The MSD assay plate was coated with 5ug/ml of the N-terminally binding HTT antibody 2B7 in coating buffer overnight. The next day, the plate was washed 3× in wash buffer Tween 20 in DBPS), blocked for 1 h at RT shaking at 350 rpm Probumin in wash buffer) and subsequently washed 3× again. The MSD plate was then incubated with the protein sample derived from the various cells for 1 h at RT shaking at 350 rpm.
Subsequently, the suspension was sonicated using a Bioruptor under the same conditions as described before. The sonicated suspension is centrifuged at 100,000×for 30 min at 4 °C again. The resulting supernatant reflects the insoluble fraction. The protein content in each fraction was quantified with bicinchoninic acid assay. Equal concentrations were applied, and loading buffer and DTT was added. The samples were incubated at 55 °C for 30 min.
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