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. 2015 Jan 10;16(1):11–23. doi: 10.1007/s10969-014-9192-z

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© The Author(s) 2015

Open AccessThis article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.

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Fig. 3 — Large-scale preparation of FLAG-tagged AdipoR1Δ88 and AdipoR2Δ99 expressed in High Five cells. In the chromatograms (a, c–h, j, k, m, n), the absorbances at 280 and 254 nm, and the NaCl concentration are shown in blue, red, and light green, respectively. a Gel filtration chromatogram of FLAG-tagged AdipoR1Δ88 expressed in High Five insect cells. The proteins were purified by FLAG-affinity chromatography, and then chromatographed on a HiLoad 16/600 Superdex 200 column. The main peak elution volume is labeled in black (58.8 ml). b SDS-PAGE analysis of the gel-filtration chromatographic fractions containing the FLAG-tagged AdipoR1Δ88. Fractions indicated by the orange and green lines along the horizontal axis in a were analyzed by SDS-PAGE. Lane M, molecular-weight markers (kDa). c Anion-exchange chromatogram of the FLAG-tagged AdipoR1Δ88, with isocratic elution by 100 mM NaCl in buffer A, and subsequently with gradient elution by 100–1,000 mM NaCl in buffer A. d, e Gel filtration chromatograms of the peak 1 (d) and peak 2 (e) fractions, indicated by the orange and green lines in c, respectively. f Anion-exchange chromatogram of the FLAG-tagged AdipoR1Δ88, with isocratic elution by 200 mM NaCl in buffer A, and subsequently with gradient elution by 200–1,000 mM NaCl in buffer A. g, h Gel filtration chromatograms of the flow-through (g) and adsorbed (h) fractions, indicated by the orange and green lines in f, respectively. i SDS-PAGE analysis of the FLAG-tagged AdipoR1Δ88. Lane M, molecular-weight markers (kDa); lane 1, the membrane fraction; lane 2, DDM-solubilized membrane proteins in the supernatant after ultracentrifugation; lane 3, the flow-through fraction from FLAG-affinity chromatography; lane 4, the eluate from FLAG-affinity chromatography; lane 5, the flow-through fraction from anion-exchange chromatography (f); lanes 6–9, the peak fractions of the FLAG-tagged AdipoR1Δ88 from gel filtration chromatography (g). j Anion-exchange chromatogram of AdipoR2Δ99, with isocratic elution by 200 mM NaCl in buffer A and subsequently with gradient elution by 200–1,000 mM NaCl in buffer A. k Gel filtration chromatogram of the flow-through fractions indicated by the orange line in j. l SDS-PAGE analysis of AdipoR2Δ99. Lane M, molecular-weight markers (kDa); lane 1, the membrane fraction; lane 2, DDM-solubilized membrane proteins in the ultracentrifugation supernatant; lane 3, the flow-through fraction from FLAG-affinity chromatography; lane 4, the eluate from FLAG-affinity chromatography; lane 5, the flow-through fraction from anion-exchange chromatography after the TEV protease digestion; lane 6, the flow-through fraction from TALON chromatography; lanes 7–11, the peak fractions of AdipoR2Δ99 from gel filtration chromatography. m, n Gel filtration chromatographic analysis of the purified FLAG-tagged AdipoR1Δ88 (m) and the purified AdipoR2Δ99 (n)