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

Published: June 29, 2023

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…

Disclosures

The authors have nothing to disclose.

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

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Cite This Article
Size-Exclusion Chromatography with Multi-Angle Light Scattering for Protein Oligomers. J. Vis. Exp. (Pending Publication), e21448, doi: (2023).

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