Fig. 1. Assay for active K-ras p21-GTP. A, lysates of 80% confluent E9 cells with 50, 100, or 300 µg of protein were incubated overnight at 4°C with 70 µg of GST-RBD attached to GSH-beads or with beads only. After pelleting and washing of the beads, the proteins were eluted, separated by electrophoresis, transferred to membranes, and probed with anti-K-ras p21 monoclonal antibody. No K-ras p21 was recovered with the beads-only control (Lane 1). The relative scan values were 250 ± 21 for 50 µg of protein, 646 ± 87 for 100 µg, and 1396 ± 221 for 300 µg. Total K-ras p21 from lysate with 300 µg of protein was recovered by immunoprecipitation (Last two lanes) and gave average scan values of 2264 ± 861. B, total K-ras p21 in nontransformed and malignant lung cells. Lysates (300 µg of protein) from five subconfluent cultures each of malignant E9 and nontransformed E10 cells were probed for total K-ras p21. This migrated slightly faster and was about 2-fold greater in amount in the E9 cells (average scan values 494 ± 68 versus 218 ± 31). C, amounts of active K-ras p21-GTP were determined in the same cultures as shown in B. An approximate 30-fold-higher level was observed in the E9 cells (average scan values, 1789 ± 208 versus 61 ± 8).