Size Exclusion Chromatography with Multiangle Light Scattering (SEC-MALS)

Concentrate the purified protein sample to 4-5 mg/ml

Instruments:

Agilent 1200 HPLC system (Agilent Technologies, Santa Clara, CA)

Wyatt DAWN HELEOS-II MALS instrument

Wyatt Optilab rEX differential refractometer (Wyatt, Santa Barbara, CA)

WTC-050S5 size-exclusion column (Wyatt)

40 ul sample loop

Equilibrate the system and column with buffer containing:

25 mM HEPES pH 7.5, 200 mM NaCl, 1 mM MgCl₂, 2 mM TCEP

Turn on the laser 1 hour before the experiment.

Confirm the readout is stable

Run 5 mg/ml BSA to calibrate the system

Inject the sample and run (takes ~1 hour/sample)

Analyze the results by ASTRA V software (Wyatt).

MALS signals, combined with the protein concentration determined by refractive index, are used to calculate the molecular mass of the complex.