Incorporation of SeHAS into extrusion liposomes induces CB quenching. A. Extrusion liposomes loaded with CB were prepared, purified and stored at 4°C under argon as described in Methods. Liposomes were suspended in 0.25 ml 0.1 M NaCl, 51 mM sodium phosphate, 3.8 mM Na citrate, pH 7.4 (~50 μM PL) with 10 μg/ml anti-CB Ab and equilibrated at 30°C in cuvettes in the fluorometer. FL was monitored at 433 nm for 5 min to obtain a stable base-line (time-zero), and then SeHAS (15 μl; purified with no DDM or glycerol in the elution buffer) was added to 200 nM (black circles; mean ± SE, n = 3) or the same volume of mock-purified empty-vector membranes (white circles) or elution buffer (white squares) was added. Samples were mixed well and FL monitoring was continued. Decreasing FL (ongoing quenching) was observed after SeHAS addition, but not after addition of elution buffer or mock-purified material from empty-vector membranes. B. Purified SeHAS was added to extrusion liposomes as in A and after 30 min the suspension was centrifuged, the supernatant was saved, and the pellet was resuspended and centrifuged. The final pellet (lane 2) was resuspended to the same volume as the original supernatant (lane 3), trichloroacetic acid was added (to 10%; w/v) to both samples and precipitated protein was centrifuged, redissolved in Laemmli buffer and analyzed by SDS-PAGE and Western blotting with anti-SeHAS Ab. Lane 1 is a sample of purified SeHAS precipitated and redissolved in parallel as a control.
Medina et al. BMC Biochemistry 2012 13:2 doi:10.1186/1471-2091-13-2