The popularization of studies in membrane protein lipid phase coexistence has prompted the development of new techniques to construct and study biomimetic systems with cholesterol-rich lipid microdomains. Here, microspheresupported biomembranes with integrated alpha-helical peptides, referred to as proteolipobeads (PLBs), were used to model peptide/protein partitioning within DOPC/DPPC/cholesterol phase-separated membranes. Due to the appearance of compositional heterogeneity and impurities in the formation of model PLB assemblies, fluorescence-activated cell sorting (FACS) was used to characterize and sort PLB populations on the basis of disordered phase (L-d) content. In addition, spectral imaging was used to assess the partitioning of FITC-labeled alpha helical peptide between fluorescently labeled Ld phase and unlabeled ordered phase (L-o) phase lipid microdomains. The apparent peptide partition coefficient, K-p,K-app was measured to 0.89 +/- 0.06, indicating a slight preference of the peptide for the L-o phase. A biomimetic motif of the L-o phase concentration enhancement of the biotinyl-peptide ligand display in proteolipobeads was also observed. Finally, peptide mobility was measured by FRAP separately in each lipid phase, yielding diffusivities of 0.036 +/- 0.005 and 0.014 +/- 0.003 mu m2/s in the L-d and L-o phases, respectively.