Cerastes cerastes snake venom was collected from the Pasteur Institute’s Serpentarium (Tunis, Tunisia) and stored at –20 °C. Crude venom (300 mg) was dissolved in 0.2 M ammonium acetate, pH 6.8, and fractionated by a Sephadex G-75 (Pharmacia, Uppsala Sweden) column. Fraction II, which contains medium-molecular-weight proteins (<30 kDa), was lyophilized subjected to a reverse phase C8 column (250 × 4.6 mm2, 5 μm; Beckman; Fullerton, CA, USA) eluted with a linear acetonitrile gradient 10–65% over 45 min at a flow rate of 0.8 mL/min. The homogeneity of CC5 and CC8 were assessed by a second step of high-performance liquid chromatography (HPLC) on a C18 column (250 × 4.6 mm2, 5 μm; Beckman) as previously described [21 (link)].
C18 column
Manufactured by Beckman Coulter
Sourced in United States
The C18 column is a type of chromatography column used in liquid chromatography (LC) and high-performance liquid chromatography (HPLC) applications. It is designed to separate and purify a wide range of organic compounds, including small molecules, peptides, and proteins. The column contains a stationary phase consisting of silica particles coated with C18 (octadecyl) functional groups, which interact with the analytes in the mobile phase, leading to their separation based on their hydrophobic properties.
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7 protocols using c18 column
1
Purification of Cerastes cerastes Venom
2
Quantifying Adenine Nucleotides via HPLC
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Adenine nucleotide levels were determined by high-performance liquid chromatography (HPLC) as described before [34] (link). Briefly, HUVEC were transferred to an ice-cold 0.6 mol/L HClO4 and immediately homogenized and centrifuged at 10,000g, 10 min, 4 °C (Eppendorf, Germany). The supernatant was neutralized with equivoluminal Na2HPO4 (1 mol/L) and centrifuged at 10,000g, 10 min, 4 °C again. The supernatant was filtered with 0.22 µm membrane and an aliquot of 50 μl were transferred into HPLC vials. Adenine nucleotides in samples were separated on a C18 column (Beckman, 5 µm, 250 mm × 4.6 mm) at a flow rate of 0.5 ml/min for 30 min and were detected at 254 nm with a ultraviolet detector (SPD-10A). The mobile phase was 96% 0.05 mol/L KH2PO4 (PH = 6.5) with 4% methanol (vol/vol). Results were adjusted by the corresponding cell numbers. Energy charge = [ATP + ADP/2]/[ATP + ADP + AMP]. Standard ATP, ADP and AMP were bought from Sigma.
3
Synthetic Peptides for Adrenergic Receptor Study
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Three peptides that corresponded to the amino acid sequence of the second extracellular loop of human β1-, β2-, and α1-AR were synthesised by Genomed (Genomed Synthesis, Inc., CA, USA). The sequences are presented in Table 1 .[17] (link)–[19] (link) The peptide was judged to be pure on the basis of a HPLC analysis on a Vydac C-18 column and via an amino acid analysis on an automated amino acid analyser (Beckman Instruments, Inc., Palo Alto, CA).[20] (link)
4
Peptide Deprotection Analysis by RP-HPLC
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RP-HPLC was carried out on a Knauer system (Knauer Wissenschaftliche Gerate Co., Berlin, Germany), with C18 column (Beckman-Coulter, Brea, CA, USA) 100 Å, 250 × 4.6 mm). One hundred μL of peptide solution was injected into the injector and a linear gradient of acetonitrile was applied from 0 to 90% (absolute acetonitrile containing 0.1% TFA) at a flow rate of 1 mL/min for 90 min. After that, the retention time of the deprotected peptide was compared to the protected one. Any alteration in the retention time of the deprotected peptide indicates that the peptide is deprotected. The deprotected peptide was collected and lyophilized as mentioned above.
