FIG. 5.
Analysis of virion-associated Vpr by two-dimensional gel electrophoresis. (A) Unlabeled virus released from infected MT-4 cells was gradient purified as described for the labeled virus preparations. A sample corresponding to 15 μg of CA was applied to an Immobiline DryStrip 3-10L (Amersham Pharmacia) and subjected to isoelectric focusing (IEF) under denaturing conditions (8 M urea) according to the manufacturer's instructions, using an IGphor unit. Separation in the second dimension was performed by SDS-PAGE; subsequently, Vpr was detected by immunoblotting using antiserum directed against synthetic Vpr. (B) Gradient-purified 32P-labeled virus equivalent to 20 ml of tissue culture supernatant was separated by IEF (Immobiline DryStrip 6-11L) followed by SDS-PAGE. Radiolabeled protein was detected by phosphorimage analysis. Arrows indicate a single phosphorylated protein spot of the apparent molecular weight of Vpr, corresponding to an IEP of 6.3 (B) and an immunoreactive spot corresponding to the same IEP (A).