reverse-phase hplc Search Results


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  • 99
    Thermo Fisher reverse phase hplc
    Reverse Phase Hplc, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 736 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore reverse phase hplc
    Reverse Phase Hplc, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 204 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Agilent technologies reverse phase hplc
    Reverse Phase Hplc, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 99/100, based on 2244 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Waters Corporation reversed phase hplc
    Turnover of <t>BODIPY-SM</t> after pulse labeling. CATH.a cells were cooled at 4 °C for 10 min, pulse-labeled with BODIPY-SM (1 μM) in serum-free culture medium for 30 min at 4 °C, washed, and chased for the indicated time periods at 37 °C. Cells were washed with HBSS, scraped, followed by lipid extraction from (A) cells and (B) the cellular supernatant. Lipid extracts were subjected to <t>HPLC</t> analysis. Results shown represent mean values from one representative experiment performed in duplicates.
    Reversed Phase Hplc, supplied by Waters Corporation, used in various techniques. Bioz Stars score: 94/100, based on 1670 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Waters Corporation reverse phase hplc
    Enzymatic assay of recombinant TcLAR. A , <t>HPLC</t> analysis of products from incubation of leucocyanidin with boiled protein as control, monitored by UV spectroscopy. B , as above, for incubation of purified recombinant TcLAR with 3 H-leucocyanidin. C , As in (A) , but monitoring radioactivity of fractions; D , As in (B) , but monitoring radioactivity of fractions; ( E ), UV spectrum of catechin product from (B) ; F , HPLC analysis of an authentic standard of (−)-catechin. mAU, milliabsorbance units; CPM, counts per minute.
    Reverse Phase Hplc, supplied by Waters Corporation, used in various techniques. Bioz Stars score: 93/100, based on 1904 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Phenomenex reversed phase hplc
    Purification of SPINK6 from human stratum corneum extracts. A , natural SPINK6 was isolated from human plantar stratum corneum extracts using first an anti-SPINK6 affinity column (not shown), followed by a <t>C18</t> reversed-phase <t>HPLC</t> column. ESI-MS analyses
    Reversed Phase Hplc, supplied by Phenomenex, used in various techniques. Bioz Stars score: 94/100, based on 1486 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Agilent technologies reversed phase hplc
    Purification of SPINK6 from human stratum corneum extracts. A , natural SPINK6 was isolated from human plantar stratum corneum extracts using first an anti-SPINK6 affinity column (not shown), followed by a <t>C18</t> reversed-phase <t>HPLC</t> column. ESI-MS analyses
    Reversed Phase Hplc, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 99/100, based on 868 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Phenomenex reverse phase hplc
    The synthesis of sugar nucleotides from 2-chloro-4-nitrophenyl glucosides. ( a ) General reaction scheme. ( b ) Structures of 2-chloro-4-nitrophenyl glycoside donors evaluated for D-sugars within this series, the differences between each member and the native OleD sugar substrate (β-D-glucose) are highlighted in red. ( c ) Maximum observed percent conversion of (U/T)DP to (U/T)DP-glucose within a 21 hour time course assay for each donor (n ≥ 2, standard deviation ≤ 5%). Standard reactions contained 7 μM TDP-16, 1 mM (U/T)DP, and 1 mM of 2-chloro-4-nitrophenyl glycoside donor ( 9 , 34 – 47 ) in Tris-HCl buffer (50 mM, pH 8.5) with a final volume of 300 μl. Over 21 hours at 25°C, aliquots taken at various times were flash frozen and analyzed by <t>HPLC</t> ( Supplementary Methods ). For reactions with <t>UDP</t> yielding
    Reverse Phase Hplc, supplied by Phenomenex, used in various techniques. Bioz Stars score: 93/100, based on 1307 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Shimadzu Corporation reversed phase hplc
    Serotonin (5-HT) and 5-hydroxyindole-3-acetic acid <t>(5-HIAA)</t> levels in neostriatum. n-3 PUFA supplementation modulates 5-HT turnover in both male and female SHR rats. Levels of ( a ) 5-HT and ( b ) 5-HIAA were used to determine ( c ) 5-HIAA/5-HT ratios. The data are presented as means ± SEM (n = 4–8) and the level of significance is symbolized with * (p-value ≤ 0.05), ** (p-value ≤ 0.01) or *** (p-value ≤ 0.001). The p-values are calculated by comparison with the mid bar, which represents control-fed SHRs. The analyses were performed by <t>HPLC</t> using electrochemical detection.
    Reversed Phase Hplc, supplied by Shimadzu Corporation, used in various techniques. Bioz Stars score: 92/100, based on 524 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Shimadzu Corporation reverse phase hplc
    The radioactive signal from <t>oligopeptides</t> being modified with radioactive acetyl CoA . The oligopeptide 1 MLGTE-RRR 24 (200 μM) was incubated with [1- 14 C] acetyl CoA (final 300 μM with specific activity 11.2 mCi/mmol) and purified MBP-hNaa30p (80 nM) in acetylation buffer for 60 minutes at 37°C. Samples were collected at indicated time points and analysed with reverse phase <t>HPLC</t> that had been connected to a radioactivity flow detector after the absorbance detector. A clear time dependent increase in the radioactivity signal is observed, verifying that the eluted oligopeptides are labelled with radioactive acetyl groups.
    Reverse Phase Hplc, supplied by Shimadzu Corporation, used in various techniques. Bioz Stars score: 92/100, based on 628 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher reversed phase hplc
    The radioactive signal from <t>oligopeptides</t> being modified with radioactive acetyl CoA . The oligopeptide 1 MLGTE-RRR 24 (200 μM) was incubated with [1- 14 C] acetyl CoA (final 300 μM with specific activity 11.2 mCi/mmol) and purified MBP-hNaa30p (80 nM) in acetylation buffer for 60 minutes at 37°C. Samples were collected at indicated time points and analysed with reverse phase <t>HPLC</t> that had been connected to a radioactivity flow detector after the absorbance detector. A clear time dependent increase in the radioactivity signal is observed, verifying that the eluted oligopeptides are labelled with radioactive acetyl groups.
    Reversed Phase Hplc, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 539 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Phenomenex reverse phase hplc column
    The radioactive signal from <t>oligopeptides</t> being modified with radioactive acetyl CoA . The oligopeptide 1 MLGTE-RRR 24 (200 μM) was incubated with [1- 14 C] acetyl CoA (final 300 μM with specific activity 11.2 mCi/mmol) and purified MBP-hNaa30p (80 nM) in acetylation buffer for 60 minutes at 37°C. Samples were collected at indicated time points and analysed with reverse phase <t>HPLC</t> that had been connected to a radioactivity flow detector after the absorbance detector. A clear time dependent increase in the radioactivity signal is observed, verifying that the eluted oligopeptides are labelled with radioactive acetyl groups.
    Reverse Phase Hplc Column, supplied by Phenomenex, used in various techniques. Bioz Stars score: 91/100, based on 320 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Agilent technologies reversed phase high performance liquid chromatography rp hplc
