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. Author manuscript; available in PMC: 2009 Jul 6.
Published in final edited form as: Biochemistry. 2008 Aug 19;47(37):9803–9810. doi: 10.1021/bi800659x

FIGURE 2.

FIGURE 2

Overexpression of GlyBP in Sf9 insect cells and binding to 2-aminostrychnine (2-AS) affinity matrix. All panels are Western immunoblots except where otherwise noted. (A) Subcellular fractionation of GlyBP. Lane 1 - total extract of Sf9 cells 4 days post-infection, Lane 2 - cytosolic fraction, Lane 3 - membrane-associated pellet. (B) Cytosolic fraction failed to bind to 2-AS resin. Lane1: total cytosolic protein, Lane 2: protein bound to 2-AS resin, Lane 3: unbound protein. (C) Membrane fraction bound to 2-AS resin. Lane 1: total membrane fraction, Lane 2: protein bound to 2-AS resin, Lane 3: unbound protein. (D) Elution of membrane-associated GlyBP from 2-AS resin. Lane 1: GlyBP bound to 2-AS resin, Lane 2: GlyBP bound to 2-AS resin after elution with 1.5 mM 2-AS; Lane 3: protein eluate after incubation with 1.5 mM 2-AS. (E) Partitioning of purified GlyBP after detergent dialysis (Lane 1) and ultracentrifugation (Lanes 2 and 3). Lane 2: aqueous form of GlyBP, Lane 3: vesicular form of GlyBP. (F) Silver staining of purified aqueous (lane 1) and vesicular (lane 2) forms of GlyBP.