FIGURE 4.

Substrate cut-up experiment to check for impurities or an incorrect radioligand stock concentration. Membrane vesicles (3.75 μL, corresponding to ∼0.4 mg/ml of total membrane protein) were added to 450 μL mixture, supplemented with 0.25% decylPEG. The mixture (380 μL) was pipetted into the upper chamber of the flow dialysis system. The curved lines represent nonlinear least-squares curve fits for K_D, E_tot, and β, for the titration experiment with uncut (solid), and 2× cut-up (dashed) 20 μM ³H-mannitol. The straight lines represents the β-values for uncut (solid), and 2× cut-up (dashed) 20 μM ³H-mannitol stocks.