Jun 09, 2025
Size-exclusion Chromatography Coupled to Multi-angle Light Scattering (SEC-MALS) for TMEM55B 80-166 2CysMUT
This protocol is a draft, published without a DOI.
- Dieter Waschbüsch1,
- Amir R. Khan1
- 1School of Biochemistry and Immunology, Trinity College Dublin, Dublin 2, Ireland
Protocol Citation: Dieter Waschbüsch, Amir R. Khan 2025. Size-exclusion Chromatography Coupled to Multi-angle Light Scattering (SEC-MALS) for TMEM55B 80-166 2CysMUT. protocols.io https://protocols.io/view/size-exclusion-chromatography-coupled-to-multi-ang-g2vabye2f
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 08, 2025
Last Modified: June 09, 2025
Protocol Integer ID: 219778
Keywords: ASAPCRN
Abstract
To determine the absolute molar mass of TMEM55B 80-166 2CysMUT construct Size-exclusion Chromatography Coupled to Multi-angle Light Scattering (SEC-MALS) was performed. It also helps to understand the purity of the protein preparation - whether the protein is in monomeric, oligomeric, or aggregated state.
Materials
1. Materials
1.1. Consumables
- 3 or 10 kDa MWCO Amicon Ultra-15 concentrator tube, Merck
1.2. Reagents
- Purified proteins for analysis
- Gel filtration buffer (20 mM Tris pH 8.0 at 4ºC, 150 mM NaCl, 1 mM DTT)
1.3. Equipment
- FPLC system (i.e., AKTA basic or AKTA purifier, Cytiva)
- MiniDAWN MALS spectrometer (Wyatt)
- Refractometer VVV (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)
Method
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 minimise mismatch.
To improve signal stabilisation, 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/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.
Analyse data using ASTRA.