Biochemical tests demonstrate presence of both binary and tertiary complexes with β2 abundance in complexes more likely than β3a at equimolar initial expression. (A) 3-step affinity purification scheme to fractionate coexpressed Slo1, β2, and β3 into binary (Slo1–β2 or Slo1–β3) and ternary (Slo1–β2–β3) complexes. (B) Dual-color FSEC chromatograms of three key fractions (as indicated in A, via arrows) monitored via eGFP (green trace) and mCherry (red trace) fluorescence. The traces are normalized, considering the intensity differences of the two fluorescence channels, and are molar proportionate. Dashed lines indicate the peak retention of Slo-TS determined in separate experiments. The black trace (right chromatogram) indicates a Gaussian fit to the primary Slo1–β3 species, separating them from lower-order (disassembled) protein complexes. Arrows from last row of A highlight origin of material for each chromatogram in B. (C) Molar ratio of β2RF-β3Gρ in cellular lysates, total Slo1 isolates, and in the triply purified ternary complex in the two transfection ratios tested (TR1-1:1:1 and TR2-1:0.2:1.8; as described in the main text). (D) Fraction of total Slo1–β complexes that are binary (bin: single type of β subunit, β2 or β3) or ternary (ter: β2 and β3) in the two cases of transfection. In C and D, error bars indicate SD of 3 independent experiments.