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

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.