In vitro modification of Nef-His protein by TPCK E. coli- expressed Nef-His protein was isolated using Ni-beads as described in Methods. Twenty μl of freshly prepared Nef-His protein at the concentration of ~0.5 μg/μl was incubated with TPCK in PBS at RT for 30 min and then resolved by SDS-PAGE. The gels were stained with Coomassie Blue. (A) TPCK-modification of the freshly prepared Nef-His protein at different TPCK concentrations. (B) TPCK-modification of the Nef-His protein pre-incubated in PBS at different temperatures for different length of times.