To isolate and purify mature albumins the desalted, albumin-rich fraction from the aforementioned size exclusion chromatography was separated further by RP-HPLC on a Shimadzu Prominence system with a semi-preparative Grace Vydac C18 column (250 mm x 10 mm, 10 µm, 300 Å) and an analytical Grace Vydac C18 column (250 mm x 4.6 mm, 5 µm, 300 Å) at a 1% gradient in which buffer A was 0.05% trifluoroacetic acid and buffer B was 90% acetonitrile 0.05% trifluoroacetic acid. Forty fractions were collected but not all contained protein so only fractions 9 to 40 (F9-F40) are shown in Fig. 3. To determine the characteristic mass spectrometric pattern, each albumin fraction was analyzed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) on an SCIEX API 2000 LC-MS/MS electrospray mass spectrometer. A volume of 10 µL from each fraction was injected at a flow rate of 0.1 mL/min with buffer A to buffer B ratio of 30:70. Buffer A consisted of 0.1% formic acid and buffer B contained 0.1% formic acid in 90% acetonitrile. Specifically LC-MS/MS instrument settings were as Declustering Potential 88; Focusing Potential 220; Entrance Potential 8; Q1 MS.
C18 grace vydac column
Manufactured by Shimadzu
Sourced in Japan
The C18 Grace Vydac column is a high-performance liquid chromatography (HPLC) column used for the separation and analysis of a wide range of compounds. It features a stationary phase consisting of silica particles with chemically bonded C18 alkyl chains, which provide reversed-phase separation. The column is designed to offer efficient and reproducible separation performance for a variety of analytes, including small molecules, peptides, and proteins.
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3 protocols using c18 grace vydac column
1
Purification of Mature Albumins
2
Isolation and Purification of Ginsenosides from Panax spp.
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Dried roots, seeds, and flowers from Panax ginseng, P. quinquefolius, or P. notoginseng (Yue Hwa Chinese Products Emporium Ltd., Singapore) were pulverized and 100 mg were extracted with 0.5 mL 50% ethanol to screen for CRPs with molecular masses of 2–6 kDa by mass spectrometry using an Applied Biosystems 4800 MALDI TOF/TOF Analyzer. To obtain sufficient ginsentides for characterization studies, ~2 kg of dried material were extracted with 10 L water. The extracts were filtered and subjected to flash chromatography using C18 powder (Grace Davison). The ginsentide-enriched fractions were subsequently eluted with 60% ethanol and concentrated using a rotary evaporator. The concentrated fractions were then purified by preparative RP-HPLC using a C18 Grace Vydac column (250 × 22 mm) at a flow rate of 8 mL/min on a Shimadzu system. A linear gradient of 1%/min of 10–80% buffer B was applied. Buffer A contained 0.05% (v/v) trifluoroacetic acid (TFA) in HPLC grade water, and buffer B contained 0.05% (v/v) TFA and 99.5% (v/v) acetonitrile (ACN). To obtain isolated ginsentides, the resulting fractions were further purified by a semi-preparative C18 Vydac column (250 × 10 mm), using the same gradient, at a flow rate of 3 mL/min.
3
Ginsentide Purification from Medicinal Plants
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Ginsentide TP1, TP3 or TP8 were extracted from the dried flowers or seeds of P. ginseng, P. quinquefolius, and P. notoginseng as previously described18 . Briefly, the plant materials were pulverized and extracted with water. The extracts were filtered and subjected to flash chromatography using C18 powder (Grace Davison). 60% ethanol was used to elute the ginsentide-enriched fractions which were then loaded onto an SP Sepharose resin column (GE Healthcare, UK) and eluted with 1 mol/L NaCl (pH 3.0). Further purification was done using preparative RP-HPLC (Shimadzu, Japan) with a C18 Grace Vydac column (250 mm × 22 mm) at a flow rate of 8 mL/min, linear gradient of 1%/min of 10%–80% buffer B. Buffer A contained 0.05% (v/v) trifluoroacetic acid (TFA) in HPLC grade water, and buffer B contained 0.05% (v/v) TFA and 99.5% (v/v) acetonitrile (ACN). Resulting fractions were further purified using a semi-preparative C18 Vydac column (250 mm × 10 mm) at a flow rate of 3 mL/min with the same linear gradient. The identity of the ginsentide containing fractions were confirmed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS; AB SCIEX 5800 MALDI-TOF/TOF).
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