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. 2013 Aug 27;8(8):e71512. doi: 10.1371/journal.pone.0071512

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© 2013 Guellouz et al

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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(A) ITC calorimetric titrations. Concentrations values are expressed in monomer concentrations. (▪): Tiration of GFP (35 μM) with bGFP-A (350 μM). (□): The bGFP-A binding specificity was tested by titration with NCS-wt (bGFP-A 30 μM, NCS-wt 350 μM). (B) The GFP-binding specificity was evaluated by ITC analysis of injection of bA3–1 (360 μM) in a solution of GFP (30 μM). (C) Size Exclusion Chromatography (Superdex 75 10/300) of the selected bGFP-A and GFP. (▾): SEC Elution profile of a mixture of GFP (2.25 nmol) and the binder bGFP-A (6.75 nmol). (∇): elution profile of the bGFP-A alone (2.25 nmol). (): elution profile of the GFP alone (6.75 nmol). (D) Affinity determination of selected bGFP-A using SPR. Different concentrations of bGFP-A (71,3; 118; 142,6; 237,6; 713; 1426 nM) were applied to flow cell with immobilized biotinylated EGFP for 120 s followed by washing buffer flow. The sensorgrams were corrected for non-specific binding by subtraction of a channel without EGFP bound (grey curve). The fits of k_on and k_off rates are indicated by black dashed line. K_d values were computed using k_off = 1.7×10⁻⁴ s⁻¹ for all concentrations and k_on = 4.3, 4, 2.2, 2.6, 2, 2×10⁴M⁻¹ s⁻¹ for the increasing concentrations respectively.

Inline graphic — (A) ITC calorimetric titrations. Concentrations values are expressed in monomer concentrations. (▪): Tiration of GFP (35 μM) with bGFP-A (350 μM). (□): The bGFP-A binding specificity was tested by titration with NCS-wt (bGFP-A 30 μM, NCS-wt 350 μM). (B) The GFP-binding specificity was evaluated by ITC analysis of injection of bA3–1 (360 μM) in a solution of GFP (30 μM). (C) Size Exclusion Chromatography (Superdex 75 10/300) of the selected bGFP-A and GFP. (▾): SEC Elution profile of a mixture of GFP (2.25 nmol) and the binder bGFP-A (6.75 nmol). (∇): elution profile of the bGFP-A alone (2.25 nmol). (): elution profile of the GFP alone (6.75 nmol). (D) Affinity determination of selected bGFP-A using SPR. Different concentrations of bGFP-A (71,3; 118; 142,6; 237,6; 713; 1426 nM) were applied to flow cell with immobilized biotinylated EGFP for 120 s followed by washing buffer flow. The sensorgrams were corrected for non-specific binding by subtraction of a channel without EGFP bound (grey curve). The fits of k_on and k_off rates are indicated by black dashed line. K_d values were computed using k_off = 1.7×10⁻⁴ s⁻¹ for all concentrations and k_on = 4.3, 4, 2.2, 2.6, 2, 2×10⁴M⁻¹ s⁻¹ for the increasing concentrations respectively.