Jul 07, 2025

Public workspaceSize-exclusion Chromatography Coupled to Multi-angle Light Scattering (SEC-MALS)

DOI
https://dx.doi.org/10.17504/protocols.io.3byl41mxolo5/v1
  • Dieter Waschbuesch1,
  • Amir Khan1
  • 1School ofBiochemistry and Immunology, Trinity College Dublin
  • AKhanLab
Dieter Waschbuesch
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DOI: https://dx.doi.org/10.17504/protocols.io.3byl41mxolo5/v1
Protocol Citation: Dieter Waschbuesch, Amir Khan 2025. Size-exclusion Chromatography Coupled to Multi-angle Light Scattering (SEC-MALS). protocols.io https://dx.doi.org/10.17504/protocols.io.3byl41mxolo5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 24, 2025
Last Modified: July 07, 2025
Protocol Integer ID: 220917
Keywords: molecular weight of protein, light scattering, size exclusion chromatography, protein complexes in solution, differential refractive index, intensity of the isotropic scattering, diverse proteins in order, protein complex, diverse protein, isotropic scattering, accurate measurements of concentration, protein, exclusion chromatography, optilab refractometer, molecular weight, concentration
Funders Acknowledgements:
Research Ireland
Grant ID: 20/FFP-A/8446
Abstract
Size exclusion chromatography coupled to light scattering (SEC-MALS) is a convenient method to determine the molecular weight of proteins and protein complexes in solution. It exploits the intensity of the isotropic scattering of proteins measured at several angles as a function of their concentration. Accurate measurements of concentration are determined in-line during the experiment by measuring the differential refractive index using an Optilab Refractometer. Here we describe a generic method for collecting data that can be exploited for diverse proteins in order to understand their size/stoichiometry in solution.
Materials
1. Consumables
  • 3 or 10 kDa MWCO Amicon Ultra-15 concentrator tube, Merck

2. Reagents
  • Purified proteins for analysis
  • Gel filtration buffer (20 mM Tris pH 8.0 at 4ºC, 150 mM NaCl, 1 mM DTT)

3. Equipment
  • FPLC system (i.e., AKTA basic or AKTA purifier, Cytiva)
  • Size exclusion chromatography column: Superdex 75 10/300 GL (Cytiva)
Note
Depending on the size of a protein or protein complex a different type of column is recommended, ie. the superdex 200 10/300GL (Cytiva)

  • MiniDAWN MALS spectrometer (Wyatt)
  • Refractometer Optilab rEX (Wyatt)
  • Wyatt Comet sonicator (cleaning device for the flow cell of the MiniDAWN spectrometer)
  • PC with the AKTA control software (UNICORN 5.11) and the SEC-MALS control and analysis software (ASTRA 4.9)
Troubleshooting
Method
Connect the “miniDAWN” light scattering spectrometer and the refractometer to the FPLC System as indicated in Fig. 1.
Figure 1: Setup SEC-MALS. The AKTA purifier is equipped with a 0.5 ml injection loop and superdex 75 10/300 GL size exclusion column. The column outlet is connected to the MiniDAWN light scattering spectrometer followed by a refractometer, which is used to collect information on the protein concentration.

Equilibrate column into Gel filtration buffer until signal on detector 2 (90° angle) of the miniDAWN is stable (signal between 0.3 and 0.4 with minimal fluctuations).
Note
This can take between 1 to 3 column volumes (1 CV = 24 ml)

During equilibration, purge cell of refractometer to minimize mismatch.
To improve signal stabilization, use the “Comet” sonicator. For conducting an experiment, it needs to be switched off.
Run the FPLC at a flow rate of 0.6 to 0.7 ml/min, maximum system pressure 1.8 MPa.
Concentrate/dilute protein to approximately 1 mg/ml.
Note
The smaller the protein is, the less it scatters. For example, our TMEM55B 80-166 2CysMUT construct has a MW of 9.8 kDa, which is at the lower end of what SLS can be used for. Smaller proteins might not scatter enough to produce a usable signal. For small proteins, higher concentrations help with the signal strength.

Attach a 0.5 ml injection loop to the FPLC System. Flush with a few ml of gel filtration buffer.
Load injection loop with the protein or protein complex.
Check settings of the ASTRA software:
- System setup: Flow Rate (i.e., 0.6 ml/min); leave the other parameters unchanged.
- Collection Setup: dn/dc (mL/g): 0.185; Collection Duration: 35 ml; Collection interval (sec) 1.
Start collecting light scattering data using the ASTRA software and set the FPLC software from 'load' sample to 'inject' directly after another.
Collect data for an appropriate time to allow proper baselining for analysis.
Analyze data using ASTRA.