This article discusses the determination of molecular weights of protein monomers and oligomers using size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS). The method allows for the separation of protein complexes based on size, enabling researchers to analyze the functional units of proteins in solution.
A protein oligomer is a complex of multiple protein subunits that are bound together to form a larger functional unit.
To determine the molecular weight of protein monomers and their oligomers in solution by SEC-MALS, a combination of size-exclusion chromatography and multi-angle light scattering, begin with a size-exclusion chromatography, or SEC, column.
The column contains porous sieve-like spherical gel beads that trap smaller-sized molecules within them. Equilibrate the column with a buffer compatible with the protein's native state.
Load a sample containing protein monomers and oligomers at the top of the column.
During the flow, the oligomers pass through the interparticle space of the column, while the monomers become entrapped within the pores of the beads. This causes the larger-sized protein oligomers to travel faster than the smaller-sized monomers.
Direct the fast-eluting oligomers, followed by the slow-eluting monomers, to the MALS detector.
As they pass through the detector, a laser light illuminates them, which is scattered at different angles. The multi-subunit oligomers scatter more light than the monomer.
The intensity of the scattered light at multiple angles is directly proportional to the molecular weight of the protein oligomer and monomer.
Begin this procedure with preparation of the SEC-MALS system as described in the text protocol. Using HPLC-grade reagents, prepare one liter of phosphate-buffered saline with 50 to 100 milliliters sodium chloride. Filter the buffer to 0.1 micron using a bottle-top polyether sulfone filter or similar.
Filter the first 50 to 100 milliliters of buffer to a waste bottle in order to eliminate particulates from the dry filters. Then, filter the remainder to a clean, sterile bottle that has been previously washed with filtered, deionized water and kept to prevent dust from entering.
Flush the column overnight at a flow rate of 0.5 milliliters per minute 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. Place the dRI flow cell in purge mode during the overnight flush.
When beginning the flush, gradually ramp the flow rate to prevent the "column shedding" effect caused by a sudden change of pressure in the column. Turn the purge off before beginning sample runs.
Check system cleanliness by lightly tapping the tubing downstream of the column to release accumulated particles. Observe the signal in the 90-degree detector on the front panel display of the MALS instrument. Verify that the peak-to-peak noise is no more than 50 to 100 microvolts. Also, verify that the refractive index or RI signal is stable to less than 1 x 10-7 refractive index units.
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. If the particle peak is no more than 1 milliliter in volume, and no more than 5 millivolts above baseline, then the system is ready for samples. Otherwise, perform additional blank injections until clean or perform maintenance to clean the injector.
Now, prepare at least 200 microliters of BSA at 1 to 2 milligrams per milliliter in the SEC buffer. Filter the protein to 0.025 microns using a syringe-tip filter. Discard the first few drops of filtrate in order to eliminate particles from the dry filters. Alternatively, centrifuge the sample at 10,000 times g for 15 minutes to enable precipitation of non-soluble aggregates and other large particles. Then, inject 100 microliters of the BSA solution into the loop.
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