αS and TDP-43PrLD were fluorescently labeled using Hilyte Flour 532/647 succinimidyl ester (Anaspec) and Hilyte Flour 647, respectively. Proteins were incubated for 16 hours at 4 °C with a 3 M excess of dye in 20 mM MES solution pH 6.0. TDP-43PrLD and αS were eluted in sterile 20 mM MES buffer pH 6.0 using two cycles of the Sephadex G-25 Spin Trap desalting column (Cytiva). For confocal imaging, TDP-43PrLd fluorescently labeled using Hilyte Flour 647 succinimidyl ester (Anaspec) and αS labeled with Hilyte Flour 532 succinimidyl ester (Anaspec) were used at 1–2% of final protein concentration.
Hilyte flour 647 succinimidyl ester
Manufactured by AnaSpec
Hilyte Flour 647 succinimidyl ester is a fluorescent labeling reagent used for covalent attachment of dye molecules to proteins and other biomolecules containing primary amine groups. The Hilyte Flour 647 dye has an excitation maximum at 651 nm and an emission maximum at 667 nm, making it suitable for applications involving red fluorescence detection.
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2 protocols using hilyte flour 647 succinimidyl ester
1
Fluorescent Labeling of αS and TDP-43PrLD
2
Fluorescent Labeling of αS and TDP-43PrLD
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αS and TDP-43PrLD were fluorescently labeled using Hilyte Flour 532/647 succinimidyl ester (Anaspec) and Hilyte Flour 647, respectively. Proteins were incubated for 16 hours at 4 °C with a 3 M excess of dye in 20 mM MES solution pH 6.0. TDP-43PrLD and αS were eluted in sterile 20 mM MES buffer pH 6.0 using two cycles of the Sephadex G-25 Spin Trap desalting column (Cytiva). For confocal imaging, TDP-43PrLd fluorescently labeled using Hilyte Flour 647 succinimidyl ester (Anaspec) and αS labeled with Hilyte Flour 532 succinimidyl ester (Anaspec) were used at 1-2% of final protein concentration.
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