    Effect of DB3 and DB3DB3 on different <t>Aβ</t> aggregation species. A) Analysis of Aβ(1–42) aggregation species with density gradient centrifugation and followed by analysis using silver-stained Tricine-SDS-PAGE to analyze the influence of DB3 and DB3DB3 on the distribution of Aβ assemblies. B) Quantification of Aβ(1–42) by <t>RP-HPLC.</t> All data were recorded in triplicate.
    Reversed Phase High Performance Liquid Chromatography Rp Hplc, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 93/100, based on 385 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Phenomenex semi preparative reversed phase hplc
    Chromatography elution profiles and calcium binding capacities of calcium-binding peptides. ( A ) <t>Sephadex</t> G-25 gel filtration chromatography of SPH; ( B ) Semi-preparative C18 <t>RP-HPLC</t> of fraction III; ( C ) RP-HPLC of fraction 17 from semi-preparative HPLC.
    Semi Preparative Reversed Phase Hplc, supplied by Phenomenex, used in various techniques. Bioz Stars score: 90/100, based on 162 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Agilent technologies reverse phase high performance liquid chromatography hplc
    ( A ) Coomassie blue staining of the purified region-1 and region-2 of mPCSK9 proteins. Region-1 (residues 152–351) and region-2 (residues 249–452) of mouse catalytic domain were cloned and expressed by pET-3a plasmids. The proteins were reduced and purified by a reverse-phase high-performance liquid chromatography <t>(HPLC)</t> on an <t>Agilent</t> 1100 HPLC system. The purified proteins were analyzed by SDS/PAGE and stained with Coomassie blue. The positions of expressed proteins are shown; ( B ) MALDI mass spectrometry on Region-1. The molecular mass of purified PCSK9 region-1 in fully reduced form (PCSK9-1R). The molecular mass of PCSK9 region-1 as determined by MALDI was 21,201 Da; ( C ) MALDI mass spectrometry on Region-2. The molecular mass of purified PCSK9 region-2 in fully reduced form (PCSK9-2R). The molecular mass of PCSK9 region-2 as determined by MALDI was 21,355 Da.
    Reverse Phase High Performance Liquid Chromatography Hplc, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 91/100, based on 298 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Waters Corporation reversed phase high performance liquid chromatography rp hplc
    Representative <t>HPLC</t> chromatogram of <t>ceftriaxone</t> sodium
    Reversed Phase High Performance Liquid Chromatography Rp Hplc, supplied by Waters Corporation, used in various techniques. Bioz Stars score: 92/100, based on 218 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Shimadzu Corporation reversed phase high performance liquid chromatography rp hplc
    <t>RP-HPLC</t> analysis of <t>fibrinopeptides.</t> Standard fibrinopeptides A ( a ) and B ( b ), used for comparative purposes ( A ) and fibrinopeptides formed by incubating human fibrinogen (3 mg/mL) with thrombin (5 µg/mL); ( B ) or Moojase (20 µg/mL); ( C ) at 37 °C for 120 min were analyzed by reversed phase HPLC at 214 nm.
    Reversed Phase High Performance Liquid Chromatography Rp Hplc, supplied by Shimadzu Corporation, used in various techniques. Bioz Stars score: 93/100, based on 202 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Phenomenex reversed phase hplc column
    <t>RP-HPLC</t> analysis of <t>fibrinopeptides.</t> Standard fibrinopeptides A ( a ) and B ( b ), used for comparative purposes ( A ) and fibrinopeptides formed by incubating human fibrinogen (3 mg/mL) with thrombin (5 µg/mL); ( B ) or Moojase (20 µg/mL); ( C ) at 37 °C for 120 min were analyzed by reversed phase HPLC at 214 nm.
    Reversed Phase Hplc Column, supplied by Phenomenex, used in various techniques. Bioz Stars score: 91/100, based on 129 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Merck & Co reversed phase hplc
    Translocation. A. Colicin E3-cleaved (closed circles) or control (open circles) pre-translocation complexes (0.1 μM) were mixed in a quench-flow apparatus with EF-G·GTP (3 μM) and Pmn (10 mM) and translocation analysed by formation of <t>fMetPhe-Pmn.</t> The amount of pre-translocation complex (PTC) was determined by <t>HPLC</t> analysis of fMetPhe. B. Translocation was monitored by the fluorescence change of fMetPhe-tRNA Phe (Prf16/17). Curve 1 – control ribosomes; curve 2 – colicin E3-cleaved ribosomes; curve 3 – control ribosomes in the presence of viomycin (200 μM); curve 4 – colicin E3-cleaved ribosomes with viomycin (200 μM). Normalized ΔF, fluorescence change normalized by the maximum extent of translocation determined by the Pmn reaction. C. Pi release upon GTP hydrolysis by EF-G (curves 1 and 2) or buffer control (curves 3 and 4) with control ribosomes (curves 1 and 3) or colicin E3-cleaved (curves 2 and 4). Pi release was measured by the fluorescence change of MDCC-PBP upon Pi binding ( Experimental procedures ). D. Multiple-turnover GTP hydrolysis by EF-G. Velocity of GTP hydrolysis was measured with catalytic concentrations of EF-G (10 nM) and increasing concentrations of colicin E3-cleaved ribosomes (closed circles) or control ribosomes (open circles) in the presence of excess [γ- 32 P]-GTP (200 μM).
    Reversed Phase Hplc, supplied by Merck & Co, used in various techniques. Bioz Stars score: 94/100, based on 220 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Waters Corporation reverse phase hplc column
    ESI/LC/MS analysis of CYP2B1 apoprotein inactivated by BPA or <t>BMP.</t> The incubation conditions, <t>HPLC,</t> and MS analysis conditions were as described under Materials and Methods. A, representative deconvoluted mass spectrum from a control incubation of CYP2B1 with either BPA or BMP in the absence of NADPH. B, representative deconvoluted mass spectrum of P450 2B1 incubated with BPA in the presence of NADPH. C, representative deconvoluted mass spectrum of CYP2B1 incubated with BMP in the presence of NADPH.
    Reverse Phase Hplc Column, supplied by Waters Corporation, used in various techniques. Bioz Stars score: 92/100, based on 273 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Phenomenex preparative reverse phase hplc
    ESI/LC/MS analysis of CYP2B1 apoprotein inactivated by BPA or <t>BMP.</t> The incubation conditions, <t>HPLC,</t> and MS analysis conditions were as described under Materials and Methods. A, representative deconvoluted mass spectrum from a control incubation of CYP2B1 with either BPA or BMP in the absence of NADPH. B, representative deconvoluted mass spectrum of P450 2B1 incubated with BPA in the presence of NADPH. C, representative deconvoluted mass spectrum of CYP2B1 incubated with BMP in the presence of NADPH.
    Preparative Reverse Phase Hplc, supplied by Phenomenex, used in various techniques. Bioz Stars score: 91/100, based on 126 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Turnover of BODIPY-SM after pulse labeling. CATH.a cells were cooled at 4 °C for 10 min, pulse-labeled with BODIPY-SM (1 μM) in serum-free culture medium for 30 min at 4 °C, washed, and chased for the indicated time periods at 37 °C. Cells were washed with HBSS, scraped, followed by lipid extraction from (A) cells and (B) the cellular supernatant. Lipid extracts were subjected to HPLC analysis. Results shown represent mean values from one representative experiment performed in duplicates.

