Characterization of the specificity and affinity of TA8 monobody.
(A) Evaluating the specificity of TA8 by probing 150 human SH3 domains. Four arrays (Panomics) were probed with TA8 monobody and scanned simultaneously with a Storm860 imager (GE Healthcare Life Sciences). The signal intensities of the spots were quantified with the ImageQuant (GE Healthcare Life Sciences). Units in the histograms represent relative comparisons, with error bars corresponding to the standard deviations of the signal intensities of duplicate spots. (B) Measurement of the dissociation constant (KD) of TA8 binding to the Lyn SH3 domain by isothermal titration calorimetry (ITC). The thermogram (top panel) and the plotted titration curve (bottom panel) were acquired with an ITC200 (GE Healthcare Life Sciences). A representative experiment is shown here and the observed N, ΔH, and ΔS values are 1.32, -7.6 x 103 cal/mol, and -1.38 cal/mol/deg, respectively. The ITC experiment was performed five times to generate an average KD value of 5.5 ± 0.8 μM.
