{"id":608,"date":"2019-10-31T18:52:05","date_gmt":"2019-10-31T22:52:05","guid":{"rendered":"https:\/\/www.lifetein.com\/blog\/?p=608"},"modified":"2024-11-16T21:40:39","modified_gmt":"2024-11-17T02:40:39","slug":"how-to-form-the-fibrillary-structure-using-beta-amyloid-peptides","status":"publish","type":"post","link":"https:\/\/www.lifetein.com\/blog\/how-to-form-the-fibrillary-structure-using-beta-amyloid-peptides\/","title":{"rendered":"How to form the fibrillary structure using beta-amyloid peptides or amylin?"},"content":{"rendered":"\n
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A\u03b2-(1\u201342) was dissolved to 1 mM in 100% hexafluoroisopropanol, hexafluoroisopropanol was removed under vacuum, and the peptide was stored at \u221220 \u00b0C. For the aggregation protocols, the peptide was first resuspended in dry Me2SO (DMSO) to 5 mM. For oligomeric conditions, F-12 (without phenol red) culture media was added to bring the peptide to a final concentration of 100 \u03bcM, and the peptide was incubated at 4 \u00b0C for 24 h. For fibrillar conditions, 10 mM HCl was added to bring the peptide to a final concentration of 100 \u03bcM, and the peptide was incubated for 24 h at 37 \u00b0C. <\/p>\n\n\n\n

ADDLS, amyloid-derived diffusible ligands.<\/p>\n\n\n\n

Preparing human islet amyloid polypeptide (hIAPP), also known as amylin, can be challenging due to its hydrophobic amino acid residues.<\/strong> <\/p>\n\n\n\n

Here’s an improved method for dissolving lyophilized hIAPP:<\/p>\n\n\n\n

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  1. Begin by dissolving lyophilized hIAPP in 80% (v\/v) HFIP containing 10 mM HCl. This step ensures complete dissolution. The CD spectrum indicates the presence of a stable alpha-helical conformation, which remains so for several days.<\/li>\n\n\n\n
  2. Next, remove the HFIP by lyophilization, leaving behind lyophilized hIAPP.<\/li>\n\n\n\n
  3. Re-dissolve the lyophilized hIAPP in 10 mM HCl, and eliminate any insoluble components by ultracentrifugation.<\/li>\n\n\n\n
  4. The resulting hIAPP solution in 10 mM HCl is ready for immediate use in experiments.<\/li>\n<\/ol>\n\n\n\n

    To initiate the formation of hIAPP fibrils, introduce the stock solution into the reaction buffer. Conditions for fibril formation were optimized under two pH conditions:<\/p>\n\n\n\n