    Journal: Biochimica et Biophysica Acta

    Article Title: Endocytosis and intracellular processing of BODIPY-sphingomyelin by murine CATH.a neurons

    doi: 10.1016/j.bbalip.2013.08.007

    Figure Lengend Snippet: Turnover of BODIPY-SM after pulse labeling. CATH.a cells were cooled at 4 °C for 10 min, pulse-labeled with BODIPY-SM (1 μM) in serum-free culture medium for 30 min at 4 °C, washed, and chased for the indicated time periods at 37 °C. Cells were washed with HBSS, scraped, followed by lipid extraction from (A) cells and (B) the cellular supernatant. Lipid extracts were subjected to HPLC analysis. Results shown represent mean values from one representative experiment performed in duplicates.

    Article Snippet: BODIPY- and PYRENE-labeled lipids were separated by reversed-phase HPLC (Waters HPLC 2690 Separations Module) on a Kromasil C18 reversed-phase column (150 × 4.6 mm, 5 μm particle size; Altmann Analytik, München, Germany) equipped with the corresponding guard column (5 × 4.6 mm, 5 μm particle size) at a flow rate of 0.7–1 ml/min.

    Techniques: Labeling, High Performance Liquid Chromatography

    Analysis of PM-located BODIPY-SM. CATH.a cells were cooled at 4 °C for 10 min and pulse-labeled with BODIPY-SM (1 μM for B. cereus SMase treatment and 2 μM for the BE protocol) in serum-free culture medium for 30 min at 4 °C. (A) Cells were washed and chased in serum-free culture medium at 37 °C for 60 min to enable BODIPY-SM internalization. Cells were then subjected to B. cereus SMase treatment (150 mU/ml) in serum-free culture medium at 37 °C up to 4 h. Cellular lipid extracts were analyzed by HPLC. (B) After pulse labeling cells were washed and chased at 37 °C. At the indicated times cells were subjected to BE at 4 °C followed by BODIPY-SL analysis in the BE fractions and cell extracts by HPLC. Results shown represent mean values from one representative experiment performed in duplicates.

    Journal: Biochimica et Biophysica Acta

    Article Title: Endocytosis and intracellular processing of BODIPY-sphingomyelin by murine CATH.a neurons

    doi: 10.1016/j.bbalip.2013.08.007

    Figure Lengend Snippet: Analysis of PM-located BODIPY-SM. CATH.a cells were cooled at 4 °C for 10 min and pulse-labeled with BODIPY-SM (1 μM for B. cereus SMase treatment and 2 μM for the BE protocol) in serum-free culture medium for 30 min at 4 °C. (A) Cells were washed and chased in serum-free culture medium at 37 °C for 60 min to enable BODIPY-SM internalization. Cells were then subjected to B. cereus SMase treatment (150 mU/ml) in serum-free culture medium at 37 °C up to 4 h. Cellular lipid extracts were analyzed by HPLC. (B) After pulse labeling cells were washed and chased at 37 °C. At the indicated times cells were subjected to BE at 4 °C followed by BODIPY-SL analysis in the BE fractions and cell extracts by HPLC. Results shown represent mean values from one representative experiment performed in duplicates.

    Article Snippet: BODIPY- and PYRENE-labeled lipids were separated by reversed-phase HPLC (Waters HPLC 2690 Separations Module) on a Kromasil C18 reversed-phase column (150 × 4.6 mm, 5 μm particle size; Altmann Analytik, München, Germany) equipped with the corresponding guard column (5 × 4.6 mm, 5 μm particle size) at a flow rate of 0.7–1 ml/min.

    Techniques: Labeling, High Performance Liquid Chromatography

    Uptake and metabolism of fluorescent SM and Cer analogs during steady state labeling. CATH.a cells were incubated with (A) BODIPY-SM, (B) PYRENE-SM, or (C) BODIPY-Cer (1 μM each) for the indicated time periods at 37 °C. Cells were washed with HBSS, scraped, and cellular lipids were extracted with CHCl 3 /MeOH, dried, dissolved in ethanol, and analyzed by HPLC. Results shown represent mean values from one representative experiment performed in duplicates.

    Journal: Biochimica et Biophysica Acta

    Article Title: Endocytosis and intracellular processing of BODIPY-sphingomyelin by murine CATH.a neurons

    doi: 10.1016/j.bbalip.2013.08.007

    Figure Lengend Snippet: Uptake and metabolism of fluorescent SM and Cer analogs during steady state labeling. CATH.a cells were incubated with (A) BODIPY-SM, (B) PYRENE-SM, or (C) BODIPY-Cer (1 μM each) for the indicated time periods at 37 °C. Cells were washed with HBSS, scraped, and cellular lipids were extracted with CHCl 3 /MeOH, dried, dissolved in ethanol, and analyzed by HPLC. Results shown represent mean values from one representative experiment performed in duplicates.

    Article Snippet: BODIPY- and PYRENE-labeled lipids were separated by reversed-phase HPLC (Waters HPLC 2690 Separations Module) on a Kromasil C18 reversed-phase column (150 × 4.6 mm, 5 μm particle size; Altmann Analytik, München, Germany) equipped with the corresponding guard column (5 × 4.6 mm, 5 μm particle size) at a flow rate of 0.7–1 ml/min.

