Microfuge tube polypropylene

HTT and chaperone proteins were thawed on ice and transferred to centrifugation tubes (Microfuge Tube Polypropylene, Beckman Coulter). Ultracentrifugation (Optima MAX Ultracentrifuge, Beckman) was carried out at 50000 rpm for 1 h at 4 °C. Protein supernatants (around 95% of the initial volume) were transferred to low-binding tubes, and molar concentration was determined via Bradford assay. Samples containing 5–10 μM total protein and 0.5–1 mM SDA were incubated for 30 min at room temperature. Cross-linking was stopped by adding 1 M Tris-base, samples were then put on ice and photoactivated for 30 min under UV light. SDA cross-linked proteins were loaded on SDS-PAGE and subjected to in-gel digestion. In brief, gel bands were reduced with 5 mM DTT at 56 °C for 30 min and alkylated with 40 mM chloroacetamide at room temperature for 30 min in the dark. Protein digestion was carried out using elastase at an enzyme-to-protein ratio of 1:20 (w/w) at 37 °C overnight.

Prior to all biochemical assays, proteins were thawed on ice and transferred to centrifugation tubes (Microfuge Tube Polypropylene, Beckman Coulter). Ultracentrifugation (Optima MAX Ultracentrifuge, Beckman) was carried out at 40.000 rpm for 40 min at 4 °C. Protein supernatants (around 95% of the initial volume) were transferred to low-binding tubes (Sorenson) and molar concentration was determined via Bradford assay.