A 500 μg aliquot of the pooled sample was fractionated using basic pH reverse-phase HPLC (as described) (66 (link)). Briefly, the sample was loaded onto a 4.6 mm × 250 mm Xbridge C18 column (Waters, 3.5 μm bead size) using an Agilent 1260 Infinity Bio-inert HPLC and separated over a 90 min linear gradient from 10 to 50% solvent B at a flow rate of 0.5 mL/min (Buffer A = 10 mM ammonium formate, pH 10.0; Buffer B = 90% ACN, 10 mM ammonium formate, pH 10.0). A total of 120 fractions were collected and non-concatenated fractions combined into 40 final fractions. The final fractions were concentrated in the SpeedVac and stored at −80°C until further analysis.
Agilent 1260 infinity bio inert hplc
Manufactured by Agilent Technologies
The Agilent 1260 Infinity Bio-inert HPLC is a high-performance liquid chromatography system designed to handle sensitive biomolecules. It features an inert flow path to minimize sample interaction and preserve sample integrity.
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4 protocols using agilent 1260 infinity bio inert hplc
1
Fractionation of Pooled Sample by HPLC
2
High-resolution HPLC Fractionation Protocol
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A 500-μg aliquot of pooled sample was fractionated using basic pH reverse-phase HPLC (as described) (42 (link)). Briefly, the sample was loaded onto a 4.6 × 250-mm Xbridge C18 column (Waters, 3.5-μm bead size) using an Agilent 1260 Infinity Bio-inert HPLC and separated over a 70-min linear gradient from 10 to 50% solvent B at a flow rate of 0.5 ml/min (buffer A = 10 mm ammonium formate, pH 10.0; buffer B = 90% acetonitrile and 10 mm ammonium formate, pH 10.0). A total of 130 fractions were collected throughout the gradient. The early-, middle-, and late-eluting fractions were concatenated and combined into 40 final fractions. The combined fractions were concentrated in the SpeedVac and stored at −80 °C until further analysis.
3
Basic pH Reverse-Phase HPLC Fractionation
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A 500 μg aliquot of pooled sample was fractionated using basic pH reverse-phase HPLC using previously established procedures [49 (link)]. Briefly, the sample was loaded onto a 4.6 mm × 250 mm Xbridge C18 column (Waters, 3.5 μm bead size) using an Agilent 1260 Infinity Bio-inert HPLC and separated over a 70 min linear gradient from 10 to 50% solvent B at a flow rate of 0.5 ml/min (Buffer A = 10 mM ammonium formate, pH 10.0; Buffer B = 90% ACN, 10 mM ammonium formate, pH 10.0). A total of 40 fractions were collected throughout the gradient. The early, middle and late eluting fractions were concatenated and combined into 10 final fractions. The combined fractions were concentrated in the SpeedVac and stored at -80°C until further analysis.
4
HPLC Fractionation of Peptide Mixture
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The pooled sample was fractionated using basic pH reverse-phase HPLC (Buffer A = 10 mM ammonium formate, pH 10.0; Buffer B = 90% ACN, 10 mM ammonium formate, pH 10.0) on a 4.6 mm × 250 mm Xbridge C18 column (Waters, 3.5 μm bead size) using an Agilent 1260 Infinity Bio-inert HPLC. The peptide mixture was separated over a 60 min linear gradient from 10 to 50% solvent B at a flow rate of 0.5 ml/min. A total of 90 fractions were collected. Combining equal volumes of early, middle and late eluting fractions the 90 fractions were concatenated into 30 final fractions. The fractions were concentrated in the Speedvac and stored at 80 °C until further analysis.
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