    Techniques: Labeling, Incubation, High Performance Liquid Chromatography

    Quantitation of BODIPY-SM endocytosis in the presence of pharmacological antagonists. CATH.a cells were incubated with BODIPY-SM (1 μM) and the corresponding inhibitors as described in Fig. 3 . Cells were scraped in ice-cold HBSS and lipids were extracted with CHCl 3 /MeOH. Dried lipids were re-dissolved in ethanol and analyzed by HPLC. Results are expressed as fluorescence intensity as % of control and represent mean ± SD (n = 3) of one representative experiment. ***p

    Journal: Biochimica et Biophysica Acta

    Article Title: Endocytosis and intracellular processing of BODIPY-sphingomyelin by murine CATH.a neurons

    doi: 10.1016/j.bbalip.2013.08.007

    Figure Lengend Snippet: Quantitation of BODIPY-SM endocytosis in the presence of pharmacological antagonists. CATH.a cells were incubated with BODIPY-SM (1 μM) and the corresponding inhibitors as described in Fig. 3 . Cells were scraped in ice-cold HBSS and lipids were extracted with CHCl 3 /MeOH. Dried lipids were re-dissolved in ethanol and analyzed by HPLC. Results are expressed as fluorescence intensity as % of control and represent mean ± SD (n = 3) of one representative experiment. ***p

    Article Snippet: BODIPY- and PYRENE-labeled lipids were separated by reversed-phase HPLC (Waters HPLC 2690 Separations Module) on a Kromasil C18 reversed-phase column (150 × 4.6 mm, 5 μm particle size; Altmann Analytik, München, Germany) equipped with the corresponding guard column (5 × 4.6 mm, 5 μm particle size) at a flow rate of 0.7–1 ml/min.

    Techniques: Quantitation Assay, Incubation, High Performance Liquid Chromatography, Fluorescence

    Uptake of HDL-associated BODIPY-SM. (A) Protein lysates obtained from porcine brain microvascular endothelial cells (positive control; lane 1) and CATH.a cells incubated for 24 h in the absence (lane 2) or presence (lane 3) of serum were separated on 10% SDS gels and transferred to PVDF membranes for subsequent detection using anti-SR-BI as a primary antibody. Immunoreactive bands were detected at an apparent molecular weight of approx. 80 and 160 kDa (arrows). (B) Cells were incubated in serum-free medium (a–d) or in the presence of hypertonic sucrose (e) for 30 min followed by addition of BODIPY-SM/DiI-labeled HDL (50 μg HDL protein/ml) for (a) 5 min, (b, d, and e) 60 min, or (c) 240 min at 37 °C. Cells in (d) received a 40-fold excess of unlabeled HDL. After washing with HBSS cells were subjected to LSM analysis. All micrographs were recorded with the same laser intensity. (C) Cells were incubated in serum-free medium (a–d) or in the presence of hypertonic sucrose (e) for 30 min followed by addition of BODIPY-SM/Cy3-labeled HDL (50 μg HDL protein/ml) for (a) 5 min, (b, d, and e) 60 min, or (c) 240 min at 37 °C. Cells in (d) received a 40-fold excess of unlabeled HDL. After washing with HBSS cells were subjected to LSM analysis. All micrographs were recorded with the same laser intensity. (D) Cells were incubated with BODIPY-SM-labeled HDL (50 μg HDL protein/ml) for the indicated times at 37 °C. Cellular lipids were extracted, dissolved in ethanol, and analyzed by HPLC. Results shown represent mean values from one representative experiment performed in duplicates.

    Journal: Biochimica et Biophysica Acta

    Article Title: Endocytosis and intracellular processing of BODIPY-sphingomyelin by murine CATH.a neurons

    doi: 10.1016/j.bbalip.2013.08.007

    Figure Lengend Snippet: Uptake of HDL-associated BODIPY-SM. (A) Protein lysates obtained from porcine brain microvascular endothelial cells (positive control; lane 1) and CATH.a cells incubated for 24 h in the absence (lane 2) or presence (lane 3) of serum were separated on 10% SDS gels and transferred to PVDF membranes for subsequent detection using anti-SR-BI as a primary antibody. Immunoreactive bands were detected at an apparent molecular weight of approx. 80 and 160 kDa (arrows). (B) Cells were incubated in serum-free medium (a–d) or in the presence of hypertonic sucrose (e) for 30 min followed by addition of BODIPY-SM/DiI-labeled HDL (50 μg HDL protein/ml) for (a) 5 min, (b, d, and e) 60 min, or (c) 240 min at 37 °C. Cells in (d) received a 40-fold excess of unlabeled HDL. After washing with HBSS cells were subjected to LSM analysis. All micrographs were recorded with the same laser intensity. (C) Cells were incubated in serum-free medium (a–d) or in the presence of hypertonic sucrose (e) for 30 min followed by addition of BODIPY-SM/Cy3-labeled HDL (50 μg HDL protein/ml) for (a) 5 min, (b, d, and e) 60 min, or (c) 240 min at 37 °C. Cells in (d) received a 40-fold excess of unlabeled HDL. After washing with HBSS cells were subjected to LSM analysis. All micrographs were recorded with the same laser intensity. (D) Cells were incubated with BODIPY-SM-labeled HDL (50 μg HDL protein/ml) for the indicated times at 37 °C. Cellular lipids were extracted, dissolved in ethanol, and analyzed by HPLC. Results shown represent mean values from one representative experiment performed in duplicates.

    Article Snippet: BODIPY- and PYRENE-labeled lipids were separated by reversed-phase HPLC (Waters HPLC 2690 Separations Module) on a Kromasil C18 reversed-phase column (150 × 4.6 mm, 5 μm particle size; Altmann Analytik, München, Germany) equipped with the corresponding guard column (5 × 4.6 mm, 5 μm particle size) at a flow rate of 0.7–1 ml/min.

    Techniques: Positive Control, Incubation, Molecular Weight, Labeling, High Performance Liquid Chromatography

    Enzymatic assay of recombinant TcLAR. A , HPLC analysis of products from incubation of leucocyanidin with boiled protein as control, monitored by UV spectroscopy. B , as above, for incubation of purified recombinant TcLAR with 3 H-leucocyanidin. C , As in (A) , but monitoring radioactivity of fractions; D , As in (B) , but monitoring radioactivity of fractions; ( E ), UV spectrum of catechin product from (B) ; F , HPLC analysis of an authentic standard of (−)-catechin. mAU, milliabsorbance units; CPM, counts per minute.

