Protein digests were fractionated using high pH reverse-phase chromatography. A C18 column (150 × 2.1 mm inside diameter Kinetex EVO [5 µm, 100 Å]) was used with an HPLC system (LC 1260 Infinity II; Agilent). Modules were controlled by Chemstation rev 01.07 SR2. Solvent A (98% water, 2% acetonitrile) and solvent B (90% acetonitrile and 10% water) were adjusted to pH 10.0 using ammonium hydroxide. Samples were injected manually through a Rheodyne valve onto the reverse phase–high performance liquid chromatography (RP-HPLC) column equilibrated with 1% solvent B and kept at this percentage for 3 min. A two-step gradient was applied at a flow rate of 200 µl/min (from 1–25% B in 42 min and then from 25–43% B in 8 min) followed by an 8-min washing step at 100% solvent B and an 8-min reequilibration step. Column eluate was monitored at 220 and 280 nm via a variable wavelength detector and collected using an Agilent 1260 infinity fraction collector. Column eluate was collected from 4 to 63 min and divided in 21 fractions.
1260 infinity fraction collector
Manufactured by Agilent Technologies
Sourced in United States
The 1260 Infinity Fraction Collector is a versatile laboratory instrument designed for the automated collection of liquid fractions. It is a key component in various analytical workflows, such as chromatography and purification processes. The 1260 Infinity Fraction Collector collects and stores liquid fractions in a controlled and precise manner, enabling efficient sample handling and subsequent analysis.
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Lab products found in correlation
1260 infinity 2 lc, by Agilent Technologies (1 mentions)
1260 infinity manual injector, by Agilent Technologies (1 mentions)
Kinetex column, by Phenomenex (1 mentions)
Nucleodur c18 isis column, by Macherey-Nagel (1 mentions)
1260 infinity diode array detector, by Agilent Technologies (1 mentions)
1260 infinity binary pump, by Agilent Technologies (1 mentions)
4 protocols using 1260 infinity fraction collector
1
High pH Fractionation of Protein Digests
2
Basic pH Fractionation of TMT-Labeled Peptides
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Mixed TMT-labeled peptides were solubilized in 10 mM ammonium hydroxide, pH 10.0 and subsequently fractionated using basic pH reverse phase HPLC. Peptides were loaded on a Gemini 3 µm C18 110A 100 × 1.0 mm column (Phenomenex) using an Agilent 1100 pump equipped with a degasser and a photodiode array (PDA) detector. Peptides were concentrated on the column at 100 µl/min using 100% buffer A (10 mM ammonium hydroxide, pH 10) after which the fractionation gradient was applied as follow: 5% solvent B (10 mM ammonium hydroxide in 90% ACN, pH 10) to 30% B in 53 mins, 70% B in 7 min and increased to 100% B in 3 min at a flow rate of 100 µl/min. In total 60 fractions of 1 min were collected using an Agilent 1260 infinity fraction collector and then concatenated into 12 final fractions. Collected fractions were vacuum-dried, reconstituted in 5% formic acid/5% DMSO and stored at −80°C prior to mass spectrometry analysis.
3
Preparative HPLC Isolation of Pure Compounds
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For the isolation of pure compounds, a preparative high-performance liquid chromatography (HPLC) device equipped with a 1260 Infinity binary pump, a 1260 Infinity manual injector, a 1260 Infinity fraction collector, a 1260 Infinity diode array detector (all Agilent Technologies, Santa Clara, USA) and a Kinetex® column (Biphenyl, 100 Å, 5 μm, 21.2 × 250 mm, Phenomenex, Aschaffenburg, Germany) at a flow rate of 21 mL/min or—for fractions M1.2C6 and M1.2C7—a NucleodurTM C18 Isis column (RP18, 5 µm, 10 × 250 mm, Macherey-Nagel, Düren, Germany) at a flow rate of 5 mL/min was used. Separation was achieved by gradients consisting of acetonitrile (A)/water (B) and peaks were detected at 200 nm. After the elimination of acetonitrile via evaporation, the water fractions were partitioned four times with diethyl ether or ethyl acetate (M1.2C6 and M1.2C7) and organic phases were dried under a nitrogen stream. The gradients used for the separation can be found in Table 5 .
4
TMT-labeled Peptide Fractionation
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Mixed TMT-labeled peptides were solubilized in 10mM ammonium hydroxide, pH 10.0 and subsequently fractionated using basic pH reverse phase HPLC. Peptides were loaded on a Gemini 3µm C18 110A 100 x 1.0 mm column (Phenomenex) using an Agilent 1100 pump equipped with a degasser and a photodiode array (PDA) detector. Peptides were concentrated on the column at 100µl/min using 100% buffer A (10mM ammonium hydroxide, pH 10) after which the fractionation gradient was applied as follow: 5% solvent B (10mM ammonium hydroxide in 90% ACN, pH 10) to 30% B in 53 mins, 70% B in 7 min and increased to 100% B in 3 min at a flow rate of 100µl/min. In total 60 fractions of 1 min were collected using an Agilent 1260 infinity fraction collector and then concatenated into 12 final fractions. Collected fractions were vacuum-dried, reconstituted in 5% formic acid/5% DMSO and stored at -80°C prior mass spectrometry analysis.
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