The crude venom of C. albostriatus was obtained by electronic stimulation, and preserved at −80°C after lyophilization. The lyophilized venom was dissolved in ddH2O to a final concentration of 5 mg/ml and subjected to the first round of semi-preparative RP-HPLC purification (C18 column, 10 mm × 250 mm, 5 μm, Welch, Shanghai, China) using linear acetonitrile gradient ranging from 10 to 55% with an increasing rate of 1% per minute (Waters e2695 Separations Module, Waters, CA, United States). The fraction containing Ca2a was then collected, lyophilized, and subjected to a second round of analytical RP-HPLC purification (C18 column, 4.6 mm × 250 mm, 5 μm, Welch, Shanghai, China). The acetonitrile gradient was increased ranging from 20 to 40% at an increasing rate of 1% per minute (Waters 2795 Separations Module, Waters, CA, United States). Fractions were lyophilized and stored at −20°C before use. The purity of the toxin was tested by MALDI-TOF MS analysis (AB SCIEX TOF/TOFTM 5800 system, Applied Biosystems, United States).
C18 column
Manufactured by Hill-Rom
Sourced in China
The C18 column is a chromatographic column used in high-performance liquid chromatography (HPLC) and other analytical techniques. It is designed to separate and purify a wide range of organic compounds, including pharmaceuticals, proteins, and other biomolecules. The column is packed with a silica-based stationary phase that is chemically modified with octadecyl (C18) functional groups, providing a hydrophobic surface for the separation of analytes.
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Lab products found in correlation
L 2400, by Hitachi (2 mentions)
Uv 2000, by Unico (1 mentions)
Waters 2795 hplc system, by Waters Corporation (1 mentions)
Waters acquity uplc xevo g2 qtof, by Waters Corporation (1 mentions)
Rp hplc, by Shimadzu (1 mentions)
E2695 separations module, by Waters Corporation (1 mentions)
2795 separations module, by Waters Corporation (1 mentions)
7 protocols using c18 column
1
Purification and Characterization of Ca2a Toxin
2
Controlled Release of PTX and DOX
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PTX/DOX–LCP powders (containing 0.78 mg PTX and 9.70 mg DOX) were dispersed in 200 mL PBS (pH =6.8) release medium. The test was operated at 37°C±0.5°C, with a paddle speed of 100 rpm. At predetermined time points, 5 mL of samples was withdrawn and replaced with 5 mL of fresh release medium. The filtrate was passed through a 0.45 µm microporous membrane filter and used as the test sample. The concentration of DOX was analyzed by UV spectroscopy (UV-2000; Unico Inc.) at 490 nm. The amount of PTX was measured at 237 nm by HPLC (L-2400; HITACHI) with a Welch C18 column (4.6×200 mm, 5 µm). Acetonitrile–water (50:50, v/v) was used as mobile phase.
3
Purification of C. longipes Venom
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The crude venom of C. longipes was obtained by electrical stimulation, freeze-dried, and stored at −80 °C until further use. Lyophilized venom was dissolved in double-distilled water (ddH2O) and purified by both reverse-phase (RP-HPLC) and ion-exchange chromatography. RP-HPLC was performed using a C18 column (10 × 250 mm, 5 μm; Welch Materials Inc., Shanghai, China) on an analytical Waters 2795 HPLC system. The acetonitrile gradient increased at a rate of 1% per minute from 5–50%, using a flow rate of 3 mL/min. Eluted fractions were lyophilized and further fractionated using ion-exchange chromatography with an XB-SCX column (4.6 mm × 250 mm, 5 μm; Welch, China) on a preparative Hanbon HPLC system. The NaCl gradient increased at a rate of 2% per minute from 0–70% at a flow rate of 1 mL/min. Collected fractions containing Cl6a and Cl6b were subjected to further desalination by RP-HPLC using a C18 column (4.6 mm × 250 mm, 5 μm; Welch, China) on an analytical Waters 2795 HPLC system. In the second round of RP-HPLC, the acetonitrile gradient increased from 27–33% at a rate of 0.5% per minute and a flow rate of 1 mL/min. For C18 RP-HPLC, solvent A was 0.1% trifluoroacetic acid (TFA) in water and solvent B was 0.1% TFA in acetonitrile. Cl6a- and Cl6b-containing fractions were lyophilized and stored at −20 °C until further use.
