Size-Exclusion Chromatography with Multi-Angle Light Scattering for Protein Oligomers

Abstract

Source: Some, D. et al., Characterization of Proteins by Size-Exclusion Chromatography Coupled to Multi-Angle Light Scattering (SEC-MALS). J. Vis. Exp. (2019).

This video describes SEC-MALS, which leverages the abilities of size-exclusion chromatography (SEC) and multi-angle light scattering (MALS). In SEC-MALS, the SEC separates proteins or protein complexes based on their size, and the MALS detector determines the average molecular weight by measuring the intensity of the light scattered by the molecule at different angles.

Protocol

1. Preparation of the system Connect the MALS and dRI detectors downstream of the FPLC's UV detector. Bypass the pH and conductivity detectors since they will add significant inter-detector volume between the UV and MALS detectors. Use capillary tubing of 0.25 mm i.d. from the column to and between the detectors, and 0.75 mm i.d. capillary tubing on the output of the detectors to waste or fraction collector. Ensure that the necessary signal connections between the FPLC and detec…

Materials

AKTA pure	GE Healthcare	29-0182-26	Fast Protein Liquid Chromatograph (FPLC)
AKTA UNICORN	GE Healthcare		FPLC control software
ASTRA	Wyatt Technology		MALS data acquisition and analysis software
Bovine serum albumin (purity>97%)	Sigma	A1900	Analyte
DAWN or miniDAWN	Wyatt Technology	WH2 or WTREOS	WH2 or WTREOS
Increase 200 10/300	GE Healthcare		Size exclusion column, 200 Å pores, 10 mm i.d., 300 mm length
OptiLab	Wyatt Technology	WTREX	Differential refractive index (RI) detector
Sodium chloride NaCl	Sigma	71382	HPLC-grade NaCl
Stericup bottle top filter polyether sulfone 0.1 µm 1000 mL	Millipore	Millipore	Mobile phase filter
Whatman Anotop 10 syringe filter 0.02 µm	GE Healthcare	6809-1002	Sample filter
Whatman Anotop 10 syringe-tip filter, 0.1 µm pore, 10 mm diameter G	GE Healthcare	6809-1012	Sample filter
Whatman Anotop 25 syringe filter 0.1 µm	GE Healthcare	6809-2012	Mobile phase filter
WyattQELS	Wyatt Technology	WIQ	Dynamic light-scattering detector