Laboratory of Molecular Biophysics
Figure 3. Purification of Nef. (A) Digestion of GST-Nef protein using a GST-tagged 3C protease. Digestion was performed at 22oC at a protein:protease ratio of 20:1. Lanes correspond to digestion at a time, t (min)=0, 1, 10, 20, 40, 60, 90, 120 and overnight.
(B) Following digestion, the GST/Nef mixture was separated on a Superdex-75 column. A sample (6mg, 10ml) was loaded and eluted at 3ml/min in 150mM NaCl, 10mM HEPES, 3mM EDTA, 0.01% monothioglycerol, pH 7.5.