Abstract Full length cDNAs of small GTP-binding proteins were inserted into PGEM1 under the control of the T7 promotor as described previously (Chavrier et al. 1990; Huber et al. 1993). GTP-binding proteins were then transiently expressed in BHK 21 cells with the T7 RNA polymeraserecombinant vaccinia virus system (Fuerst et al. 1986; Chavrier et al. 1990; Huber et al. 1993, 1994b). Cells were scraped and fractionated 5 hours after infection/transfection and membrane fractions applied to high resolution 2D-gel electrophoresis. After renaturing transfer to nitrocellulose the blots were overlaid with [a-32P] GTP and the GTP-binding profiles visualized by autoradiography.