Crude α-syn protein after GST tag cleavage was purified using reversed phase HPLC. Columns used were an analytical Phenomenex Jupiter C4 (250 3 4.6 mm) and a preparative Phenomenex Jupiter C4 (250 3 10 mm). α-Syn was loaded into the column and eluted with a linear gradient (30-80%) of acetonitrile (70% acetonitrile, 0.1% trifluoroacetic acid,) versus water (0.1% trifluoracetic acid) in 38 minutes, at a flow rate of 0.5 ml/minute. (Fig. 2 ). Homogeneity of α-syn protein was ascertained by analytical HPLC and SDS-page (Fig. 2 ).
Jupiter c4
Manufactured by Phenomenex
Sourced in Germany
The Jupiter C4 is a high-performance liquid chromatography (HPLC) column manufactured by Phenomenex. It is designed for the separation and analysis of a wide range of analytes, including small molecules, peptides, and proteins. The column features a C4 stationary phase, which provides suitable retention and selectivity for a variety of compounds.
Automatically generated - may contain errors
Lab products found in correlation
Microflex lt sh, by Brucker (2 mentions)
2707 autosampler, by Waters Corporation (1 mentions)
Jupiter c18, by Phenomenex (1 mentions)
Preparative hplc, by Waters Corporation (1 mentions)
Histrap column, by GE Healthcare (1 mentions)
Human serum, by Merck Group (1 mentions)
Automated peptide synthesizer, by Thermo Fisher Scientific (1 mentions)
Microflex lt sh, by Bruker (1 mentions)
Stearic acid, by Merck Group (1 mentions)
Kinetex c18 column, by Phenomenex (1 mentions)
15 protocols using jupiter c4
1
Purification of α-Synuclein Protein by HPLC
2
Purification of Transcriptional Machinery Proteins
3
Purification of Peptides by Preparative HPLC
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For purification of peptides and peptide derivatives, a Waters preparative HPLC consisting of a 1525 pump module, 2707 autosampler, fraction collector III and a dual wavelength detector 2489 was used. The mobile phase was H2O/ACN containing 0.1% TFA (v/v). Columns Jupiter C18, 250 × 21.2 mm, 300 Å, 10 μm; Jupiter C18, 250 × 10.0 mm, 300 Å, 10 μm and Jupiter C4, 250 × 10.0 mm, 300 Å, 10 μm (all from Phenomenex, Aschaffenburg, Germany) were used as stationary phases.
4
Purification of Recombinant Mouse Prion Protein
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The plasmid pET-11a (Novagen) encoding for the full-length MoPrP(23–230) was kindly provided by Prof. J.R. Requena (University of Santiago de Compostela, Santiago de Compostela, Spain). An overnight culture of E. coli Rosetta 2 (DE3) (Novagen) freshly transformed with the plasmid was added at 37°C to 2 L of Zym-5052 medium [40] (link). Cells were grown in a 2 L fermenter system (Sartorius), harvested after 24 hours and lysed by homogenizer (Panda plus, GEA Niro Soavi). Inclusion bodies were washed and solubilized according to [41] (link). MoPrP was purified using its octapeptide repeat sequence as natural affinity tag for nickel. MoPrP was loaded onto a 5-mL HisTrap column (GE Healthcare) equilibrated in binding buffer (2 M GndHCl, 500 mM NaCl, 20 mM Tris, pH 8) and eluted with 500 mM imidazole. Subsequently, the protein was purified by reverse phase (Jupiter C4, Phenomenex) and separated using a gradient of 0–95% acetonitrile and 0.1% trifluoroacetic acid. The purified protein was lyophilized and refolded in acetate buffer (25 mM NaOAc, 0.005% NaN3, pH 5.5) according to [41] (link).
5
Capsid Protein Analysis by RP-HPLC
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The RP-HPLC method provides a capsid protein map. The samples were denatured with ACN and TFA, then separated on a Phenomenex Jupiter C4, 5µm, 2 × 150mm, 300Å analytical column. Detection is performed with UV at 214 nm to provide chromatograms that can be visually compared for peak consistency.
