Accurate measurement of activity of wild-type K-ras protein is important because of its tumor suppressor action in tissues such as lung. A novel non-radioactive method to detect activated (GTP-bound) K-ras protein has been described previously (Taylor et al., 1996, 2000). The method uses a glutathione-S-transferase / raf-1 ras binding domain (GST-RBD) fusion protein attached to glutathione beads to separate activated from inactivated ras protein based on the affinity of the activated protein for raf. The recovered protein is subjected to gel electrophoresis, blotted, and visualized by detection with specific ras antibodies. Prior to using this method for our studies on lung tissue, we desired to optimize the shelf-life of the GST-RBD beads we prepared, and increase the yield of the GST-RBD fusion protein. We also compared our GST-RBD beads with two commercial kits used to detect activated ras. We first altered the incubation temperature used in production of the fusion protein, changing it from 37° C, as originally described, to 30° C. This resulted in a consistently greater yield of fusion protein, and eliminated the problem of occasional ineffective lots of GST-RBD beads. To improve stability of the GST-RBD beads in order to perform large scale experiments, we added NaN3 at 0.1% to the GST-RBD beads. This allowed the beads to retain full affinity for at least 14 days. We also compared our GST-RBD beads with two commercial assay kits (Upstate and Pierce). In these comparisons, our beads had superior sensitivity and reduced variability compared to the commercial kits. In summary, our modification of the GST-RBD affinity method to recover activated K-ras greatly increases the yield of fusion protein and allows use of a single batch of GST-RBD beads for at least 14 days. These improvements allow much easier use of the method in large or longer-term experiments.
[Proc Amer Assoc Cancer Res, Volume 46, 2005]