| Assay Method Information | |
| | Isothermal Titration Calorimetry Experiments |
| Description: | ITC experiments were performed at 25° C. on a MicroCal iTC200 system (Malvern, PA, USA). hRpn13 Pru, XL5, XL5 derivative, or RA190 were prepared in buffer C. One aliquot of 0.5 μL followed by 17 or 18 aliquots of 2.1 μL of 200 μM hRpn13 Pru was injected at 750 r.p.m. into a calorimeter cell (volume 200.7 ml) that contained 20 μM XL5, XL5 derivative, or RA190. Blank experiments were performed by replacing XL5, XL5 derivative, or RA190 with buffer in the cell and the resulting data subtracted from the experimental data during analyses. The integrated interaction heat values were normalized as a function of protein concentration and the data were fit with MicroCal Origin 7.0-based software implementing the One Set of Sites model to yield binding affinity Ka (1/Kd), stoichiometry, and other thermodynamic parameters. |
| Affinity data for this assay | |
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