Detailed information on the probes and chemical compounds is listed in Suppl. Table 1 [60 (link), 61 (link)]. Recombinant K18 4R tau was expressed and produced by Escherichia coli as described previously [62 , 63 ] (Supplementary material ). Details on recombinant K18 tau fibril production and characterization are in SFig. 1 and supplementary methods. The absorbance of the compounds was measured with a spectrofluorometer. Thioflavin T assays against K18 tau fibrils using a fluorometer (Fluoromax 4, Horiba Scientific, Japan) were performed as described previously [62 ], with two independent experiments and three technical replicates. PBB5 (excitation peak 630 nm, concentration 1.6 μM) was dissolved in Milli-Q H2O or dimethyl sulfoxide and further diluted in 1 × PBS (Gibco). PBB5 was then mixed with 5 μL of tau K18 fibril solution in a 45-µL quartz cuvette (quartz SUPRASIL Ultra Micro Cell, Hellma). The solution was incubated for 1 min at room temperature and resuspended, and fluorescence was measured with a spectrofluorometer using the corresponding excitation wavelength.
Suprasil ultra micro cell
Manufactured by Hellma
The SUPRASIL Ultra Micro Cell is a compact and versatile laboratory instrument designed for precision measurements. It features a quartz cuvette with a small volume capacity, allowing for efficient sample utilization and accurate spectrophotometric analysis.
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Lab products found in correlation
2 protocols using suprasil ultra micro cell
1
Characterization of Tau Fibril Binding Probes
2
Fluorescent Dye Binding Assay for Amyloid Proteins
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Recombinant Aβ42, K18 tau, and αSyn were expressed and produced by E. coli as described previously [34 (link)–36 (link)]. The fluorescent dyes were dissolved in Milli-Q H2O or dimethyl sulfoxide (DMSO) and further diluted in 1×PBS pH 7.4. The absorbance of the compounds was measured. Thioflavin T assays against Aβ42 and K18 tau and αSyn fibrils were performed as described previously [34 (link), 36 (link)], with two independent experiments and three technical replicates (Fluoromax 4, Horiba Scientific, Japan). The dyes were then mixed with either 2 μL of αSyn, 5 μL of K18 tau (380 μg/ml) or 5 μL of Aβ42 fibril (80 μg/ml) solution in a 45 μL quartz cuvette (quartz SUPRASIL Ultra Micro Cell, Hellma). The solutions were incubated for 1 min at room temperature and resuspended, and fluorescence was measured with a spectrofluorometer (FluoroMax-4, Horiba Jobin Yvon, Japan) using a known excitation wavelength for the ligands.
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