Commercially available 27-alkyne cholesterol (800 μg, 2 μM) and Azide-PEG3-biotin (400 μg, 0.9 μM) were mixed in 100 mL dimethyl sulfoxide (DMSO) and water solution (v/v: 1/1). DMSO/water (50 μL, v/v: 1/1) containing CuSO4 (0.25 μM) and sodium L-ascorbate (0.5 μM) was added in the reaction mixture. The solution was incubated at room temperature under N2 protection for 12 h. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure. The product was purified using a reversed-phase high performance liquid chromatography (HPLC) instrument (Shimadzu, Kyoto, Japan) equipped with an SPD-M40 photo diode array detector (Shimadzu, Kyoto, Japan and an Agilent poroshell 120 EC-C18 column (2.7 mm, 4.6 × 250 mm), using water/methanol/formic acid = 20/80/0.1 (v/v/v) as the mobile phase, at a flow rate of 0.3 mL/min to afford biotinylated cholesterol as a white solid powder. The structure of the resulting biotinylated cholesterol conjugate was characterized by an 800 MHz nuclear magnetic resonance spectroscopy (Bruker Corporation, Rheinstetten, Germany) an LTQ-Orbitrap XL FT-MS spectrometer (Thermo Scientific, Bremen, Germany).
Reversed phase high performance liquid chromatography
Manufactured by Shimadzu
Sourced in Japan
Reversed-phase high performance liquid chromatography (RP-HPLC) is an analytical technique used to separate, identify, and quantify components in a mixture. It utilizes a polar mobile phase and a non-polar stationary phase to separate analytes based on their polarity and interactions with the stationary phase.
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3 protocols using reversed phase high performance liquid chromatography
1
Synthesis of Biotinylated Cholesterol
2
Peptide-Conjugated Gold Nanoparticles Synthesis
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The peptide (Ac-CILPWKWPWWPWRR-CONH2) was synthesized using an automatic synthesizer (Syro I MultiSynThec, Wullener, Germany), as previously reported.33 (link) The peptide was characterized using reversed-phase high-performance liquid chromatography (Shimadzu Corporation, Kyoto, Japan), on a column equipped with an ultraviolet Lambda-Max Model 481 detector, and liquid chromatography–mass spectrometry (Finnigan/Thermo Electron Corporation, San Jose, CA, USA).
Purified peptide was conjugated, in the presence of 10 mmol/L citrate buffer (pH 6), to Au-NPs (Cod. 752.568; Sigma-Aldrich, St Louis, MO, USA) with a diameter of 5 nm (5.47×1013 NPs/mL), as previously reported.34 (link) The functionalized NPs were purified by centrifugation (14,000 g for 30 min) and the pellet was resuspended in PBS in the presence of 0.05% (v/v) Triton X-100. To calculate the amount of peptide coupled to AuNPs, fluorimetric measurements were carried out using a Varian Carry Eclipse spectrometer (Agilent Technologies, Santa Clara, CA, USA). The fluorimetric and zeta potential (ZP) measurements have been previously reported.35 (link) The final peptide concentration coupled to AuNPs was 250 µM.
Purified peptide was conjugated, in the presence of 10 mmol/L citrate buffer (pH 6), to Au-NPs (Cod. 752.568; Sigma-Aldrich, St Louis, MO, USA) with a diameter of 5 nm (5.47×1013 NPs/mL), as previously reported.34 (link) The functionalized NPs were purified by centrifugation (14,000 g for 30 min) and the pellet was resuspended in PBS in the presence of 0.05% (v/v) Triton X-100. To calculate the amount of peptide coupled to AuNPs, fluorimetric measurements were carried out using a Varian Carry Eclipse spectrometer (Agilent Technologies, Santa Clara, CA, USA). The fluorimetric and zeta potential (ZP) measurements have been previously reported.35 (link) The final peptide concentration coupled to AuNPs was 250 µM.
3
Purification of Tityus fasciolatus Venom Peptide
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Crude venom of Tityus fasciolatus was fractioned by RP-HPLC (Reversed Phase High Performance Liquid Chromatography) (Shimadzu Co., Kyoto, Japan), using a C18 column (Synergi Fusion RP 4 μ, 80 Å, 250 × 4.6 mm (Phenomenex, Inc., Torrance, CA, USA). Components were separated using a linear gradient of solvent A (0.12% TFA in water) and solvent B (0.10% TFA in acetonitrile) from 0 to 60% for 60 min at a 1 mL/min flow rate as described previously [20 (link)]. Three extra steps of RP-HPLC were conducted to purify Tf1a, the first with 0.5%[B]/min, second purification step with 0.5%[B]/min at 45 °C, and the last purification step with 0.3%[B]/min at 45 °C.
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