Imagequant las 4000

Fifty μM of each expanded TrxHttex1-polyQ with and without equimolar SERF1a were incubated at 37 °C with continuous shaking at 200 rpm. Samples (4 μl) were collected at different timepoints and dotted onto nitrocellulose membrane with two replicates. Membranes were blocked with 5% skim milk in TBS buffer and incubated with 1C2 (1:3,000; Millipore), MW7 (1:3,000; DSHB), A11 (1:1,000; Invitrogen), or OC (1:10,000; Millipore) antibodies separately at 4 °C overnight. The secondary antibody was horseradish peroxidase conjugated anti-mouse or anti-rabbit IgG (1:5,000; Millipore, Billerica, MA, USA), and it was probed for 2 h at room temperature. The membranes were developed with electrochemiluminescence reagent (Millipore) and the signals were detected by ImageQuant LAS 4000.

Western blotting was performed using Hoefer Scientific Instrument apparatus and BioRad Western Blot electrophoresis system. 20-30 μg of protein was resolved using sodium dodecyl sulfate – polyacrylamide gel electrophoresis. Proteins were transferred onto a nitrocellulose membrane (Millipore-Immobilon), which was then blocked in 5% milk-TBST for 1 hour at room temperature (RT) and incubated overnight at 4°C with the relevant primary antibody (see Key Resources Table). The following day, 3 washes in TBS-T were performed before incubation with the appropriate HRP-conjugated secondary antibody (anti-mouse, GE Healthcare NA931; antirabbit, GE Healthcare NA934; anti-chicken, Sigma-Aldrich AP194P) for 1 hour at RT at 1/5000. After 3 additional washes in TBS-T, proteins of interest were detected with Pierce- ECL western blot substrate (Thermo Scientific) or Luminata Crescendo Western HRP substrate (EMD-Millipore) and images acquired on the Imagequant LAS 4000.

Bacterial strains were grown in THY medium until OD₅₈₀ 0.6. Cells were then pelleted by centrifugation for 20 min at 3,000 rpm, washed twice in PBS, before the cell pellet was sonicated on ice using three 30 s bursts at 200–300 W. Cell lysates were then centrifuged to pellet all cellular debris, and protein concentrations measured in the supernatant using the BioRad DC^TMProtein Assay. 6.25 μl of LDS Sample Buffer (4X) and 2.5 μl of Sample Reducing Agent (10X) was added to a known lysate concentration, making up to total volume of 25 μl using water, denatured at 70°C for 10 min prior to analyses using SDS-PAGE and Western blotting using pre-prepared 4–12% Bis-Tris Plus SDS-PAGE gels. Electrophoresis was carried out using 1X MES running buffer at a constant 200 V for 30 min, and the protein gels were transferred to PVDF membrane using a dry transfer iBlot system. Membranes were blocked in 5% milk in 1X TBS-Tween (TBS-T) for 2 h at room temperature, then incubated in primary antibody (1 h at room temperature or overnight at 4°C). Membranes were washed 3 times in TBS-T for 45 min and then incubated in secondary antibody for 1 h at room temperature. After washing again 3 times, membranes were developed using Luminata^TMCrescendo HRP substrate (Merck Millipore) and imaged using the ImageQuant LAS 4000.

Western blotting was performed using Hoefer Scientific Instrument apparatus and Bio-Rad western blot electrophoresis system. 20–30 μg of protein was resolved using a sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein was transferred onto nitrocellulose membrane (Millipore-Immobilon) and blocked for 1 hr at RT using 5% milk-TBST. The membrane was incubated with primary antibodies overnight at 4°C. The following day, the membrane was washed three times with TBST, followed by incubation with the appropriate HRP conjugated secondary antibody. Subsequently, membranes were washed three times with TBS-T before detection of proteins of interest with Pierce-ECL western blot substrate (Thermo Scientific) or Luminata Crescendo Western HRP substrate (EMD-Millipore) on the Imagequant LAS 4000.