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Isolation of hGLUT9b by size-exclusion chromatography with Western blot, silver stain, and single particle analysis.

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posted on 06.10.2014 by Benjamin Clémençon, Benjamin P. Lüscher, Michael Fine, Marc U. Baumann, Daniel V. Surbek, Olivier Bonny, Matthias A. Hediger

(A) Superose 6 gel filtration chromatography shows two peaks. The first peak corresponds to the dead volume of the gel filtration where large molecular weight proteins or aggregates do not interact with the beads and quickly pass through the column. The second peak corresponds principally to the monomeric (sharp peak, right side) and oligomeric (shoulder, left side) states. Western blot representation of fractions 30 to 43 after gel filtration showing clearly the separation of the second peak into hGLUT9b oligomers and monomer. (B) Silver-staining of hGLUT9b monomers obtained from fraction 35. (C) Micrograph of hGLUT9 particles obtained from negatively-stained TEM. Scale bar is 50 nm. Gallery representation of the 9 class-averages obtained from 1439 particles. Each representation is scaled to a 26 nm square.