6
Serum Stability Assay for Peptides
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Human serum (Sigma Aldrich-Italy) was centrifuged at 13000 rpm for 10 min to remove lipids;the supernatant was collected and incubated at 37°C for at least 15 min. The assay started upon the addition of the peptides to the 25% serum for a final peptide concentration of 0.3 μM.
200 μL aliquots of the samples were taken after the following time points: 0, 60, 120, 180, 360 and 900 min. The aliquots were mixed with 100 μL of 15% TFA and incubated at 4°C for at least 15 min to induce the precipitation of the serum proteins. The supernatants were collected for each sample after centrifugation at 13000 rpm for 10 min and stored at -20°C.
The serum stability of peptides was followed by LC-MS analysis. using a Phenomenex Jupiter C4 (150 × 2 mm; 5 μm; 300 Å) and a method consisting of an isocratic step with H2O (0.1% TFA)/CH3CN (0.1% TFA) (70:30) for 10 min and a linear gradient of H2O (0.1% TFA)/CH3CN (0.1% TFA) from 30 to 80% of CH3CN (0.1% TFA) in 20 min at flow rate of 200 μL/min.
The assays were performed in triplicate.
200 μL aliquots of the samples were taken after the following time points: 0, 60, 120, 180, 360 and 900 min. The aliquots were mixed with 100 μL of 15% TFA and incubated at 4°C for at least 15 min to induce the precipitation of the serum proteins. The supernatants were collected for each sample after centrifugation at 13000 rpm for 10 min and stored at -20°C.
The serum stability of peptides was followed by LC-MS analysis. using a Phenomenex Jupiter C4 (150 × 2 mm; 5 μm; 300 Å) and a method consisting of an isocratic step with H2O (0.1% TFA)/CH3CN (0.1% TFA) (70:30) for 10 min and a linear gradient of H2O (0.1% TFA)/CH3CN (0.1% TFA) from 30 to 80% of CH3CN (0.1% TFA) in 20 min at flow rate of 200 μL/min.
The assays were performed in triplicate.
7
Fibrinogen Purification and Mass Spectrometry
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Fibrinogen was purified by ammonium sulphate precipitation [17] (link) from Li heparin plasma that had been used for acute phase assessment of C reactive protein (CrP). After reduction (4 h at 37 °C) in 8 M urea, 0.1 M Tris/HCl pH 8, 15 mM dithiothreitol individual fibrinogen chains were isolated by reverse phase HPLC on a Jupiter C-4 (25 × 0.46 cm) column (Phenomenex) [8] (link), [17] (link). Approximately 150 μg of fibrinogen was injected, and ~ 200 μl of individual Bβ and γ peak crests was collected and 20 μl directly injected into a VG Platform quadrupole analyser at 10 μl/min. The probe was charged at + 3000 V, and the electrospray source was maintained at 60 °C. The mass range 1050–1600 m/z was scanned with a cone ramp of 50–70. Up to 120 scans were averaged and transformed using Mass-Links software [8] (link), [17] (link).
8
Synthesis and Characterization of Cationic Peptides
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Peptides were synthesized on an automated peptide synthesizer (Applied Biosystems) using fluorenylmethyloxycarbonyl (Fmoc) solid-phase peptide synthesis strategy with Rink-amide methylbenzylhydrylamine resin (0.41 mmol/g loading) to obtain C-terminally amidated peptides. The stearic acid was coupled manually to the N terminus of the peptide overnight, at room temperature with 5 eq. stearic acid. For the synthesis of NF51 and NF57 Boc-L-Orn(Fmoc)-OH (Iris Biotech) was used. Reaction was carried out using HOBT/HBTU as coupling reagents in DMF with DIEA as an activator base. For the synthesis of PF6, a chloroquine analog trifluoromethylquinoline (QN) was coupled to the lysine tree in the side chain of TP10 as described in Andaloussi et al.22 (link)
Cleavage was performed with trifluoroacetic acid (TFA), 2.5% triisopropylsilane, and 2.5% water for 2 hr at room temperature. Peptides were purified by reversed-phase high-performance liquid chromatography on C4 column (Phenomenex Jupiter C4, 5 μm, 300A, 250 × 10 mm) using a gradient of acetonitrile/water containing 0.1% TFA. The molecular weight of the peptides was analyzed by matrix-assisted laser desorption-ionization/time of flight mass spectrometry (The Voyager-DE PRO Biospectrometry System). Concentration of the peptides was determined based on dilutions of accurately weighed substances.
