Determination of the dissociation binding constant (K_D) for the binding of E-selLs from HSPC-enriched lysates to E-Ig. The sensorgrams show binding of consecutive injections of E-Ig at 15 μL/min for 240 seconds each at concentrations of 3.9, 7.8, 15.6, 31.3, 62.5, 125, 250, 500, 1000, and 2000 nM that are each spaced by a 60-second buffer washing step (A) over captured E-selLs (CD34, CD44, CD43, and PSGL-1) expressed in KG1a cell lysate (150 mM NaCl) and (B) over captured CD34 from CD34^pos-BM lysate (50 mM NaCl). 563-mAb (10 800 RU for KG1a lysate or 6952 RU for CD34^pos-BM lysate) or MsIgG₁ isotype control (8320 RU for KG1a and 7500 RU for CD34^pos-BM lysate) was immobilized to capture CD34 (left upper panel). Hermes-3-mAb (9300 RU) or MsIgG_2a isotype control (7700 RU) was immobilized to capture CD44 (right upper panel). KPL-1-mAb (11 400 RU) or MsIgG₁ isotype control (8090 RU) was immobilized to capture PSGL-1 (right lower panel). A polyclonal CD43 Ab (15 800 RU) or Goat isotype control (14450 RU) was immobilized to capture CD43 (left lower panel). The sensorgrams presented are corrected for the bulk refractive index and nonspecific interactions using the isotype controls. K_D was determined by fitting the binding isotherm using a steady-state model and the RU_max values just prior to the start of the buffer injection where steady-state conditions were nearly met (inset) and k_off-apparent as described in Figure 3B. Data are representative of n = 3 independent experiments.