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Preparation of αSyn PFFs

JB Jin-Song Bian

Last updated date: Mar 30, 2021 Views: 878 Forks: 0

original An abbreviated version of this protocol was published in Science Advances in Jan, 2021
Anti–Na+/K+-ATPase immunotherapy ameliorates α-synuclein pathology through activation of Na+/K+-ATPase α1–dependent autophagy
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  1. Escherichia coli BL21 (DE3) competent cells (Ampicillin-resistant) were transformed with a pPK172 human α-synuclein plasmid
    1. Add 1μl plasmid to BL21 (DE3) and put on ice for 30 minutes
    2. Put BL21 (DE3) into 42℃ water for 90 seconds and take it out on ice for 5 minutes
    3. Add 500μl LB medium to BL21 (DE3), 200rpm, 40 minutes, 37℃ incubator
    4. Centrifuge at 4000rpm for 5 minutes, room temperature
    5. Resuspend the pellet into 100μl LB medium and add to agar plates (containing Ampicillin, warm up to room temperature or incubate in 37℃ incubator before use)
    6. Gently mix by flicking the plates with beads and incubate at 37℃ for 30 minutes (upward)
    7. Place upside down and incubate at 37℃ overnight
    8. Pick clones from Agar plates into different tubes with 3ml LB medium, incubate at 37℃ incubator, 200rpm, overnight
    9. Extract cell lysis from 2.5ml LB medium with lysis buffer (10 mM Tris, pH 7.6, 750 mM NaCl, 1μg/mL pepstatin, 20μg/mL aprotinin, 1mM benzamidine, 1mM PMSF, 1mM EDTA and 0.25 mg/mL lysozyme), sonicate for 10 minutes, boil at 95℃ for 10 minutes, centrifuge at 4℃, 13,000g for 40 minutes
    10. Collect the supernatant and run Western Blot (15% gel)
    11. Coomassie blue staining to find the best clone for α-synuclein extraction
  2. BL21 (DE3) cells expressing human α-synuclein were cultured in tubes with LB medium containing Ampicillin at 37℃ incubator, 200rpm, overnight
  3. Dilute cells (1/1000) into 2L conical flask with LB medium containing Ampicillin at 37℃ incubator, 200rpm, overnight
  4. Centrifuge cells at 4000rpm for 15 minutes, 4℃, and resuspend in PBS, centrifuge again
  5. The pellet can be stored at -80℃. Resuspend the pellet in Lysis buffer (10 mM Tris, pH 7.6, 750 mM NaCl, 1μg/mL pepstatin, 20μg/mL aprotinin, 1mM benzamidine, 1mM PMSF, 1mM EDTA and 0.25 mg/mL lysozyme), sonicate for 10 minutes, boil at 95℃ for 10 minutes, centrifuge at 4℃, 13,000g for 40 minutes
  6. The supernatant was collected and dialyzed with 10 mM Tris (pH 7.6), 50 mM NaCl and 1 mM EDTA
  7. The supernatant was applied to Anion exchange column HiTrap Q HP equilibrated with Buffer A (20mM Tris, pH8.0)
  8. Wash the column with 50mM NaCl in Buffer A, 5 column volume (CV) and collect the fractions
  9. Wash the column with 100mM NaCl in Buffer A, 5CV and collect the fractions
  10. Wash the column with 200mM NaCl in Buffer A, 5CV and collect the fractions
  11. Wash the column with 300mM NaCl in Buffer A, 5CV and collect the fractions
  12. Wash the column with 400mM NaCl in Buffer A, 5CV and collect the fractions
  13. Wash the column with 500mM NaCl in Buffer A, 5CV and collect the fractions
  14. These fractions were analyzed by SDS PAGE and Coomassie staining to choose pure factions with bands corresponding to 17 kDa
  15. The pure fractions were concentrated by 3.5 kDa MWCO Amicon Ultra Centrifuge filter devices (Millipore), centrifuge at 4℃, 4000rpm for 60 minutes. BCA was used to determine the protein concentration
  16. The purified αSyn monomers were dissolved in buffer B (50 mM Tris–HCl, pH 7.5, 150 mM KCl) into 5 mg/ml
  17. The PFF was formed by incubating the monomers at 37°C for 7 days with continuous shaking at 1000 rpm in a thermomixer (Eppendorf, Germany)
  18. Thioflavin T binding assay and Transmission Electron Microscopy were used to determine the PFF formation.
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How to cite：
Readers should cite both the Bio-protocol preprint and the original research article where this protocol was used:
  1. Bian, J(2021). Preparation of αSyn PFFs. Bio-protocol Preprint. bio-protocol.org/prep984.
  2. Cao, L., Xiong, S., Wu, Z., Ding, L., Zhou, Y., Sun, H., Zhu, M., Lee, W. T., Nie, X. and Bian, J.(2021). Anti–Na+/K+-ATPase immunotherapy ameliorates α-synuclein pathology through activation of Na+/K+-ATPase α1–dependent autophagy . Science Advances 7(5). DOI: 10.1126/sciadv.abc5062

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