Fifty-microliter-aliquots of recombinant arylsulfatases in 100 mM Tris, pH 7.5, were mixed with 50 µl of 5 mM GS solution or 50 µl of GS extracts for 2 h at ambient temperature. Reactions were stopped with 500 µl methanol and mixtures were centrifuged for 5 min. Two hundred microliters of supernatants were diluted 3-fold with distilled water and subjected to high performance liquid chromatography on an Agilent 1100 HPLC system using a reversed phase C-18 column (Nucleodur Sphinx RP, 250 × 4.6 mm, 5 µm, Macherey-Nagel, Düren, Germany) with a water (A)/acetonitrile (B) gradient (1 min: 1.5% B; 5 min: 1.5–5% B; 2 min: 5–7% B; 10 min: 7–21% B; 5 min, 21–29% B; 0.1 min: 29–100% B; 0.9 min: 100% B; 4 min: 1.5% B; flow rate: 1.0 ml min−1). Detection was performed with a photodiode array detector and peaks were integrated at 229 nm. Desulfoglucosinolates were identified based on ultraviolet absorption spectra, retention time, and mass spectra from liquid chromatography-mass spectrometry (LC-MS) analysis conducted with a Bruker Esquire 6000 IonTrap mass spectrometer.
Nucleodur sphinx rp
Manufactured by Macherey-Nagel
Sourced in Germany
Nucleodur Sphinx RP is a reversed-phase HPLC column for the separation of a wide range of organic compounds. The column features a spherical silica gel with a proprietary bonded phase, providing high efficiency and resolution.
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4 protocols using nucleodur sphinx rp
1
Quantification of Desulfoglucosinolates via HPLC
2
Glucosinolate Profiling in Plant Seedlings
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Fresh seedlings (20 to 100 mg) were harvested, weighted and freeze-dried until constant weight and ground to fine powder. GSLs were extracted with 1 mL of 80% methanol solution containing 0.05 mM of Sinalbin as internal standard. After centrifugation, 700 µL of extract was loaded onto DEAE Sephadex A 25 columns and treated with arylsulfatase for desulfation (Sigma-Aldrich). The eluted desulfo-GSLs were separated using high performance liquid chromatography (Agilent 1100 HPLC system, Agilent Technologies) on a reversed phase column (Nucleodur Sphinx RP, 250 × 4.6 mm, 5 µm, Macherey–Nagel, Düren, Germany) with a water (A)–acetonitrile (B) gradient: 0–1.0 min, 1.5% B; 1.0–6.0 min, 1.5–5% B; 6.0–8.0 min, 5–7% B; 8.0–18.0 min, 7–21% B; 18.0–23.0 min, 21–29% B; 23.0–23.1 min, 29–100% B; 23.1–24.0 min 100% B and 24.1–28.0 min 1.5% B; flow 1.0 mL min−1. Detection was performed with a photodiode array detector and peaks were integrated at 229 nm. Desulfated GSLs were identified by comparison of their retention time and UV spectra to those of purified standards previously extracted from A. thaliana42 (link). We used the following molar response factors for quantification of individual GSL relative to the internal standard Sinalbin: 2.0 for aliphatic GSLs and 0.5 for indolic GSLs43 (link).
3
Quantification of Glucosinolates by HPLC
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Fresh seedlings (20 to 100 mg) were harvested, weighted and freeze-dried until constant weight and ground to fine powder. GSLs were extracted with 1 mL of 80% methanol solution containing 0.05 mM of Sinalbin as internal standard. After centrifugation, 700 μL of extract was loaded onto DEAE Sephadex A 25 columns and treated with arylsulfatase for desulfation (Sigma-Aldrich). The eluted desulfo-GSLs were separated using high performance liquid chromatography (Agilent 1100 HPLC system, Agilent Technologies) on a reversed phase column (Nucleodur Sphinx RP, 250 x 4.6 mm, 5 μm, Macherey-Nagel, Düren, Germany) with a water (A) -acetonitrile (B) gradient: 0 -1.0 min, 1.5% B; 1.0 -6.0 min, 1.5-5% B; 6.0 -8.0 min, 5 -7% B; 8.0 -18.0 min, 7 -21% B; 18.0 -23.0 min, 21 -29% B; 23.0 -23.1 min, 29 -100% B; 23.1 -24.0 min 100% B and 24.1 -28.0 min 1.5% B; flow 1.0 mL min -1 . Detection was performed with a photodiode array detector and peaks were integrated at 229 nm. Desulfated GSLs were identified by comparison of their retention time and UV spectra to those of purified standards previously extracted from A. thaliana (Brownet al. , 2003) . We used the following molar response factors for quantification of individual GSL relative to the internal standard Sinalbin: 2.0 for aliphatic GSLs and 0.5 for indolic GSLs (Burowet al. , 2006) .
4
Quantification of Glucosinolates by HPLC
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Fresh seedlings (20 to 100 mg) were harvested, weighted and freeze-dried until constant weight and ground to fine powder. GSLs were extracted with 1 mL of 80% methanol solution containing 0.05 mM of Sinalbin as internal standard. After centrifugation, 700 μL of extract was loaded onto DEAE Sephadex A 25 columns and treated with arylsulfatase for desulfation (Sigma-Aldrich). The eluted desulfo-GSLs were separated using high performance liquid chromatography (Agilent 1100 HPLC system, Agilent Technologies) on a reversed phase column (Nucleodur Sphinx RP, 250 x 4.6 mm, 5 μm, Macherey-Nagel, Düren, Germany) with a water (A) -acetonitrile (B) gradient: 0 -1.0 min, 1.5% B; 1.0 -6.0 min, 1.5-5% B; 6.0 -8.0 min, 5 -7% B; 8.0 -18.0 min, 7 -21% B; 18.0 -23.0 min, 21 -29% B; 23.0 -23.1 min, 29 -100% B; 23.1 -24.0 min 100% B and 24.1 -28.0 min 1.5% B; flow 1.0 mL min -1 . Detection was performed with a photodiode array detector and peaks were integrated at 229 nm. Desulfated GSLs were identified by comparison of their retention time and UV spectra to those of purified standards previously extracted from A. thaliana (Brownet al. , 2003) . We used the following molar response factors for quantification of individual GSL relative to the internal standard Sinalbin: 2.0 for aliphatic GSLs and 0.5 for indolic GSLs (Burowet al. , 2006) .
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