Two times recrystallized, dialyzed, and lyophilized HewL was purchased from Worthington Biochemicals (Lakewood, NJ) and used for all experiments. Ultrapure grade ThT was obtained from Anaspec (Freemont, CA) and standard grade ThT from Sigma Aldrich. All other chemicals were from Fisher Scientific (Pittsburgh, PA) and were reagent grade or better.
Ultrapure grade tht
Manufactured by AnaSpec
Sourced in United States
Ultrapure grade Thioflavin T (ThT) is a fluorescent dye used in the detection and quantification of amyloid fibrils. It exhibits an enhanced fluorescence signal upon binding to amyloid structures. The Ultrapure grade ensures high purity and consistency for reliable results in amyloid research applications.
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6 protocols using ultrapure grade tht
1
Purification and Characterization of HewL
2
Thioflavin T Aggregation Assay
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A black-walled clear-bottom 384-well plate
(Costar) was sterilized under UV light for 15 min in a laminar flow
hood. UltraPure grade ThT (AnaSpec, Fremont, CA) was dissolved in
deionized water at a concentration of 1 mM and then filter-sterilized.
Wells for 2D and 3D samples were prepared as above but contained 20
μM ThT. The wells were sealed with black TopSeal-A membranes
to prevent evaporation. The ThT experiment was analyzed on a SpectraMax
M5 (Molecular Devices, San Jose, CA) spectrophotometer set to ex.
450 nm, em. 480 nm, at 37 °C, taking measurements every 30 min
for 72 h and reading from the bottom of the plate. Replicates were
averaged, Aβ data were corrected with ThT control data, and
corrected curves were normalized. As in standard practice in Aβ
aggregation studies,85 (link),86 (link) because of the stochastic nature
of aggregation, curves representative of at least 10–20 experiments
are presented here.
(Costar) was sterilized under UV light for 15 min in a laminar flow
hood. UltraPure grade ThT (AnaSpec, Fremont, CA) was dissolved in
deionized water at a concentration of 1 mM and then filter-sterilized.
Wells for 2D and 3D samples were prepared as above but contained 20
μM ThT. The wells were sealed with black TopSeal-A membranes
to prevent evaporation. The ThT experiment was analyzed on a SpectraMax
M5 (Molecular Devices, San Jose, CA) spectrophotometer set to ex.
450 nm, em. 480 nm, at 37 °C, taking measurements every 30 min
for 72 h and reading from the bottom of the plate. Replicates were
averaged, Aβ data were corrected with ThT control data, and
corrected curves were normalized. As in standard practice in Aβ
aggregation studies,85 (link),86 (link) because of the stochastic nature
of aggregation, curves representative of at least 10–20 experiments
are presented here.
3
Thioflavin T Interaction with Amyloid Fibrils
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The samples of “UltraPure Grade” ThT from AnaSpec (Fremont, CA, USA) were used without further purification. ThT was dissolved in 2 mM Tris-HCl buffer (pH 7.7) with 150 mM NaCl. All subsequent studies of the interaction of ThT with amyloid fibrils were performed strictly in the conditions in which the fibrils were obtained (in 150 mM Gly-HCl buffer (pH 2.5)). At the same time, a series of preliminary studies was made, which resulted in the conclusion that the properties of the dye in both of these buffers are identical. Fluorescent dye ATTO-425 from ATTO-TEC (Siegen, Germany) and the buffer components from Sigma (Louis, MO, USA) were used without additional purification. To determine the pH of tested solutions, HI 9024 pH meter (HANNA Instruments, Wensoket, RI, USA) was used.
4
Characterizing Amyloid Fibrils with ThT
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ThT UltraPure Grade (AnaSpec, Fremont, CA) without after-purification was used. ThT-fibrils–tested solutions were prepared by equilibrium microdialysis using a Harvard Apparatus/Amika device (Harvard Apparatus, Holliston, MA). Equilibrium microdialysis was performed with a concentration of aggregates approximately 0.5 mg/mL and initial concentration of ThT approximately 32 μM. Spectroscopic study of the sample and reference solutions prepared by the proposed approach allowed us to determine the photophysical characteristics of ThT bound to tested amyloids [36 (link)].
For obtaining the fluorescence images of the ThT-stained fibrillar structures confocal laser scanning microscope Olympus FV 3000 (Olympus, Japan) was used. We used the oil immersion objective with a 60× magnification, numerical aperture NA 1.42, and laser with excitation line 405 nm.
For obtaining the fluorescence images of the ThT-stained fibrillar structures confocal laser scanning microscope Olympus FV 3000 (Olympus, Japan) was used. We used the oil immersion objective with a 60× magnification, numerical aperture NA 1.42, and laser with excitation line 405 nm.
5
Thioflavin T Amyloid Binding Analysis
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ThT “UltraPure Grade” (AnaSpec, Fremont, CA, USA) without after-purification was used. ThT-fibril-tested solutions were prepared by equilibrium microdialysis using the Harvard Apparatus/Amika device (Harvard Apparatus, Holliston, MA, USA). Equilibrium microdialysis was performed with the concentration of aggregates ~0.5 mg/mL and initial concentration of ThT ~32 μM. The spectroscopic study of the sample and reference solutions prepared by proposed approach allowed us to determine the photophysical characteristics ThT bound to tested amyloids [45 (link)]. For obtaining fluorescence images of the ThT-stained fibrillar structures, we used the confocal laser scanning microscope Olympus FV 3000 (Olympus, Tokyo, Japan) equipped with the 60× oil immersion objective with a numerical aperture NA 1.42 and laser with excitation line 405 nm.
6
Spectroscopic Study of Amyloid-Bound ThT
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ThT UltraPure Grade (AnaSpec, Fremont, CA, USA) without after-purification was used. ThT-fibrils tested solutions were prepared by equilibrium microdialysis using a Harvard Apparatus/Amika device (Harvard Apparatus, Holliston, MA, USA). Equilibrium microdialysis was performed with a concentration of aggregates of about 0.5 mg/ml and an initial concentration of ThT of about 32 μM. Spectroscopic study of the sample and reference solutions prepared by the proposed approach allowed us to determine the spectral and photophysical characteristics ThT bound to tested amyloids (Kuznetsova et al., 2012b (link)).
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