A QUICK GUIDE TO PREPARE FIBRILS OF AMYLOIDOGENIC PROTEINS
(Please do not consider this to be a protocol for ThT assay)
Insulin fibril preparation:
Make up 50 mM KCl/HCl in MilliQ. Adjust pH to 1.6 by adding HCl. Filter this solution using 0.02 μm Anotop filter. Use small filters as the loss of solution for 1-5 mL lots is less than 1 mL. Heat the solution at 60 °C. Weigh out insulin to make 1 mg/mL solution in 0.5 mL. Add the hot HCl/KCl solution onto powder protein, mix by vortexing and icubate for 9 h. (Biophys J, 90, 589–597 (2006)). Longer incubations may produce more mature fibrils.
For atomic-force microscopy (AFM), this solution was diluted 1:1000. We could try 1:500 and 1:1000 dilutions for EM. Dilute the solution and apply to grids. Prepare for EM. Set aside 10-μL aliquots for AAA when needed or before dot-blotting experiments to ascertain the amount blotted.
Lysozyme fibril preparation:
Make up 10 mM glycine at pH 2.0. Adjust the pH with HCl. Add 0.2 % azide. Filter through Anotop. Make up lysozyme at 10 mg/mL in 50 μL and incubate at 37 or 57 °C. Incubate for 1 week to produce fibrils (J Struct Biol 130: 339–351 (2000)). Dilute 1:100 and use 8 μL for EM grid preparations.
According to Protein Sci, 9:867–877 (2000), disulfide bonding between Cys1–Cys7 is essential for calcitonin (MW=3417.9 Da) fibril formation. The preparation we received from American Peptide Company, Inc, is already disulfide bonded.
Filter the solution through Anotop 0.02 μm filters before dissolving the protein. Make up calcitonin at 1 mg/mL (0.2-0.4 mL) in 50 mM Tris/HCl pH 7.4 containing 0.02% azide. Make up 1% sodium azide in MilliQ and dilute from this solution. Be careful when handling azide; do not discard down the sink.
Fibrillation time is dependent on concentration. At this concentration, human calcitonin forms fibrils after 21 min in this buffer (JBC, 268:6415–6422 (1993)). Use 8 μL of this solution for EM grid preprations.
Molecular weight: 1912.3 Da. Make up 20 mM citrate buffer (pH 5.5). To make this, add 1 g citric acid and 4.5 g sodium citrate in one liter to have the buffer at that pH according to this java program http://www.columbia.edu/~scb2001/tools/citric/cit.htm. Or make up 20 mM sodium citrate and adjust the pH by adding liquid citric acid. Scale down as needed. Add azide (0.02 %), filter through Anotop 0.02 μm filters. Make up Prion protein at 1 mg/mL in 200 μL and incubate at room temperature for 24 h. PBS at pH 7.4 with azide could also be used (PNAS 90:9678–9682 (1993)). Use 5 μL of this solution for EM.
Be careful using TTR. This is a human product purified from serum. Make up sodium acetate buffer at 50 mM and adjust the pH with acetic acid to pH 3.6. Filter through Anotop filters. Make up TTR at 2 mg/mL in 200 μL of the buffer and incubate at room temperature for 72 h. Centrifuge this sample at 4 °C for 30 min at top speed, wash the pellet in MilliQ and finally suspend in MilliQ and incubate at 37 °C for more than 11 days. Dilute this solution 1:100 and apply 8 μL on EM grid for microscopy (J Mol Biol 317:683–695 (2002)).
Prepare a stock solution of IAPP (FW=3905.3 Da) at 400 μM in HFIP. Dilute this stock to 4 μM in 10 mM sodium acetate at pH 6.5. Make up sodium acetate solution and adjust the pH to 6.5 using acetic acid. Thrity to 48 h is enough for fibrils to form. For EM, apply 10 μL of this solution on the grid for 1-5 min (Biochemistry, 43:14454–14462 (2004)).