Cleavage was performed with trifluoroacetic acid (TFA), 2.5% triisopropylsilane, and 2.5% water for 2 hr at room temperature. Peptides were purified by reversed-phase high-performance liquid chromatography on C4 column (Phenomenex Jupiter C4, 5 μm, 300A, 250 × 10 mm) using a gradient of acetonitrile/water containing 0.1% TFA. The molecular weight of the peptides was analyzed by matrix-assisted laser desorption-ionization/time of flight mass spectrometry (The Voyager-DE PRO Biospectrometry System). Concentration of the peptides was determined based on dilutions of accurately weighed substances.
9
Purification and Characterization of VHPK-Conjugated Anti-miR-712-FAM
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Lipo-PEG-peptide (LPP) containing VHPK (VHPK-LPP) was dissolved in chloroform and subsequently dried under a stream of nitrogen. CCL-anti-miR-712-FAM was then added to the dried LPP and stirred slowly. The resulting VHPK-CCL-anti-miR-712-FAM and nontargeted CCL-anti-miR-712-FAM were purified using Sepharose CL-4B columns. LPP incorporation was analyzed by HPLC. The HPLC column used for the analysis was Phenomenex Jupiter C4, 5 μm, 250 × 4.6 mm). A standard curve was obtained from a known concentration of LPP solution by area integration from HPLC chromatogram.
10
Peptide Synthesis and Characterization
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The peptides were synthesized on an automated peptide synthesizer (Biotage Initiator+ Alstra) using the fluorenylmethyloxycarbonyl (Fmoc) solid phase peptide synthesis strategy with Rink-Amide ChemMatrix resin (0.45 mmol g−1 loading) to obtain C-terminally amidated peptides. For fluorescently labelled peptides, the fluorescent label (FAM) 5(6)-Carboxyfluorescein was coupled manually to the N-terminus of the peptide overnight at room temperature with 5 eq. The reaction was carried out using HOBT/HBTU as coupling reagents in DMF, with DIEA as an activator base.
Cleavage was performed with trifluoroacetic acid, 2.5% triisopropylsilane, and 2.5% water for 2 h at room temperature. The peptides were purified by reversed-phase high-performance liquid chromatography on a C4 column (Phenomenex Jupiter C4, 5 μm, 300 Å, 250 × 10 mm) using a gradient of acetonitrile/water containing 0.1% TFA. The molecular weight of the peptides was analyzed by matrix-assisted laser desorption-ionization/time of flight mass spectrometry (Bruker microflex LT/SH, United States). The concentration of the peptides was determined based on the dilutions of accurately weighed substances.
Cleavage was performed with trifluoroacetic acid, 2.5% triisopropylsilane, and 2.5% water for 2 h at room temperature. The peptides were purified by reversed-phase high-performance liquid chromatography on a C4 column (Phenomenex Jupiter C4, 5 μm, 300 Å, 250 × 10 mm) using a gradient of acetonitrile/water containing 0.1% TFA. The molecular weight of the peptides was analyzed by matrix-assisted laser desorption-ionization/time of flight mass spectrometry (Bruker microflex LT/SH, United States). The concentration of the peptides was determined based on the dilutions of accurately weighed substances.
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