    Journal: BMC Plant Biology

    Article Title: Proanthocyanidin synthesis in Theobroma cacao: genes encoding anthocyanidin synthase, anthocyanidin reductase, and leucoanthocyanidin reductase

    doi: 10.1186/1471-2229-13-202

    Figure Lengend Snippet: Enzymatic assay of recombinant TcLAR. A , HPLC analysis of products from incubation of leucocyanidin with boiled protein as control, monitored by UV spectroscopy. B , as above, for incubation of purified recombinant TcLAR with 3 H-leucocyanidin. C , As in (A) , but monitoring radioactivity of fractions; D , As in (B) , but monitoring radioactivity of fractions; ( E ), UV spectrum of catechin product from (B) ; F , HPLC analysis of an authentic standard of (−)-catechin. mAU, milliabsorbance units; CPM, counts per minute.

    Article Snippet: Catechin and epicatechin content was determined by reverse-phase HPLC using an Alliance separations module (Model 2695; Waters, Milford, MA, USA) equipped with a multi λ fluorescence detector (Model 2475; Waters, Milford, MA, USA).

    Techniques: Enzymatic Assay, Recombinant, High Performance Liquid Chromatography, Incubation, Spectroscopy, Purification, Radioactivity

    Complementation of the Arabidopsis PA-deficient ldox mutant by constitutively expressing TcLAR . A , Catechin and epicatechin standards analyzed by HPLC. B , HPLC analysis of Col-0 young siliques. C , HPLC analysis of ldox 35S:TcLAR Line14 young siliques. D , HPLC analysis of ldox mutant young siliques. E . TcLAR and AtUbiquitin transcripts in total RNA from leaves of ldox 35S:TcLAR transgenic plants, (line 9, line 14 and line 36), Col-O and ldox muant plants by RT-PCR. PCR products from the TcLAR-PGEM plasmid were loaded on the last lane as a positive control for the TcLAR primer set and as a negative control for the AtUbiquitin primer set. F , Catechin and epicatechin levels in young siliques of plants that were the source of the total RNA used in (E) . Catechin and epicatechin levels were determined by extraction and HPLC analysis. The data are presented as means ± SE, n = 3. *P

    Journal: BMC Plant Biology

    Article Title: Proanthocyanidin synthesis in Theobroma cacao: genes encoding anthocyanidin synthase, anthocyanidin reductase, and leucoanthocyanidin reductase

    doi: 10.1186/1471-2229-13-202

    Figure Lengend Snippet: Complementation of the Arabidopsis PA-deficient ldox mutant by constitutively expressing TcLAR . A , Catechin and epicatechin standards analyzed by HPLC. B , HPLC analysis of Col-0 young siliques. C , HPLC analysis of ldox 35S:TcLAR Line14 young siliques. D , HPLC analysis of ldox mutant young siliques. E . TcLAR and AtUbiquitin transcripts in total RNA from leaves of ldox 35S:TcLAR transgenic plants, (line 9, line 14 and line 36), Col-O and ldox muant plants by RT-PCR. PCR products from the TcLAR-PGEM plasmid were loaded on the last lane as a positive control for the TcLAR primer set and as a negative control for the AtUbiquitin primer set. F , Catechin and epicatechin levels in young siliques of plants that were the source of the total RNA used in (E) . Catechin and epicatechin levels were determined by extraction and HPLC analysis. The data are presented as means ± SE, n = 3. *P

    Article Snippet: Catechin and epicatechin content was determined by reverse-phase HPLC using an Alliance separations module (Model 2695; Waters, Milford, MA, USA) equipped with a multi λ fluorescence detector (Model 2475; Waters, Milford, MA, USA).

    Techniques: Mutagenesis, Expressing, High Performance Liquid Chromatography, Transgenic Assay, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Plasmid Preparation, Positive Control, Negative Control

    Expression analysis of key PA biosynthesis genes, and PA composition in various tissues of Theobroma cacao . A , Gene expression levels of TcANR , TcLAR1 and TcLAR1 . Expression was determined by semi-quantitative RT-PCR normalized relative to the expression of TcActin in each sample. B , Levels of soluble PAs expressed as mg PA per g of dry weight. C , Levels of insoluble PAs expressed as mg PA per g of dry weight. D , Catechin and epicatechin standards analyzed by HPLC. E , Representative HPLC analysis of flavan-3-ols extracted from cacao leaves. F , Flavan-3-ol accumulation and composition analyzed by HPLC. All data are presented as means ± SE, for gene expression data, n ≥ 3, for PA level data, n ≥ 5. DW, dry weight.

    Journal: BMC Plant Biology

    Article Title: Proanthocyanidin synthesis in Theobroma cacao: genes encoding anthocyanidin synthase, anthocyanidin reductase, and leucoanthocyanidin reductase

    doi: 10.1186/1471-2229-13-202

    Figure Lengend Snippet: Expression analysis of key PA biosynthesis genes, and PA composition in various tissues of Theobroma cacao . A , Gene expression levels of TcANR , TcLAR1 and TcLAR1 . Expression was determined by semi-quantitative RT-PCR normalized relative to the expression of TcActin in each sample. B , Levels of soluble PAs expressed as mg PA per g of dry weight. C , Levels of insoluble PAs expressed as mg PA per g of dry weight. D , Catechin and epicatechin standards analyzed by HPLC. E , Representative HPLC analysis of flavan-3-ols extracted from cacao leaves. F , Flavan-3-ol accumulation and composition analyzed by HPLC. All data are presented as means ± SE, for gene expression data, n ≥ 3, for PA level data, n ≥ 5. DW, dry weight.

    Article Snippet: Catechin and epicatechin content was determined by reverse-phase HPLC using an Alliance separations module (Model 2695; Waters, Milford, MA, USA) equipped with a multi λ fluorescence detector (Model 2475; Waters, Milford, MA, USA).

    Techniques: Expressing, Quantitative RT-PCR, High Performance Liquid Chromatography

    Isolation of Tγ−X product from ROS membranes. (A) Immunoblot patterns of the extracted Tγ−X species. The GTPγS-wash and phosducin-extraction procedures were repeated three and two times, respectively. These aliquots of supernatants (s) and pellets (p) were electrophoresed and transferred to a PVDF membrane for immunostaining. Shown is the clipped image of the anti-Tγ blot at the gel front region. (B) Immunoblot patterns of the fractions eluted from the reverse-phase HPLC as described in Experimental Procedures. Each fraction was subjected to SDS−polyacrylamide gel (12.5%) electrophoresis and immunostaining by anti-Tγ antibody. The fractions containing the Tγ−X product (fractions 25−34) are shown.

