files of the full entry zip archive.

Carbonic anhydrase 2 (CAH)
Mol. type   Protein
Organism   Bos taurus
Olig. state   Monomer
Mon. MW   29.0 kDa
 
UniProt   P00921
Sequence   FASTA
 
PDB code   1V9E