Enhanced αSyn SAA

Materials:

  • Human αSyn protein [Amprion, cat# S2020]
  • 1X Synthetic CSF (sCSF) [Amprion, cat# 2022]
  • Nuclease Free Water [Growcells, cat# NUPW-0500]
  • 1,4-Piperazinediethanesulfonic acid (PIPES) [MilliporeSigma, cat# 80635]
  • 5M Sodium Chloride (NaCl) [Lonza, cat# 51202]
  • Thioflavin T (ThT) [MilliporeSigma, cat# T3516]
  • 30% sodium lauroyl sarcosinate (sarkosyl) [MilliporeSigma, cat# 61747-100ML]
  • 96-well ELISA clear bottom plates [Greiner, cat# 655906]
  • ELISA plate lids with condensation rings [Greiner, cat# 656170]
  • 1/8” silicon nitride (Si3N4) beads [Mascera, grade 5, 1 ultrasonic wash, MSF/SN-001]
  • X-Pierce™ Sealing Films, Non-sterile [Excel Scientific, cat# XP-100]
  • Applied Biosystems™ MicroAmp™ Optical Adhesive Film [ThermoFisher. cat# 4311971]
  • TipOne® RPT pipette tips, 100μL to 1000mL [USA Scientific]
  • Protein LoBind Tubes, PCR Clean, colorless, various sizes [Eppendorf]
  • 50 mL conical tubes [Thomas Scientific, cat# 1158R10]
  • 12-well V-bottom reservoir [USA Scientific, cat# 3823-3512]
  • Sodium hydroxide (NaOH) [Fisher Chemicals, cat# S318-3]
  • Absorbent Underpads [Fisherbrand, cat# 1420663]
  • Growscell Water
  • FLUOstar Reader [BMG Lab Technologies]
  • 5 Inch rubber roller [Amazon]
  • Bench Top Vortex
  • Mini Centrifuge

 Stocks

  • 500mM PIPES pH 6.5
  • 5M NaCl
  • 5mM ThT
  • 10% Sarkosyl
  • Alpha Synuclein Monomer 6.5mg/ml (can vary batch to batch)
  • Growscell Water

Master Mix (Final Concentrations)

  • Final well Volume 100ul
  • 100mM PIPES pH 6.5
  • 500mM NaCl
  • 10uM ThT
  • 1% Sarkosyl
  • Alpha Synuclein Monomer 0.3mg/ml

Procedure:

  • Prepare 96 Well plate by dispensing 2 of the 1/8th silicon nitride bead into each well. This can be done by using a pair of tweezers, or if available, a bead dispenser.
  • After 2 beads have been dispensed in the wells, place one ELISA plate lid with condensation ring. Place the plate on a box of ice. (Use a “dummy plate” to sculpt the ice to fit the plate)
  • Preparation master mix (before sample loading) in a 50 ml conical tube, mix the 0.5M PIPES pH 6.5 for the final is 100mM, 5M NaCl so that the final is 0.5M, and the Growscell water that is calculated. Swirl to mix the reagent. Place the 50ml after mixing on ice.
  • Place 12-well V-bottom reservoir on ice with the lid on.
  • Thaw CSF/samples in water, briefly vortex the samples for 10 seconds, and spin them done for 3-5seconds with a small microcentrifuge. Place samples on ice.
  • Load 40ul of CSF/Sample in each of the well in triplicates.
  • Remove the ELISA plate lid, with clean gloves, grab a X-Pierce film on the plate, and carefully place the film on the plate so that the wells match with the corresponding film.
  • Add rest of the materials into the master mix.
  • Add the calculated 10% Sarkosyl into the master mix gently to avoid any bubbles.
  • Add the calculated 50mM ThT to the 50ml conical tube.
  • Thaw the recombinant alpha synuclein monomer in water, gently vortex the substrate 3-5 seconds at 70%-80% intensity. Spin for 3 seconds, on mini centrifuge.
  • Add the calculated amount needed for the experiment in to the 50ml conical tube for the master mix. Gently swirl, avoid making any bubbles.
  • Pour the master mix in the reservoir and add 60ul of master mix into each well using multichannel pipette.
  • Gently mix by pipetting up and down 2-3 times, before dispensing the total volume. (Try to avoid making bubbles as much as possible.)
  • After that, carefully remove the X-Pierce film from plate and place a MicroAmp film on the plate to cover all the wells.
  • Place the plate into the Ω at 42°C, making sure that column A1 is on the top left of the plate tray.
  • Start the protocol on the Ω, using 1min shaking at 800RPM, and 14 minutes rest.