Dried calyces (1 kg) of Hibiscus sabdariffa were extracted for 15 min with water and centrifuged at 9,000 rpm for 10 min at 4 °C (Beckman Coulter, USA) and the supernatant was filtered through 1 μM pore size glass fiber filter paper (Sartorius, Singapore). The filtrate was then loaded onto a C18 flash column (Grace Davison, US) and eluted with 60% ethanol/0.01% TFA. The eluted fractions were then loaded onto an SP Sepharose resin column (GE Healthcare, UK), eluted with 1 M NaCl (pH 3.0), and followed by ultrafiltration (ViVaflow 200, 2000 MWCO hydrostat). Further purification was performed by reversed-phase high performance liquid chromatography (RP-HPLC) (Shimadzu, Japan). A linear gradient of mobile phase A (0.05% TFA/H2O) and mobile phase B (0.05% TFA/ACN) was used on the C18 column (250 × 22 mm, 5 μm, 300 Å) (Grace Davison, US). MALDI-TOF MS was used to identify the presence of roseltide rT1 in the eluted fractions. The eluted fractions were lyophilized for storage at room temperature.
Sp sepharose resin column
Manufactured by GE Healthcare
Sourced in United Kingdom
The SP Sepharose resin column is a chromatography media used for the purification of biomolecules. It consists of cross-linked agarose beads with covalently attached sulfopropyl (SP) functional groups, which act as a strong cation exchange material. This resin is designed to capture and separate positively charged molecules based on their differences in ionic interactions with the SP ligands.
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3 protocols using sp sepharose resin column
1
Purification of Roseltide rT1 from Hibiscus
2
Extraction and Purification of Bleogen pB1
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Fresh Pereskia bleo leaves (1 kg) were blended for 15 min with water and centrifuged at 9000 rpm for 10 min at 4°C (Beckman Coulter, United States). The supernatant was filtered through 1 μM pore size glass fiber filter paper (Sartorius, Singapore). The filtrate was loaded onto a C18 flash column (Grace Davison, United States) and eluted with 60% ethanol. The eluted fractions were then loaded onto an SP Sepharose resin column (GE Healthcare, United Kingdom), eluted with 1 M NaCl (pH 3.0), followed by ultrafiltration (ViVaflow 200, 2000 MWCO hydrostat). Further purification was performed by RP-HPLC (Shimadzu, Japan). A linear gradient of mobile phase A (0.05% TFA/H2O) and mobile phase B (0.05% TFA/ACN) was used with a C18 column (250 mm × 22 mm, 5 μm, 300Å; Grace Davison, United States). MALDI-TOF MS was used to identify the presence of bleogen pB1 in the eluted fractions. The eluted fractions containing bleogen pB1 were lyophilized for storage at room temperature. The MALDI spectra were acquired in the m/z range of 500–6000, with a focus m/z 3500. Total laser shots were 2250 with a laser intensity of 3500.
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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