Improved membrane protein solubilization and clean-up for optimum two-dimensional electrophoresis utilizing GLUT-1 as a classic integral membrane protein

K. Devraj, R. Geguchadze, M. E. Klinger, W. M. Freeman, A. Mokashi, R. A. Hawkins, Ian Simpson

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)119-123
Number of pages5
JournalJournal of Neuroscience Methods
Volume184
Issue number1
DOIs
StatePublished - Oct 30 2009

Fingerprint

Electrophoresis
Membrane Proteins
Facilitative Glucose Transport Proteins
Isoelectric Point
Proteins
Trichloroacetic Acid
Neurosciences
Acetone
Microvessels
Hydrophobic and Hydrophilic Interactions
Detergents
Proteomics
Solubility
Amino Acid Sequence
Glycoproteins
Coloring Agents
Western Blotting
Gels
Membranes
Water

All Science Journal Classification (ASJC) codes

  • Neuroscience(all)

Cite this

Devraj, K. ; Geguchadze, R. ; Klinger, M. E. ; Freeman, W. M. ; Mokashi, A. ; Hawkins, R. A. ; Simpson, Ian. / Improved membrane protein solubilization and clean-up for optimum two-dimensional electrophoresis utilizing GLUT-1 as a classic integral membrane protein. In: Journal of Neuroscience Methods. 2009 ; Vol. 184, No. 1. pp. 119-123.
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Improved membrane protein solubilization and clean-up for optimum two-dimensional electrophoresis utilizing GLUT-1 as a classic integral membrane protein. / Devraj, K.; Geguchadze, R.; Klinger, M. E.; Freeman, W. M.; Mokashi, A.; Hawkins, R. A.; Simpson, Ian.

In: Journal of Neuroscience Methods, Vol. 184, No. 1, 30.10.2009, p. 119-123.

Research output: Contribution to journalArticle

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