4
Latroeggtoxin-V Recombinant Protein Purification
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The Latroeggtoxin-V fusion protein was cleaved by enterokinase at 25 °C overnight in a cleavage buffer (20 mM Tris-HCl, 50 mM NaCl, 2 mM CaCl, pH8.0) at an enzyme: substrate ratio of 1:50 (Sangon Biotech, Shanghai, China). The cleaved products were separated with RP-HPLC (SHIMADZU, Kyoto, Japan) using a C18 column (4.6 mm × 250 mm, 5 μm particle size, Welch XtimateTM, Shanghai, China). The cleaved products were eluted with a linear gradient of acetonitrile containing 0.1% TFA from 0% to 70% in 40 min at a flow rate of 1.0 mL/min. The molecular weight of the components in each elution peak was determined by electrospray ionization mass spectrometry (Waters ACQUITY UPLC/Xevo G2 QTOF, Waters, Milford, MA, USA) and thus the peak containing recombinant Latroeggtoxin-V (rLatroeggtoxin-V) was selected and lyophilized.
5
HPLC Determination of Retinol and Retinal
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Retinol and retinal were determined by HPLC (Waters 2010) equipped with an UV detector and C-18 column (Welch, 30 m × 250 µm × 0.25 µm). The HPLC conditions were listed as follows: Temperature, 40 °C; the wavelength of UV detector, 340 nm; mobile phase, methanol:acetonitrile = 95:5; flow rate, 1 ml/min. The retention times for retinol and retinal were 5.237 min and 5.618 min, respectively.
6
Thermostability Analysis of Transamination Enzymes
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The activities of MvTA and its mutants were assayed in 100 mM PBS (pH 6.0) containing 0.1 mg/mL purified enzyme, 0.1 mM PLP, 25 mM (R)-α-methylbenzylamine ((R)-MBA) as amino donor, and 10 mM d -fructose as amino acceptor. Reactions were performed at 50°C for 2 h and terminated by adding an equal volume of acetonitrile:HCl (19:1) solution. One unit (U) of enzymatic activity was defined as the amount of enzyme catalyzing the production of 1 μmol acetophenone (APO) from (R)-MBA. The APO product was analyzed using an HPLC system equipped with a C18 column (4.6 mm × 250 mm, Welch, Shanghai, China) and an ultraviolet detector at 254 nm. The mobile used was solution A (acetonitrile with 0.1% formic acid) and solution B (H2O with 0.1% formic acid), with a flow rate of 1 mL/min and the following gradient: 0–15 min, 30–80% A.
For the determination of thermostability, WT and mutant enzymes were incubated in 100 mM PBS (pH 6.0) at temperatures of 30, 40, 50°C and 60°C, respectively. Residual activities were measured at different time intervals using HPLC. The half-life (t1/2) was calculated based on the first-order deactivation function: ln(E/E0) = −kdt; t1/2 = ln 2/kd, where kd represents the deactivation rate constant.
For the determination of thermostability, WT and mutant enzymes were incubated in 100 mM PBS (pH 6.0) at temperatures of 30, 40, 50°C and 60°C, respectively. Residual activities were measured at different time intervals using HPLC. The half-life (t1/2) was calculated based on the first-order deactivation function: ln(E/E0) = −kdt; t1/2 = ln 2/kd, where kd represents the deactivation rate constant.
7
Quantifying Indomethacin in Plasma Samples
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To the plasma samples (400 mL), 200 mg/mL methanolic indomethacin solution was added as an internal standard (20 mL), followed by methanol (20 mL) and 1 mol/L HCl aqueous solution (400 mL) with vortex mixing for 3 min. Then, 3 mL diethyl ether was added to the mixture and vortexed for another 10 min. The organic layer was dried using a centrifugal drying machine at room temperature after centrifuging at 4250  g for 10 min and the residues were reconstituted in 50 mL mobile phase. Then each sample was vortexed for 5 min and centrifuged at 12,750  g for 10 min and 20 mL samples were subjected to HPCL (L-2400; HITACHI, Japan). The mobile phase was a mixture of methanol and 0.1% glacial acetic acid (65:35, vol/vol) 24 and detection was carried out at 25 C with a flow rate of 1 mL/min. The retention time of FNB and indomethacin was 18.01 and 25.98 min, respectively, and the UV-vis detector was operated at 286 nm and separation was carried out using a Welch C18 column (4.6 mm  200 mm, 5 mm).
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