Sec analytical column

Size Exclusion chromatography for multiple angle laser light scattering (SEC-MALS) measurements were performed at 25 °C using a Dawn HELEOS-II MALS with QELS DSL from Wyatt Technology (Santa Barbara, CA), with Agilent (HP) 1100 series pumps, Agilent autosampler, diode-array UV/Vis and the inline fluorescence detector. Running buffer was 50 mM sodium acetate pH 5.0, 50 mM NaCl. Flow rate was 0.4 ml/min. Column fractionation range was 500 −150,00 g/mol. The refractive index detector was the Optilab T-rEX from Wyatt Technology. Data acquisition and evaluation was performed using Astra from Wyatt Technology. Bovine serum albumin (Sigma, St Louis, MO) was used to normalize detectors. Molar absorptivity (280nm) of 1.9 M-1cm-1 was used to calculate concentration. Separations were carried out using a SEC Analytical Column, 5μm 150Å 7.8mm ID, Standard (Wyatt Technologies, WTC-015S5.). The buffer used for gel filtration was 50 mM sodium acetate pH 5.0, 50 mM NaCl and the solution concentration was 0.6 mg/mL.

To assess whether the Tat₈₆ protein is monodisperse, 50 μg protein sample in a buffer containing 50 mM sodium acetate (pH 4), 100 mM NaCl, and 5 mM TCEP was injected onto a SEC analytical column (5 mm, 300Å, 4.6 mm; Wyatt Technology) on a Shimadzu HPLC system coupled to a DAWN 8 Ambient detector equipped with a light scattering module and an Optilab refractometer (Wyatt Technology). Data were analyzed using the ASTRA 8 software (Wyatt Technology).