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

Overview

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

1. 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.
2. Ensure that the necessary signal connections between the FPLC and detectors have been established, including analog output from the UV detector to the MALS analog input, and digital output from the FPLC to the MALS Autoinject, via the FPLC's I/O Box.
3. Install a suitable analytical SEC column covering a fractionation range of at least 20 kDa to 500 kDa. Check the product info to determine if the column is suitable for the range of MW, pH, and other properties of the sample and mobile phase.

2. Preparation of buffer, flushing the system overnight, and checking cleanliness

1. Using HPLC-grade reagents, prepare 1 L of phosphate-buffered saline with 50-100 mM NaCl. Filter the buffer to 0.1 µm using a bottle-top polyether sulfone filter or similar. Filter the first 50-100 mL of buffer to a waste bottle and discard, in order to eliminate particulates from the dry filters, and then filter the remainder to a clean, sterile bottle that has been washed thoroughly with filtered, de-ionized water and capped to prevent dust from entering.
   NOTE: Other mobile phase solvents such as a Tris buffer may be used if additional proteins that are preferentially dissolved in those solvents are to be analyzed.
2. Flush overnight at a flow rate of 0.5 mL/min, or as otherwise recommended by the column manufacturer, to equilibrate the column in the buffer and remove particulates. Use the FPLC's Continuous flow mode and ensure that the flow does not stop until all SEC-MALS runs are complete.
   1. Place the dRI flow cell in Purge mode during the overnight flush. Turn the purge off before beginning sample runs.
   2. When beginning the flush, gradually ramp the flow rate to prevent the "column shedding" effect (or release of particles) caused by a sudden change of pressure in the column.
   3. If the system is known to be quite stable and particle-free, and in equilibrium with the desired mobile phase, replace the overnight flush with a shorter, 2-3 h flush.
3. Check system cleanliness by lightly tapping the tubing downstream of the column to release accumulated particles and observing the signal in the 90° detector on the front-panel display of the MALS instrument. Verify that the peak-to-peak noise is no more than 50 -100 µV.
4. Perform a 'blank' injection to verify that the injector is clean of particles. A 'blank' is simply the running buffer, prepared in a fresh, sterile vial.
   1. If the particle peak is no more than 1 mL in volume and no more than 5 mV above baseline, then the system is ready for samples. Otherwise, perform additional blank injections until clean, or perform maintenance to clean the injector.

3. Preparing and loading the sample

1. Prepare at least 200 µL of BSA at 1-2 mg/mL in the SEC buffer.
   NOTE: In order to prevent precipitation, BSA should never be dissolved in pure water.
2. Filter the protein to 0.02 µm using a syringe-tip filter.
3. Discard the first few drops of filtrate in order to eliminate particles from the dry filters.
4. Alternatively, centrifuge the sample at 10,000 x g for 15 min to enable precipitation of non-soluble aggregates and other large particles.
5. Inject 100 µL of the BSA solution into the loop.
   NOTE: This is the recommended amount of material, and more or less may be injected according to the circumstances of the sample such as stability or availability. The quantity of protein required per injection varies inversely with molecular weight twice as much protein mass is needed if the molecular weight is 33 kDa or half that of BSA.

4. Preparation of the MALS software

1. Open New | Experiment from Method in the MALS software menu and select the Online method from the Light Scattering system methods folder. If a DLS detector is present, select the Online method from the Light Scattering | With QELS folder.
2. In the Configuration section, set the parameters of the sample and mobile phase.
   1. In the Generic Pump view, set the flow rate to that used in the FPLC.
   2. In the Generic Pump view, Solvent branch, Name field, select PBS.
   3. In the Injector view, Sample branch, enter the Name as BSA, and set dn/dc = 0.185 (the standard value for unmodified proteins), A2 = 0, and UV extinction coefficient = 0.667 mL/(mg-cm).
      NOTE: For other proteins, the UV₂₈₀ nm extinction coefficient may be found in the literature or calculated from its sequence using various public-domain software tools.
3. In the Procedures section, Basic Collection view, select the checkbox Trigger on Autoinject and set the duration of the run to 70 min so that data are collected for the entire elution until the total permeation volume of the SEC column is reached.
   NOTE: The necessary amount of time may vary with the column and the flow rate - 35 min of the collection is required for a standard 7.8 mm x 300 mm HPLC-SEC column at 0.5 mL/min.
4. Start the experiment in the MALS software by clicking on the Run button. It will start reading the data after receiving the pulse signal from the FPLC instrument via the MALS detector.
5. Zero the dRI signal by clicking the Autozero button on the instrument's front panel.

5. Preparation of the FPLC software

1. Insert the name of the protein and the run in the FPLC software, in Manual | Execute manual instructions | Set mark.
2. Switch the injection valve from Manual load to Inject under Flow path | Injection valve.
3. Include a pulse signal by inserting a 0.5 s pulse under the I/O box | Pulse digital out. This will trigger data collection in the MALS software.

6. Inject the sample into the loop. Click Execute in the FPLC software to start the experiment run.

Materials

Name	Company	Catalog Number	Comments
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