SDS PAGE showing chaperone binding to alpha-synuclein amyloid fibres for different conditions (+/- Apg2, WT or truncated mutant of DNAJB1, different dilutions of Apg2).

MONISTROL, Jim and Saibil, Helen

Birkbeck Schools and Research Centres > School of Science > Biological Sciences

SDS PAGE, alpha-synuclein, HSP70, DNAJB1, Apg2

Three independent repeats were performed for each condition.

All chaperone aliquots (Hsc70, DNAJB1, Apg2) were centrifuged at 17,000 g for 30 minutes at 4°C. αSyn amyloid fibres were sonicated for 15 minutes at high frequency using a CPX 2800 Bransonic Ultrasonic bath (Branson). αSyn amyloid fibres (40 µM, monomer concentration) were incubated with Hsc70 (8 µM), DNAJB1 or ΔJ-DNAJB1 (4 µM), and Apg2 (different concentrations as indicated) in the disaggregation buffer for 1 hour at 30°C. The samples were then centrifuged for 45 minutes at 17,000 g to separate the pellet, containing the fibres and bound chaperones, from the supernatant with unbound chaperones. The supernatant was then collected and both fractions were incubated in 4X NuPAGE LDS Sample buffer (Thermo Fisher Scientific) for at least 30 minutes at 90°C. Samples were then loaded on BOLT 4-12% Bis-Tris gels (Thermo Fisher Scientific) and analysed by SDS-PAGE.

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