Jupiter c18 lc column

Polyethyleneimine (PEI, branched, M_w 25000), 3‐(2‐pyridyldithio)propionic acid N‐hydroxysuccinimide ester (SPDP) were obtained from Sigma‐Aldrich. Methoxy poly(ethyleneglycol) propionic acid N‐hydroxysuccinimide (Methoxy‐PEG‐NHS, M_w 5000) was purchased from Nanocs. CpG1826 was obtained from Integrated DNA Technology. Antigen peptides used in this study were synthesized by Genemed Synthesis, which include epitopes of ovalbumin peptide SIINFEKL and CSSSIINFEKL, neo‐epitopes of MC38 colon carcinoma ASMTNMELM (Adpgk) and CSSASMTNMELM (CSS‐Adpgk), neo‐epitopes of B16F10 melanoma LCPGNKYEM (M27), VDWENVSPELNSTDQ (M30), and CSSVDWENVSPELNSTDQ (CSS‐M30). All other reagents were received from Fisher scientific unless otherwise indicated. UV–Vis absorption and fluorescence spectra were obtained using BioTek synergy neo microplate reader. GPC and HPLC were performed using Shimadzu HPLC system equipped with TSKgel G3000SWxl column (Tosoh Bioscience LLC) and Jupiter® C18 LC Column (Phenomenex), respectively. TEM images were acquired using JEOL 1400‐plus. Hydrodynamic size and zeta potential were measured using Zetasizer Nano ZSP (Malvern Panalytical). Flow cytometry was performed using ZE5 Cell Analyzer (Bio‐Rad) and the data were analyzed using FlowJo 10.5 software.

Freeze-dried crude P. patagoniensis venom (∼2 mg/ml) was homogenized in a 0.05% aqueous solution of TFA-trifluoroacetic acid and subjected to the reverse-phase high performance liquid chromatography (RP-HPLC) Shimadzu LC-10 system. In the first step, the sample was used for semipreparative Jupiter® RP-C18 LC-Column (10 μm, 300 Å, 10 mm × 129 250 mm, Phenomenex™) that had previously been balanced with A solvent (0.05% TFA). Elution was performed in a linear acetonitrile (ACN) gradient equilibrated in 0.05% TFA of 0–80% for 80 min at a flow rate of 2 ml/min. The absorbance was monitored at 225 and 280 nm, and the fractions were manually collected. Then, lyophilized fractions were suspended in 500 µL ultrapure water and an aseptically filtered in Millipore® 0.22 μm filters for antimicrobial assay.
The molecule here described (fraction 33) was submitted to a second step of fractionation in a linear gradient of ACN (20–55%) with 0.05% TFA for 80 min, at a flow rate of 1.0 ml/min and an analytic column Jupiter® C18 LC-Column (10 μm, 300 Å, 4.60 × 250 mm, Phenomenex™), with ACN equilibrated in 0.05% TFA. The hand-collected fractions were dried (Savant Instruments Inc.®) and suspended in 500 µL ultrapure water for MIC and MBC.