5
Quantification of Adenine Nucleotides
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Frozen LVs were transferred to ice‐cold 0.6M HClO4 (4 mL/g), and the tissue was immediately homogenized and centrifuged (10 000g, 4°C, 10 minutes). The supernatant was neutralized with an equal volume of 1M Na2HPO4 and centrifuged again at 10 000g and 4°C for 10 minutes. The supernatant was filtered through a 0.22 µm filter. Next, 50 µl aliquots were analyzed using a high performance liquid chromatography (HPLC) method with a Beckman C18 column (5 μm, 250 × 4.6 mm). Analytes were isocratically eluted using 96% 0.05M KH2PO4 (pH 6.5) and 4% methanol for 30 minutes. Concentrations of adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) were determined at 254 nm using an external standard method for quantification. The energy charge was defined as (ATP + ADP/2)/(ATP + ADP + AMP).
6
Salivary Gland Protein Isolation
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The extract of five pairs of salivary glands (0.5 μg/μl) was dissolved in PBS (100 μl), and the fractions were separated using an HPLC instrument (Knauer-Germany). Then 10 μl of the prepared extract (5μg) was manually injected into C18 column (5μm 100Å -250 × 4.6mm, Beckman, USA) and eluted in a linear gradient of acetonitrile containing 0.05% TFA (solution A) and 0.05% TFA in water (solution B) at a flow rate of 0.2 ml/ min. In order to isolate the proteins, the column was eluted by a linear gradient of solution A from 0 to 60 percent for 95 minutes at 0.2 ml/min. The eluted peaks were monitored at 214nm and 280nm and collected manually 23 . The isolated fractions were lyophilized by a freeze dryer (Christ, Alpha 1-2 LD plus-Germany). The purity of the isolated proteins was subsequently checked by SDS-PAGE.
7
Detailed NMR and Spectral Characterization
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Starting materials, reagents and solvents were from Sigma-Aldrich (Saint Quentin Fallavier, France), Fluka, SDS and Acros Organics (Fisher Scientific, Illkirch, France) and were of analytical grade quality. 1 H and 13 C NMR spectra were recorded on a Bruker DPX-300 (at 300 MHz) or Bruker Avance III nanobay-400 (at 400 MHz) spectrometers in CDCl3 or DMSO-d6 solution (from Euriso-Top, Saint Aubin, France). Chemical shifts (δ) for 1 H and 13 C are reported in parts per million (ppm) relative to tetramethylsilane (TMS) as internal standard and coupling constants (J) are given in Hertz (Hz) .
Multiplicity abbreviations used are as follows: br, broad; s, singlet; d, doublet; dd, doublet of doublets; t, triplet; dt, doublet of triplets; td, triplet of doublets; m, multiplet. Melting points were obtained using a Büchi Melting Point B-540 apparatus and are not corrected. High-resolution mass spectrometry (HRMS) in electron spray ionization (ESI) was performed at the Spectropole (Analytical Laboratory) at Campus St. Jérôme (Marseille, France) on a Q-STAR Elite instrument (Applied Biosystems, USA). Purity of final compounds was determined at 254 nm by HPLC (Agilent 1200 series, USA) using a C18 column (Beckman Coulter Inc., USA) and was 95% or greater.
Multiplicity abbreviations used are as follows: br, broad; s, singlet; d, doublet; dd, doublet of doublets; t, triplet; dt, doublet of triplets; td, triplet of doublets; m, multiplet. Melting points were obtained using a Büchi Melting Point B-540 apparatus and are not corrected. High-resolution mass spectrometry (HRMS) in electron spray ionization (ESI) was performed at the Spectropole (Analytical Laboratory) at Campus St. Jérôme (Marseille, France) on a Q-STAR Elite instrument (Applied Biosystems, USA). Purity of final compounds was determined at 254 nm by HPLC (Agilent 1200 series, USA) using a C18 column (Beckman Coulter Inc., USA) and was 95% or greater.
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