Prior to initiation of the binding experiment, protein samples containing Fe(II) were spotted on pH paper to ensure that the overall sample pH had not been altered as a result of spiking the sample with iron prepared in HCl.
If you are hoping to see a conformational change when you add a component like a small ligand or other binding partner to the protein sample, be careful to maintain exactly matching buffer.
To test for O2 binding, a protein sample was flushed with argon for 20 min and subsequently transferred to an argon-saturated cuvette.
For the Ca2+-binding experiments, protein samples (50, 50, 450 and 500 µM of Lig A3, Lig A4, Lig A9 and Lig A10) were titrated against 5, 5, 10 and 10 mM CaCl2 respectively, all in the same buffer (25 mM HEPES, pH 7.0, 50 mM KCl) at 30°C.
For differential scanning calorimetry (DSC) measurements and for analyzing the pH-dependence of binding to Her2, aliquots of the protein samples were also dialyzed in PBS, pH 6.0.
The binding assay for insect protein samples was conducted as described [ 5].
Likewise, non-specific Ab binding to tissue proteins due to hydrophobic interaction between proteins or ionic and electrostatic interactions does not take place in routinely fixed cell and tissue samples.
In order to check the putative binding capacity of MC to RubisCO the protein sample was preincubated at 30°C for 30 min. Finally the sample was split into two vials and microcystin-LR was added to one of the samples in a final concentration of 10 mg/L−10
Our initial ITC measurements showed variable binding constants, indicating that the purified protein samples might contain different amounts of cAMP (verified by our partial apo structure).
