Gel filtration and light scattering

Gel filtration

Gel filtration is performed at 4 °C on a Superdex 75 (26/60) (CABM Rutgers) or Superdex 75 Hiload 16/60 (U of Toronto) equilibrated with the corresponding NMR buffer at a flow rate of 2.5 ml/min. The column is calibrated with LMW gel filtration calibration kit (17-0442-01) from GE Healthcare. A calibration curve is prepared by measuring the elution volumes of several standards, calculating their corresponding K_av values and plotting their K_av values versus the logarithm of their molecular weight. The molecular weight of a protein is determined from the calibration curve once its K_av value is calculated from its measured elution volume.

<math>K_{av}=\frac{V_e-V_0}{V_t-V_0}</math>

where V_e is the elution volume for the protein, V₀ is the column void volume (elution volume for Blue Dextran 2000) and V_t is the total bed volume.

The oligomerization state of protein is estimated from the ratio of measured to calculated molecular weight. Typically, a protein momomer or dimer will run "larger" than predicted, but never "smaller".

Static light scattering

The MW of protein can also be determined by analytical size exclusion chromatography combined with multi-angle light scattering (SEC-MALS). The measurements are performed on an Agilent 1100 HPLC system (Agilent) connected to a tri-angle light scattering detector and a differential refractometer (miniDAWN Tristar and Optilab, Wyatt Technology). A Shodex KW-802.5 column (Shodex) is equilibrated with the corresponding NMR buffer at a flow rate of 0.5 ml/min. A volume of 30 μl is injected. Data is processed using ASTRA software (Wyatt Technology) assuming a specific refractive index increment (dn/dc) of 0.185 ml/g. To determine the detector delay volumes and the normalization coefficients for the MALS detector, a BSA sample (Sigma) is used as a reference.