View source for 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<sub>av</sub> values and plotting their K<sub>av</sub> values versus the logarithm of their molecular weight. The molecular weight of a protein is determined from the calibration curve once its K<sub>av</sub> value is calculated from its measured elution volume. 

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

where V<sub>e</sub> is the elution volume for the protein, V<sub>0</sub> is the column void volume (elution volume for Blue Dextran 2000) and V<sub>t</sub> 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.