    Journal: Biochemistry

    Article Title: Interacting Targets of the Farnesyl of Transducin ?-Subunit

    doi: 10.1021/bi800359h

    Figure Lengend Snippet: Isolation of Tγ−X product from ROS membranes. (A) Immunoblot patterns of the extracted Tγ−X species. The GTPγS-wash and phosducin-extraction procedures were repeated three and two times, respectively. These aliquots of supernatants (s) and pellets (p) were electrophoresed and transferred to a PVDF membrane for immunostaining. Shown is the clipped image of the anti-Tγ blot at the gel front region. (B) Immunoblot patterns of the fractions eluted from the reverse-phase HPLC as described in Experimental Procedures. Each fraction was subjected to SDS−polyacrylamide gel (12.5%) electrophoresis and immunostaining by anti-Tγ antibody. The fractions containing the Tγ−X product (fractions 25−34) are shown.

    Article Snippet: For the transfer reaction of POG-PP to Tβγ, a mixture of unmodified Tβγ-VIS (1 μM, final concentration) and 10 μg of FTase was prewarmed at 37 °C and then mixed with 100 μM final concentration of POG-PP in buffer P, adjusting the total volume to 10 mL with buffer P. The transfer reaction was carried out at 37 °C for 2 h. To monitor the transfer yield of the POG moiety to Cys71 of Tγ-VIS, aliquots of the reaction mixture were subjected to a reverse-phase HPLC system (model 600E; Waters) equipped with a Develosil 300 C4-HG-5 column (4.6 × 150 mm; Nomura Chemical, Kyoto, Japan); the elution was carried out with a linear gradient of acetonitorile (30−60%, 0.5%/min) in 0.05% trifluoroacetic acid at a flow rate of 1.0 mL/min.

    Techniques: Isolation, Immunostaining, High Performance Liquid Chromatography, Electrophoresis

    (A) Structures of POG-PP [(3-azidophenoxy)geranyl pyrophosphate] and farnesyl pyrophosphate. (B) HPLC analysis of Tγ in each modification step. Unmodified Tβγ-VIS was incubated with yeast FTase in the presence of POG-PP, and aliquots of the reaction mixtures were subjected to reverse-phase HPLC analysis before (trace a) and after (trace b) the POG-transfer reaction. The analysis was also performed following Rce1 treatment (trace c) and Icmt treatment in the presence of AdoMet (trace d). Retinal Tγ was analyzed as a standard (trace e). HPLC analysis was performed as described in Experimental Procedures. Each peak fraction detected by the absorbance at 214 nm was collected and analyzed by MALDI-TOF mass spectrometry with the Voyager-DE (Applied Biosystems) spectrometer. The singly and doubly protonated ion signals of cytochrome c (from horse heart, M r = 12359.7) were used as an internal standard of mass calibration. The observed [M + H] + values of the peak fractions were 8411.0 (trace a), 8681.1 (trace b), 8381.6 (trace c), and 8395.4 (trace d), in good agreement with the calculated [M + H] + values of Tγ-VIS (8411.4), POG-Tγ-VIS (8680.8), POG-Tγ (8381.4), and POG-Tγ-OMe (8395.4), respectively. Due to adsorption, Tβ was not eluted from the column. (C) Time courses of prenyl transfer (panel a), VIS cleavage (panel b), and carboxyl methylation (panel c) reactions catalyzed by FTase, Rce1, and Icmt, respectively.

    Journal: Biochemistry

    Article Title: Interacting Targets of the Farnesyl of Transducin ?-Subunit

    doi: 10.1021/bi800359h

    Figure Lengend Snippet: (A) Structures of POG-PP [(3-azidophenoxy)geranyl pyrophosphate] and farnesyl pyrophosphate. (B) HPLC analysis of Tγ in each modification step. Unmodified Tβγ-VIS was incubated with yeast FTase in the presence of POG-PP, and aliquots of the reaction mixtures were subjected to reverse-phase HPLC analysis before (trace a) and after (trace b) the POG-transfer reaction. The analysis was also performed following Rce1 treatment (trace c) and Icmt treatment in the presence of AdoMet (trace d). Retinal Tγ was analyzed as a standard (trace e). HPLC analysis was performed as described in Experimental Procedures. Each peak fraction detected by the absorbance at 214 nm was collected and analyzed by MALDI-TOF mass spectrometry with the Voyager-DE (Applied Biosystems) spectrometer. The singly and doubly protonated ion signals of cytochrome c (from horse heart, M r = 12359.7) were used as an internal standard of mass calibration. The observed [M + H] + values of the peak fractions were 8411.0 (trace a), 8681.1 (trace b), 8381.6 (trace c), and 8395.4 (trace d), in good agreement with the calculated [M + H] + values of Tγ-VIS (8411.4), POG-Tγ-VIS (8680.8), POG-Tγ (8381.4), and POG-Tγ-OMe (8395.4), respectively. Due to adsorption, Tβ was not eluted from the column. (C) Time courses of prenyl transfer (panel a), VIS cleavage (panel b), and carboxyl methylation (panel c) reactions catalyzed by FTase, Rce1, and Icmt, respectively.

    Article Snippet: For the transfer reaction of POG-PP to Tβγ, a mixture of unmodified Tβγ-VIS (1 μM, final concentration) and 10 μg of FTase was prewarmed at 37 °C and then mixed with 100 μM final concentration of POG-PP in buffer P, adjusting the total volume to 10 mL with buffer P. The transfer reaction was carried out at 37 °C for 2 h. To monitor the transfer yield of the POG moiety to Cys71 of Tγ-VIS, aliquots of the reaction mixture were subjected to a reverse-phase HPLC system (model 600E; Waters) equipped with a Develosil 300 C4-HG-5 column (4.6 × 150 mm; Nomura Chemical, Kyoto, Japan); the elution was carried out with a linear gradient of acetonitorile (30−60%, 0.5%/min) in 0.05% trifluoroacetic acid at a flow rate of 1.0 mL/min.

    Techniques: High Performance Liquid Chromatography, Modification, Incubation, Mass Spectrometry, Adsorption, Methylation

    Purification of SPINK6 from human stratum corneum extracts. A , natural SPINK6 was isolated from human plantar stratum corneum extracts using first an anti-SPINK6 affinity column (not shown), followed by a C18 reversed-phase HPLC column. ESI-MS analyses

    Journal: The Journal of Biological Chemistry

    Article Title: Isolation of SPINK6 in Human Skin

    doi: 10.1074/jbc.M109.091850

    Figure Lengend Snippet: Purification of SPINK6 from human stratum corneum extracts. A , natural SPINK6 was isolated from human plantar stratum corneum extracts using first an anti-SPINK6 affinity column (not shown), followed by a C18 reversed-phase HPLC column. ESI-MS analyses

    Article Snippet: Material that was strongly bound to the affinity column was further purified by reversed-phase HPLC (Jupiter C18 column, Phenomenex).

