Optilab t rex ri detector

Complex formation between eIF2Bα, -β and -δ was studied by size exclusion chromatography (SEC) on an analytical Superdex 200 (10/300) column (GE Healthcare). For standard runs of the subunits, 50 μg protein in a total volume of 200 μl was loaded onto the column equilibrated in running buffer (90 mM KCl, 10 mM NaCl, 20 mM Tris/HCl (pH 7.5) and 2 mM DTT). For the analysis of binary complex formation between two subunits, 50 μg of each protein were mixed in 200 μl running buffer and incubated for 5 min at 20°C before loading onto the column. To study complex formation between all three subunits, eIF2Bβ (50 μg) was first mixed with a ∼2-fold excess of eIF2Bα (100 μg), followed by the addition of eIF2Bδ in ∼1.5-fold excess over the β-subunit (80 μg). The mixture was incubated for 5 min at 20°C in 200 μl running buffer before loading onto the column. Runs were monitored at an absorption wavelength of 280 nm. Apparent molecular weights (MW_app) were estimated using a protein standard giving MW_app = 10^{−0.179·V+4.631} (where V is the elution volume).
The absolute molecular weight (MW) of the proteins and protein complexes was determined by multi angle light scattering (MALS). Samples were run on the SEC column, connected to a miniDAWN TREOS LS detector and an Optilab T-rEX RI detector (Wyatt Technology). MW values were calculated using the ASTRA software (Wyatt Technology).

Size-exclusion high-performance liquid chromatography with light scattering detection (SE-HPLC-LS) was performed using a 1100 HPLC system (Agilent) with TSK-GEL G3000SWxl, 5-μm particle size, 7.8 mm ID × 300-mm length column (Tosoh Biosep, Merck KGaA). Samples were injected neat into the SE-HPLC-LS system. Runs were performed at room temperature with 100 mM sodium phosphate, 250 mM sodium chloride, and pH 6.8 buffer used as mobile phase; flow rate was 0.5 mL/min. Detectors included a HELEOS MALS detector (Wyatt Technology), an Optilab TrEX RI detector (Wyatt), and a UV detector (Agilent) set at 280 nm. The refractive index increment, dn/dc = 0.185 (mL/g), was used for calculation of molecular mass. Results were reported as the molar mass of monomer and HMW species using Wyatt ASTRA software.