Figure 2
Solubility and fluorescence of MBP:GFP and MBP:GFP1-10. (A) Electrophoretic separation by 10% SDS-PAGE of 2, 4, and 8 µL of the soluble fraction of E. coli Stellar (crude extracts) containing MBP:GFP (lanes 3–5) or MBP:GFP1-10 (lanes 8–10). Lanes 1 and 6 and 2 and 7 were loaded with 10 µL of total cellular lysate taken before and after IPTG induction, respectively. The insoluble fraction represents the pellet produced after cell disruption and removal of unlysed cells, resuspended in 6 M urea. (B) Increasing amounts of the two purified proteins analyzed by 10% SDS-PAGE. The full uncropped gel pictures are shown in the Supplementary file, Fig. S1. (C) The fluorescence of 1, 5, 10, 25, 50, and 100 pmol of either MBP:GFP (green) or MBP:GFP1-10 (gray) was measured in 100 μL of Storage buffer using a Black & White Isoplate 96-well Wallac microtiter plate in the FLUOstar Omega reader. Fluorescence emitted by control samples missing the proteins has been subtracted. (D) The complementation reaction was conducted in 500 μL of Storage buffer with 500 pmol of MBP:GFP (green), 500 pmol of MBP-GFP1-10 (gray) or 500 pmol of MBP-GFP1-10 + 1 nmole of GFP11 peptide (red). The spectrum was recorded between 500 and 600 nm at room temperature with a Shimadzu RF-5301PC (Shimadzu, Japan) (slit = 5, PMT = 950).
