Optimization of a nonradioactive method for consistent and sensitive determination of activated K-ras protein.

Accurate measurement of activity of wild-type K-ras protein is important due to its tumor suppressor action in tissues such as lung. A published method by Taylor and co-workers uses plasmid-containing Escherichia coli cells to produce a glutathione-S-transferase/raf-1 ras binding domain (GST–RBD) fusion protein attached to glutathione beads to isolate activated ras protein. We systematically optimized the method before use on lung tissues. Changing the GST–RBD protein induction temperature from the original 37 to 30°C produced a consistently greater yield of fusion protein. To improve stability of the GST–RBD beads so as to perform large-scale experiments, 0.1% NaN3 was added. NaN3-treated beads retained full affinity for at least 24 days. Sensitivity was improved by using a polyvinylidene difluoride membrane rather than nitrocellulose for immunoblotting. We also compared our GST–RBD beads with two commercial assay kits and found that our beads had both superior sensitivity and reduced variability. In summary, our modification of the GST–RBD affinity method to recover activated K-ras greatly increased the yield of fusion protein, prolonged the useful life of GST–RBD beads to at least 24 days, and enhanced detection sensitivity.