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CDCl3) d 167.68, 152.46, 138.35, 135.24, 129.74, 129.10, 128.89,
128.76, 127.05, 125.03, 124.97, 124.69, 123.40, 41.81, 22.93,
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4.2. Fluorescent conjugate formation
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WT-TTR was expressed and purified from an E. coli expression
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S2) or the non-covalent TTR ligand Amide 3 (5
lL of a 1.44 mM
stock solution in DMSO, final concentration: 7.2
lM) were added
to 1 mL of a solution of WT-TTR (0.2 mg/mL, final concentration:
3.6 M) in 10 mM phosphate, 100 mM KCl and 1 mM EDTA (pH
l
7.0) in a 2 mL Eppendorf tube. The samples were vortexed, and
incubated for 24 h at 25 °C. The fluorescence changes were moni-
tored using a Varian Cary 50 spectrofluorometer at 20 °C in a
1 cm path length quartz cell. The excitation slits was set at 5 nm
and the emission slits was set at 10 nm. The samples were excited
at 328 nm and the emission spectra were collected from 330 to
550 nm (Fig. 2). The time-dependent fluorescence changes were
monitored in similar manner (Fig. 3).
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One microliter of a candidate kinetic stabilizer/amyloidogenesis
inhibitor (0.72 mM stock solution in DMSO, final concentration:
7.2 lV) was added to 100 lL of recombinant WT-TTR (3.6 lM) in
10 mM sodium phosphate, 100 mM KCl and 1 mM EDTA (pH 7.0)
in a 96-well plate (Costar black, clear bottom). The plate was sealed
and vortexed slowly for 4 h at 25 °C and then, 1
modifier S1 (0.72 mM stock solution in DMSO, final concentration:
7.2 V) was added to each well. The fluorescence changes were
lL of covalent TTR
l
monitored every 10 min using a microplate spectrophotometer
reader (Gemini SpectraMaxÒ, Molecular Devices, Sunnyvale, CA)
for 8 h at 37 °C. The fluorescence (kex = 328 nm and kem = 384 nm)
was measured from the bottom of the plate without shaking.
For the competition assay with human serum, 100
serum (Sigma–Aldrich), 1 L of test compounds (1.5 mM stock
solution in DMSO, final concentration: 15 V), and 1 L of covalent
fluorogenic modifier S2 (0.5 mM stock solution in DMSO: final con-
centration: 5 V) were employed. The fluorescence changes every
lL of human
l
l
l
l
10 min were monitored using a microplate spectrophotometer
reader (Gemini SpectraMaxÒ, Molecular Devices, Sunnyvale, CA)
for 8 h at 25 °C.
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Acknowledgments
We thank the NIH (DK46335) as well as the Skaggs Institute for
Chemical Biology and the Lita Annenberg Hazen Foundation for
financial support. Technical support from Mike Saure is also greatly
appreciated.
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51. Johnson, S. M.; Petrassi, H. M.; Palaninathan, S. K.; Mohamedmohaideen, N. N.;
Purkey, H.; Nichols, C.; Chiang, K. P.; Walkup, T.; Sacchettini, J. C.; Sharpless, K.
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Supplementary data
Supplementary data associated with this article can be found, in
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