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Assay Method Information

Assay Name:  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
 

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Last update November 1, 2007
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