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).