کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
10824933 | 1063312 | 2005 | 11 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Modified cell ELISA to determine the solubilization of cell surface proteins: Applications in GPI-anchored protein purification
دانلود مقاله + سفارش ترجمه
دانلود مقاله ISI انگلیسی
رایگان برای ایرانیان
کلمات کلیدی
TTBSB7-1TX-100DRMGPiPMSFICAM-1CMCmAbMonoclonal antibody - آنتی بادی مونوکلونالSolubility - انحلال پذیریTriton X-100 - تریتون X-100 Protein purification - خالصسازی یا تخلیص پروتئینcalf serum - سرم گوسالهdetergent-resistant membrane - غشای مقاوم در برابر مواد شویندهcritical micelle concentration - غلظت متیل مهمphenylmethylsulfonyl fluoride - فنیل متیل سولفونیل فلورایدglycosylphosphatidyl inositol - گلیکوزیل فسفاتیدیل آنزیم
موضوعات مرتبط
علوم زیستی و بیوفناوری
بیوشیمی، ژنتیک و زیست شناسی مولکولی
زیست شیمی
پیش نمایش صفحه اول مقاله
چکیده انگلیسی
A major step in purifying membrane bound proteins involves the solubilization of the protein of interest from the cell membranes. Glycosylphosphatidyl inositol (GPI)-anchored proteins pose a singular problem in this solubilization step since they are found in detergent-resistant membrane complexes and accordingly are insoluble in cold Triton X-100. In this study we have developed a modified cell ELISA that determines the solubility of these cell surface proteins under various solubilization conditions. Using this non-radioactive method we show that the combination of saponin/Triton X-100 at 4 °C solubilized GPI-anchored proteins more efficiently than Triton X-100 at 4 °C. The combination of saponin/Triton X-100 at 4 °C avoids the potential of activating proteases that occurs when using Triton X-100 at 37 °C. Furthermore, our method also shows the saponin/Triton X-100 solubilized GPI-anchored proteins equivalent to the more expensive octyl β-glucoside. This is a particularly important consideration in large-scale protein purification. This method obviates the need to use radioactivity, gel electrophoresis and immunoblotting procedures. The solubilization conditions determined by this modified ELISA are readily translated to the practical application of large-scale protein purification as demonstrated in the purification of two different recombinant GPI-anchored proteins, GPI-hB7-1 (CD80) and GPI-mICAM-1 (CD54).
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Journal of Biochemical and Biophysical Methods - Volume 64, Issue 2, 31 August 2005, Pages 99-109
Journal: Journal of Biochemical and Biophysical Methods - Volume 64, Issue 2, 31 August 2005, Pages 99-109
نویسندگان
Gary W. Bumgarner, Jamie C. Zampell, Shanmugam Nagarajan, Neil J. Poloso, Amanda S. Dorn, Martin J. D'Souza, Periasamy Selvaraj,