SUMO and SIM were buffer-exchanged
using a Superdex75Increase 10/300 or Superdex Peptide 10/300 gel filtration
column (Cytiva Life Sciences) into 20 mM HEPES, 150 mM KCl, 1 mM MgCl2, 1 mM DTT, 1 mM EGTA (pH 7.0 or 8.0) or 20 mM MES, 150 mM
KCl, 1 mM MgCl2, 1 mM DTT, and 1 mM EGTA (pH 6.5). ITC
experiments were performed using a Microcal ITC200 isothermal calorimeter
using 50–100 μM SIM peptide in the cell and 750–1.2
mM SUMO in the syringe. Baseline corrections and integrated heats
were performed using NITPIC.68 (link) Affinity
was determined using a 1:1 binding model in SEDPHAT.69 (link) Reported error is the standard deviation from the Monte
Carlo simulation based on the experimental noise.
using a Superdex75Increase 10/300 or Superdex Peptide 10/300 gel filtration
column (Cytiva Life Sciences) into 20 mM HEPES, 150 mM KCl, 1 mM MgCl2, 1 mM DTT, 1 mM EGTA (pH 7.0 or 8.0) or 20 mM MES, 150 mM
KCl, 1 mM MgCl2, 1 mM DTT, and 1 mM EGTA (pH 6.5). ITC
experiments were performed using a Microcal ITC200 isothermal calorimeter
using 50–100 μM SIM peptide in the cell and 750–1.2
mM SUMO in the syringe. Baseline corrections and integrated heats
were performed using NITPIC.68 (link) Affinity
was determined using a 1:1 binding model in SEDPHAT.69 (link) Reported error is the standard deviation from the Monte
Carlo simulation based on the experimental noise.
