کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
10533155 | 961851 | 2005 | 10 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Optimization of a nonradioactive method for consistent and sensitive determination of activated K-ras protein
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کلمات کلیدی
موضوعات مرتبط
مهندسی و علوم پایه
شیمی
شیمی آنالیزی یا شیمی تجزیه
پیش نمایش صفحه اول مقاله
چکیده انگلیسی
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.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Analytical Biochemistry - Volume 343, Issue 2, 15 August 2005, Pages 283-292
Journal: Analytical Biochemistry - Volume 343, Issue 2, 15 August 2005, Pages 283-292
نویسندگان
Richard J. Calvert, Wafa Kammouni, Keith D. Kikawa,