    Techniques: Purification, Isolation, Affinity Column, High Performance Liquid Chromatography, Mass Spectrometry

    The synthesis of sugar nucleotides from 2-chloro-4-nitrophenyl glucosides. ( a ) General reaction scheme. ( b ) Structures of 2-chloro-4-nitrophenyl glycoside donors evaluated for D-sugars within this series, the differences between each member and the native OleD sugar substrate (β-D-glucose) are highlighted in red. ( c ) Maximum observed percent conversion of (U/T)DP to (U/T)DP-glucose within a 21 hour time course assay for each donor (n ≥ 2, standard deviation ≤ 5%). Standard reactions contained 7 μM TDP-16, 1 mM (U/T)DP, and 1 mM of 2-chloro-4-nitrophenyl glycoside donor ( 9 , 34 – 47 ) in Tris-HCl buffer (50 mM, pH 8.5) with a final volume of 300 μl. Over 21 hours at 25°C, aliquots taken at various times were flash frozen and analyzed by HPLC ( Supplementary Methods ). For reactions with UDP yielding

    Journal: Nature chemical biology

    Article Title: Using Simple Donors to Drive the Equilibria of Glycosyltransferase-Catalyzed Reactions

    doi: 10.1038/nchembio.638

    Figure Lengend Snippet: The synthesis of sugar nucleotides from 2-chloro-4-nitrophenyl glucosides. ( a ) General reaction scheme. ( b ) Structures of 2-chloro-4-nitrophenyl glycoside donors evaluated for D-sugars within this series, the differences between each member and the native OleD sugar substrate (β-D-glucose) are highlighted in red. ( c ) Maximum observed percent conversion of (U/T)DP to (U/T)DP-glucose within a 21 hour time course assay for each donor (n ≥ 2, standard deviation ≤ 5%). Standard reactions contained 7 μM TDP-16, 1 mM (U/T)DP, and 1 mM of 2-chloro-4-nitrophenyl glycoside donor ( 9 , 34 – 47 ) in Tris-HCl buffer (50 mM, pH 8.5) with a final volume of 300 μl. Over 21 hours at 25°C, aliquots taken at various times were flash frozen and analyzed by HPLC ( Supplementary Methods ). For reactions with UDP yielding

    Article Snippet: Following, samples were thawed at 4°C and filtered through a MultiScreen Filter Plate (Millipore, Billerica, MA, USA) according to manufacturer’s instructions and evaluated for formation of UDP- ( 33a ) or TDP-α-D-glucose ( 33b ) by analytical reverse-phase HPLC with a 250 mm × 4.6 mm Gemini-NX 5μ C18 column (Phenomenex, Torrance, CA, USA) using a linear gradient of 0% to 15% CH3 CN (solvent B) over 15 minutes (solvent A = aqueous 50 mM triethylammonium acetate buffer [Sigma-Aldrich, St. Louis, MO, USA], flow rate = 1 ml min−1 , with detection monitored at 254 nm).

    Techniques: Standard Deviation, High Performance Liquid Chromatography

    Evaluation of putative donors for sugar nucleotide synthesis. ( a ) General reaction scheme. ( b ) Structures of the β-D-glucopyranoside donors which led to (U/T)DP-glucose formation. ( c ) Percent conversion of (U/T)DP to (U/T)DP-glucose with various donors (n ≥ 2, standard deviation ≤ 5%). Reactions contained 2.1 μM (10 μg) OleD variant, 1 mM of (U/T)DP, and 1 mM of aromatic donor ( 1–9 ) in Tris-HCl buffer (50 mM, pH 8.5) with a final volume of 100 μl. After one hour at 25°C, reactions were flash frozen and analyzed by HPLC ( Supplementary Methods ). The pK a for each corresponding donor aglycon is highlighted in parentheses. ( d ) Plot depicting the relative Gibbs free energy of selected donors/acceptors in relation to 33a . Small glycoside donors display large shifts in relative free energy, transforming formation of UDP-Glc ( 33a ) from an endo- to an exothermic process. The ΔG° pH8.5 for 1 , 2, 4, 7, and 9 with UDP in Tris-HCl buffer (50 mM, pH 8.5) at 298K relative to 33a were determined in this study ( Supplementary Methods ). The ΔG° for 61a was previously determined (at pH 9.0 and 310K) ( 5 ) .

    Journal: Nature chemical biology

    Article Title: Using Simple Donors to Drive the Equilibria of Glycosyltransferase-Catalyzed Reactions

    doi: 10.1038/nchembio.638

    Figure Lengend Snippet: Evaluation of putative donors for sugar nucleotide synthesis. ( a ) General reaction scheme. ( b ) Structures of the β-D-glucopyranoside donors which led to (U/T)DP-glucose formation. ( c ) Percent conversion of (U/T)DP to (U/T)DP-glucose with various donors (n ≥ 2, standard deviation ≤ 5%). Reactions contained 2.1 μM (10 μg) OleD variant, 1 mM of (U/T)DP, and 1 mM of aromatic donor ( 1–9 ) in Tris-HCl buffer (50 mM, pH 8.5) with a final volume of 100 μl. After one hour at 25°C, reactions were flash frozen and analyzed by HPLC ( Supplementary Methods ). The pK a for each corresponding donor aglycon is highlighted in parentheses. ( d ) Plot depicting the relative Gibbs free energy of selected donors/acceptors in relation to 33a . Small glycoside donors display large shifts in relative free energy, transforming formation of UDP-Glc ( 33a ) from an endo- to an exothermic process. The ΔG° pH8.5 for 1 , 2, 4, 7, and 9 with UDP in Tris-HCl buffer (50 mM, pH 8.5) at 298K relative to 33a were determined in this study ( Supplementary Methods ). The ΔG° for 61a was previously determined (at pH 9.0 and 310K) ( 5 ) .

    Article Snippet: Following, samples were thawed at 4°C and filtered through a MultiScreen Filter Plate (Millipore, Billerica, MA, USA) according to manufacturer’s instructions and evaluated for formation of UDP- ( 33a ) or TDP-α-D-glucose ( 33b ) by analytical reverse-phase HPLC with a 250 mm × 4.6 mm Gemini-NX 5μ C18 column (Phenomenex, Torrance, CA, USA) using a linear gradient of 0% to 15% CH3 CN (solvent B) over 15 minutes (solvent A = aqueous 50 mM triethylammonium acetate buffer [Sigma-Aldrich, St. Louis, MO, USA], flow rate = 1 ml min−1 , with detection monitored at 254 nm).

