Gel filtration and light scattering

Gel filtration and static light scattering at Rutger's University

Oligomerization Analysis by Gel Filtration
The molecular weight of protein is determined by gel filtration. It is performed at 4 °C on a Superdex 75 (26/60) column equilibrated with the NMR buffer pH 6.5 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 Kav values and plotting their Kav values versus the logarithm of their molecular weight. The molecular weight of a protein is determined from the calibration curve once its Kav value is calculated from its measured elution volume.

         Kav =(Ve - Vo)/(Vt - Vo)
         where Ve = elution volume for the protein
         Vo = column void volume = elution volume for Blue Dextran 2000
         Vt = total bed volume     

The oligomerization state of protein is estimated from the value of MW(Measured)/MW(Predicted). Typically a protein momomer or dimer will run "larger" than predicted, but never "smaller".

Static Light Scattering Analysis
The MW of protein was also determined by analytical size exclusion chromatography combined with multi-angle light scattering (SEC-MALS). The measurements were 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) was equilibrated in 10 mM MES, pH 6.5, 100 mM NaCl, 5.0 mM CaCl2, 10 mM DTT, and 0.02% NaN3 at a flow rate of 0.5 ml/min. A volume of 30 μl was injected. Data were 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) was used as a reference.

Gel filtration at University of Toronto

Gel filtration chromatography is used to determine the protein oligomerization state in solution.

equipment: biologic duo flow; column: Superdex 75 Hiload 16/60