| Assay Method Information | |
| | Isothermal Titration Calorimetry (ITC) |
| Description: | Synthetic peptides were purchased from Genicbio Ltd. Peptides were dissolved in 25 mM HEPES, pH 7.4, 100 mM NaCl (ITC buffer) to obtain 4 mM stock solutions, which were flash-frozen and stored at −80 °C. The isothermal titration experiments were performed using either a MicrocalTm ITC200 or an autoITC200 (GE Healthcare). The syringe was loaded with titrant peptide at concentrations of 2 or 4 mM (DsbB P2 peptide) or 200 μM (EF-Tu switch I peptide). The sample cell was loaded with oxidized AbDsbA with concentrations of 100 μM (for titrations with DsbB P2 peptide) or oxidized/reduced/mixed AbDsbA of 10 μM (for titrations with EF-Tu switch I peptide) in ITC buffer. To assess whether EF-Tu switch I peptide competes for the DsbB P2 peptide binding site, 100 μM oxidized AbDsbA was incubated with 125 μM EF-Tu switch I peptide for 60 min in ITC buffer before titration with DsbB P2 peptide at 2 mM concentration as described above. Titrations were executed at 25 °C using 19 titrations of 2 μl each separated by 180 s and at a constant stirring speed of 1000 rpm. A pre-injection of 0.5 μl was performed to limit slow diffusion of titrant into the cell before the titration, and the corresponding data point was removed from subsequent analysis. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |