Improved membrane protein solubilization and clean-up for optimum two-dimensional electrophoresis utilizing GLUT-1 as a classic integral membrane protein.
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 55kD 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-beta-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.
Devraj
K
K
Department of Neural & Behavioral Sciences, College of Medicine, Pennsylvania State University, United States.
Geguchadze
R
R
Klinger
M E
ME
Freeman
W M
WM
Mokashi
A
A
Hawkins
R A
RA
Simpson
I A
IA
D000818:Animals; D001622:Betaine / Q000031:analogs & derivatives / Q000494:pharmacology; D015153:Blotting, Western / Q000379:methods; D001921:Brain / Q000098:blood supply / Q000187:drug effects / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D051272:Glucose Transporter Type 1 / Q000737:chemistry / Q000378:metabolism*; D005960:Glucosides / Q000494:pharmacology; D008565:Membrane Proteins / Q000378:metabolism*; D055806:Microvessels / Q000187:drug effects / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D012995:Solubility
D003902:Detergents; D051272:Glucose Transporter Type 1; D005960:Glucosides; D008565:Membrane Proteins; C495679:Slc2a1 protein, rat; C506587:amidosulfobetaine-14; D001622:Betaine; C040358:dodecyl maltoside