کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
4335698 1295174 2009 5 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Improved membrane protein solubilization and clean-up for optimum two-dimensional electrophoresis utilizing GLUT-1 as a classic integral membrane protein
موضوعات مرتبط
علوم زیستی و بیوفناوری علم عصب شناسی علوم اعصاب (عمومی)
پیش نمایش صفحه اول مقاله
Improved membrane protein solubilization and clean-up for optimum two-dimensional electrophoresis utilizing GLUT-1 as a classic integral membrane protein
چکیده انگلیسی
Two-dimensional (2-D) electrophoresis remains a primary resolving tool for proteomic analyses. The final number of proteins resolved by 2-D electrophoresis depends on their respective solubility, size, charge, and isoelectric point. While water-soluble cytosolic proteins have often been well represented in 2-D maps, the same is not true with membrane proteins. Highly hydrophobic in nature, membrane proteins are poorly resolved in 2-D gels due to problems associated primarily with sample preparation. This is of especial concern in neuroscience studies where many proteins of interest are membrane bound. In the current work, we present a substantially improved sample preparation protocol for membrane proteins utilizing the GLUT-1 glucose transporter from brain microvessels as an example of a typical membrane protein. GLUT-1 (SLC2A1; solute carrier family 2 (facilitated glucose transporter), member 1) is a 55 kD glycoprotein that contains 12 membrane-spanning alpha helices that impart the protein its characteristic hydrophobicity. GLUT-1 based on its amino acid sequence has a theoretical isoelectric point (pI) of 8.94. Using a combination of the non-ionic detergents, n-dodecyl-β-maltoside (DDM) and amido sulphobetaine-14 (ASB-14) for sample solubilization, and a modification of the Bio-Rad 2-D clean-up protocol involving trichloroacetic acid (TCA)/acetone, we obtained near complete solubilization of GLUT-1 and greater than 90% recovery of this membrane protein in 1-D and 2-D Western blots. The total number of proteins resolved also increased dramatically in Deep Purple™ total protein stains using our improved protocol.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Journal of Neuroscience Methods - Volume 184, Issue 1, 30 October 2009, Pages 119-123
نویسندگان
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