Slid a lyzer

Purified cdHDAC7 was biotinylated to facilitate binding to the super streptavidin coated sensor (SSC sensor) for BLI analysis. The purified cdHDAC7 protein (0.5 mL of a 60 μM solution) was incubated with biotin-NHS-PEG4 (1.5 μL of a 20 mM solution, Thermo Fisher, cat #A39259) with a 1:1 protein-to-biotin ratio (0.3 μM of each) in a total of 0.5 mL to prevent over-biotinylation. The reaction was incubated on ice for 2 h before dialysis against HEPES buffer containing 10% glycerol using a slid-A-lyzer (10 kDa cut off, Thermo Fisher, cat #66380) to remove the biotin-NHS-PEG4 reagent. Dialyzed samples were fast frozen with liquid nitrogen in aliquots and stored at −80°C before use.

In preparation for BLI analysis, the biotinylated cdHDAC7 protein was thawed on ice and subjected to dialysis against HEPES buffer using a slid-A-lyzer (10 kDa cut off, Thermo Fisher, cat #66380) to remove glycerol. BLI analysis was carried out using an Octet Red 96 instrument (FortéBio). All kinetics experiments were performed at 25°C while stirred at 1000 rpm to create homogeneous mixtures. Biotinylated cdHDAC7 (0.1 mg/mL) was loaded onto the SSC sensor (FortéBio, cat #18–5057) until the binding signal reached 7 nm. Acetylated LGK(Ac) peptide was used at 20 nM, 1 μM, and 5 μM concentrations, whereas the unacetylated control peptide LGK was used only at a 5 μM concentration. The binding experiments were performed as follows: initial equilibration after loading was set to 1200 s and the baseline was then stabilized for 60 s. Peptide was then allowed to associate for 1200 s, followed by dissociation for 1200 s. FortéBio Octet Data Acquisition and Analysis Software version 7.0 was utilized to perform data acquisition and processing. Global fitting applied to acetylated LGK(Ac) peptide binding curves.