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 degrees 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% NaN(3) was added. NaN(3)-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.
Calvert
Richard J
RJ
U.S. Food and Drug Administration, Office of Nutritional Products, Labeling, and Dietary Supplements, Division of Research and Applied Technology, College Park, MD 20740, USA. calvert@mail.ncifcrf.gov
Kammouni
Wafa
W
Kikawa
Keith D
KD
D000818:Animals; D002623:Chemistry Techniques, Analytical / Q000379:methods; D003101:Collodion / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000235:genetics; D005982:Glutathione Transferase / Q000737:chemistry; D008168:Lung / Q000737:chemistry; D051379:Mice; D011145:Polyvinyls / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism; D011933:Reagent Kits, Diagnostic; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012680:Sensitivity and Specificity; D018631:ras Proteins / Q000032:analysis*; D005465:Fluorocarbon Polymers
D003101:Collodion; D005982:Glutathione Transferase; D011145:Polyvinyls; D019908:Proto-Oncogene Proteins c-raf; D011933:Reagent Kits, Diagnostic; D011993:Recombinant Fusion Proteins; D018631:ras Proteins; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers