Capillary and rotating-tube isoelectric focusing of a transmembrane protein, the human red cell glucose transporter

Englund, Ann-Kristin; Lundahl, Per; Elenbring, K.; Ericson, Christer

doi:10.1016/0021-9673(95)00197-u

Cited by 12 publications

(5 citation statements)

References 34 publications

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“…These results demonstrate that GLUT-1 positive spots increased substantially when the partial clean-up protocol was followed. The spots in the pI range of 8–9 where most of the GLUT-1 has been shown to be concentrated (Englund et al, 1995) were present in much higher intensity when partial clean-up was utilized.…”

Section: Resultsmentioning

confidence: 99%

“…Apart from these, we also report GLUT-1 positive spots at the acidic end of the scale around 3.0 pI and also at 6.0 pI (Fig 2b), potentially resulting from the excessive sialic acid glycosylations of GLUT-1. It is also possible that the diffuse spots at these pI could be a result of oligomerization of GLUT-1 monomers as suggested by Englund and Lundahl, (1995) for spots around 6.0 pI. We are currently assessing the various covalent modifications by using deglycosylation and dephosphorylation in combination with chymotrypsin digestion and mass spectrometry (MS).…”

Section: Resultsmentioning

confidence: 99%

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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

Geguchadze

Klinger

et al. 2009

Journal of Neuroscience Methods

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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 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 sulfobetaine-14 (ASB-14) for sample solubilzation, and a modification of the Biorad 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.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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

Devraj

Geguchadze

Klinger

et al. 2009

Journal of Neuroscience Methods

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“…The inner and outer surfaces rotate together at about 1 rpm as a solid body, ostensibly to help distribute the cooling effect of the finger. The associated literature seems to suggest that the role of the slow rotation is in some way related to the abatement of natural convection engendered in Hjertén's rotating tube apparatus [10,11], but this connection has never been rigorously established. Aside from the engineering aspects of the Rotofor design, this device has a number of attractive features, not least of which is its affordable price.…”

Section: General Aspectsmentioning

confidence: 98%

Preparative free‐flow electrofocusing in a vortex‐stabilized annulus

Ivory¹

2004

Electrophoresis

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This paper describes the development of an annular chamber designed for preparative free-flow electrophoresis which can operate at voltages up to 20 kV, electric fields up to 65 kV/m, and power densities as high as 10 MW/m(3). This apparatus uses counter-rotating fluid vortices to stabilize the aqueous medium in the annulus against natural convection while improving heat and mass transfer over vortex-free Couette flow. The vortices are generated by rotating the inner surface of the annulus and, if the rotor and stator have complementary shapes, dispersion induced by electrokinetic and electrohydrodynamic flows can be mitigated even at the highest operating voltages. Following a brief overview of contemporary annular free-flow instrumentation, the theoretical principles for momentum and heat transport in the vortices are discussed in some detail and then the results of several electrofocusing experiments are provided to illustrate the resolving power of this instrument.

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“…Thus, identification of rhinoceros horns has become an urgent task for convicting and prosecuting people suspected of selling and smuggling horns from this endangered animal. Owing to the merit of on-column detection and the capability of attaining high-resolution separation comparable to the conventional isoelectric focusing, capillary isoelectric focusing (CIEF) method has recently attracted considerable attention as a fast analytical tool for profiling of biomolecules [2][3]. The use of narrow diameter (25-50 gm ID) capillary with CIEF further offers the advantage of minute sample requirement (ng).…”

Section: Introductionmentioning

confidence: 99%

Analysis of α-keratins in the horns of rhinoceros and buffalo by non-native capillary isoelectric focusing

Huang¹,

Yu²

1997

Chromatographia

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SummaryA capillary isoelectric focusing (CIEF) method under denaturing condition has been developed for separation of a-keratins, highly cross-linked biomacromolecules, in horns of rhinoceros and buffalo. The a-keratins were denatured in 8 M urea and separated in the presence of ampholytes with applicable pH ranges of 3-7 or 3-10. In the preliminary results, it was demonstrated that the o~-keratins in the horns of buffalo or rhinoceros might have their own unique isoelectric focusing profiles. The analyses of more samples from different rhinoceros and buffalo are presently underway, with the final goal to establish a fast and convenient method for identification of the source (buffalo or rhinoceros) of an unknown powdered sample.

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Capillary and rotating-tube isoelectric focusing of a transmembrane protein, the human red cell glucose transporter

Cited by 12 publications

References 34 publications

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

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

Preparative free‐flow electrofocusing in a vortex‐stabilized annulus

Analysis of α-keratins in the horns of rhinoceros and buffalo by non-native capillary isoelectric focusing

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