or simultaneously fitting apparent NSB helps identify low-affinity specific binding. A. Data sets with maximum S/N = 36 (SD = 0.0041 nM), R1T = 0.13 nM, α = 0, and various values of [3H]EBmax were fitted with one site modeltotal and two sites modeltotal. The y-axis shows p values from comparisons. Total binding data only (○) or total binding along with apparent NSB (●) were fit. Dashed and solid lines connect averages of log(p) values. At [3H]EBmax = 100 nM and total binding only, the CIs included true values (Kd1, 11.9-14.0 pM, mean = 12.9 pM, Kd2, 3.4-12.2 nM, mean = 6.5 nM, R1T, 0.128-0.131 nM, mean = 0.129 nM; R2T, 0.014-0.021 nM, mean = 0.018 nM) (n = 5 for each CI). With [3H]EBmax = 22 nM and explicitly fitting apparent NSB, CIs included true values (Kd1, 10.6-13.2 pM, mean = 11.8 pM; Kd2, 3.9-17.4 nM, mean = 8.2 nM; R1T, 0.126-0.131 nM, mean = 0.128 nM; R2T, 0.020-0.023 nM, mean = 0.021 nM) (n = 5 for each CI). B, C. With zero NSB and highly precise data (SD = 1 × 10-4 nM), simultaneously fitting total binding data and apparent NSB helps identify low-affinity specific binding. Total binding data in B (40 points) were generated with R1T = 0.13 nM and [3H]EBmax = 2.37 nM. The one site modeltotal and two sites modeltotal appear to fit total binding data equally well up to [3H]EBmax. The two sites modeltotal, however, fits apparent NSB values (●) in C significantly better than does the one site modeltotal, leading to p = 2 × 10-33 comparing models with simultaneous fitting. One site modeltotal: Kd = 0.014 nM, RT = 0.13 nM, α = 5.9 × 10-4; two sites modeltotal: Kd1 = 0.013 nM, Kd2 = 10.5 nM, R1T = 0.013 nM, R2T = 0.022 nM, α = 1.0 × 10-7.