Bitan:fibrils



       Insulin fibril preparation:



A QUICK GUIDE TO PREPARE FIBRILS OF AMYLOIDOGENIC PROTEINS

<span style='font-size:14.0pt'>(Please do not consider this to be a protocol for ThT assay)</o:p>

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<p class=MsoNormal> Insulin fibril preparation:</o:p></b>

<p class=MsoNormal>Make up 50 mM KCl/HCl in MilliQ. Adjust pH to 1.6 by adding HCl. Filter this solution using 0.02 &#956;m 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.<span style="mso-spacerun: yes"> Add the hot HCl/KCl solution onto powder protein, mix by vortexing and icubate for 9 h. (Biophys J, 90, 589&#8211;597 (2006)). Longer incubations may produce more mature fibrils.

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<p class=MsoNormal>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-&#956;L aliquots for AAA when needed or before dot-blotting experiments to ascertain the amount blotted.

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<p class=MsoNormal> Lysozyme fibril preparation:</o:p></b>

<p class=MsoNormal>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 &#956;L and incubate at 37 or 57 °C. Incubate for 1 week to produce fibrils (J Struct Biol 130: 339&#8211;351 (2000)). Dilute 1:100 and use 8 &#956;L for EM grid preparations.

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<p class=MsoNormal> Calcitonin:</o:p>

<p class=MsoNormal>According to Protein Sci, 9:867&#8211;877 (2000), disulfide bonding between Cys1&#8211;Cys7 is essential for calcitonin (MW=3417.9 Da) fibril formation. The preparation we received from American Peptide Company, Inc, is already disulfide bonded. </o:p>

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<p class=MsoNormal>Filter the solution through Anotop 0.02 &#956;m 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. </o:p>

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<p class=MsoNormal>Fibrillation time is dependent on concentration. At this concentration, human calcitonin forms fibrils after 21 min in this buffer (JBC, 268:6415&#8211;6422 (1993)). Use 8 &#956;L of this solution for EM grid preprations.</o:p>

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<p class=MsoNormal> Prion (106-126):<o:p></o:p>

<p class=MsoNormal>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 <a href="http://www.columbia.edu/~scb2001/tools/citric/cit.htm">http://www.columbia.edu/~scb2001/tools/citric/cit.htm</a>. 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 &#956;m filters. Make up Prion protein at 1 mg/mL in 200 &#956;L and incubate at room temperature for 24 h. PBS at pH 7.4 with azide could also be used (PNAS 90:9678&#8211;9682 (1993)). Use 5 &#956;L of this solution for EM.

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<p class=MsoNormal> Transthyretin (TTR):<o:p></o:p>

<p class=MsoNormal>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.<span style="mso-spacerun: yes"> Make up TTR at 2 mg/mL in 200 &#956;L 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 &#956;L on EM grid for microscopy (J Mol Biol 317:683&#8211;695 (2002)).

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<p class=MsoNormal> Amylin (IAPP):<o:p></o:p>

<p class=MsoNormal>Prepare a stock solution of IAPP (FW=3905.3 Da) at 400 &#956;M in HFIP. Dilute this stock to 4 &#956;M 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 &#956;L of this solution on the grid for 1-5 min (Biochemistry, 43:14454&#8211;14462 (2004)). <o:p></o:p>