    Techniques: Standard Deviation, Variant Assay, High Performance Liquid Chromatography, Gas Chromatography

    Evaluation of 2-chloro-4-nitrophenyl glycosides as sugar donors in coupled GT-catalyzed transglycosylation reactions. ( a ) The scheme for a single enzyme (TDP-16) coupled system with 4-methylumbelliferone ( 58) as the final acceptor (left) and a representative HPLC analysis (right) using the donor for 6-azido-6-deoxy-D-glucose ( 37 ). Reactions contained 1 mM glycoside donor, 1 mM 58 , 1 mM UDP, and 11 μM TDP-16 in a total volume of 100 μl with Tris-HCl buffer (50 mM, pH 8.5) at 25°C for 24 hour and were subsequently analyzed by HPLC ( Supplementary Methods ). For the representative reaction: (i) control reaction lacking TDP-16; (ii) control reaction lacking UDP; (iii) full reaction where 37 is donor, 58 is acceptor, 59d is desired product and ⋄ represents 2-chloro-4-nitrophenolate. ( b ) The scheme for a double enzyme (TDP-16 and GtfE) coupled system with vancomycin aglycon ( 60 ) as the final acceptor (left) and a representative HPLC analysis (right) using the donor for 6-azido-6-deoxy-D-glucose ( 37 ). Reactions contained 1 mM glycoside donor, 0.1 mM 60 , 1 mM UDP, 11 μM TDP-16, and 11 μM GtfE in a total volume of 100 μl with Tris-HCl buffer (50 mM, pH 8.5) at 25°C for 24 hour and were subsequently analyzed by HPLC ( Supplementary Methods ). For the representative reaction: (i) control reaction lacking TDP-16; (ii) control reaction lacking GtfE; (iii) full reaction where 37 is donor, 60 is acceptor, 61e is desired product and ⋄ represents 2-chloro-4-nitrophenolate. Sample preparation and HPLC parameters, along with chromatograms ( Supplementary Fig. 14 and 17 ), conversion rates, and mass characterization ( Supplementary Table 4 and 5 ) for all products are presented in supporting online material .

    Journal: Nature chemical biology

    Article Title: Using Simple Donors to Drive the Equilibria of Glycosyltransferase-Catalyzed Reactions

    doi: 10.1038/nchembio.638

    Figure Lengend Snippet: Evaluation of 2-chloro-4-nitrophenyl glycosides as sugar donors in coupled GT-catalyzed transglycosylation reactions. ( a ) The scheme for a single enzyme (TDP-16) coupled system with 4-methylumbelliferone ( 58) as the final acceptor (left) and a representative HPLC analysis (right) using the donor for 6-azido-6-deoxy-D-glucose ( 37 ). Reactions contained 1 mM glycoside donor, 1 mM 58 , 1 mM UDP, and 11 μM TDP-16 in a total volume of 100 μl with Tris-HCl buffer (50 mM, pH 8.5) at 25°C for 24 hour and were subsequently analyzed by HPLC ( Supplementary Methods ). For the representative reaction: (i) control reaction lacking TDP-16; (ii) control reaction lacking UDP; (iii) full reaction where 37 is donor, 58 is acceptor, 59d is desired product and ⋄ represents 2-chloro-4-nitrophenolate. ( b ) The scheme for a double enzyme (TDP-16 and GtfE) coupled system with vancomycin aglycon ( 60 ) as the final acceptor (left) and a representative HPLC analysis (right) using the donor for 6-azido-6-deoxy-D-glucose ( 37 ). Reactions contained 1 mM glycoside donor, 0.1 mM 60 , 1 mM UDP, 11 μM TDP-16, and 11 μM GtfE in a total volume of 100 μl with Tris-HCl buffer (50 mM, pH 8.5) at 25°C for 24 hour and were subsequently analyzed by HPLC ( Supplementary Methods ). For the representative reaction: (i) control reaction lacking TDP-16; (ii) control reaction lacking GtfE; (iii) full reaction where 37 is donor, 60 is acceptor, 61e is desired product and ⋄ represents 2-chloro-4-nitrophenolate. Sample preparation and HPLC parameters, along with chromatograms ( Supplementary Fig. 14 and 17 ), conversion rates, and mass characterization ( Supplementary Table 4 and 5 ) for all products are presented in supporting online material .

    Article Snippet: Following, samples were thawed at 4°C and filtered through a MultiScreen Filter Plate (Millipore, Billerica, MA, USA) according to manufacturer’s instructions and evaluated for formation of UDP- ( 33a ) or TDP-α-D-glucose ( 33b ) by analytical reverse-phase HPLC with a 250 mm × 4.6 mm Gemini-NX 5μ C18 column (Phenomenex, Torrance, CA, USA) using a linear gradient of 0% to 15% CH3 CN (solvent B) over 15 minutes (solvent A = aqueous 50 mM triethylammonium acetate buffer [Sigma-Aldrich, St. Louis, MO, USA], flow rate = 1 ml min−1 , with detection monitored at 254 nm).

    Techniques: High Performance Liquid Chromatography, Sample Prep

    Utilizing a colorimetric screen for glycosyl transfer. ( a ) Scheme for colorimetric screen using the single enzyme (TDP-16) coupled format. ( b ) Evaluation of the colorimetric assay with 58 as the final acceptor. The reactions contained 0.5 mM 9 as donor, 0.5 mM 58 as acceptor, 5 μM UDP, and 11μM TDP-16 in a final total volume of 100 μl with Tris-HCl buffer (50 mM, pH 8.5) in a 96-well plate incubated at 25°C for one hour. ( i ) Qualitative color change after one hour for the full reaction (yellow square), a control lacking the final acceptor 58 (white circle), and a control lacking UDP (red triangle). ( ii ) Δ410 nm over one hour for the full reaction (yellow squares), a control lacking the final acceptor 58 (white circles), and a control reaction lacking UDP (red triangles). ( iii ) HPLC chromatograms of full reaction at 1, 5, and 60 min where 1 is desired product, 9 is the donor, 58 is the target aglycon and ⋄ represents 2-chloro-4-nitrophenolate. (c) The absorbance data and HPLC chromatograms of three representative hits [( i ) 62 (genistein), ( ii ) 79 (tyrphostin), or ( iii ) 92 (ciprofloxacin)] from the broad 50 compound panel screen using the single enzyme (TDP-16) coupled format. In HPLC chromatograms 9 indicates donor; 62 , 79 or 92 represent target aglycon; ⋄ indicates 2-chloro-4-nitrophenolate; and ● depicts glucosylated product(s). For the overall results of the 50 compound screen, additional representative absorbance plots and chromatograms, and combined HPLC and LC/MS characterization, see Supplementary Fig. 19–21 and Supplementary Table 6 .

    Journal: Nature chemical biology

    Article Title: Using Simple Donors to Drive the Equilibria of Glycosyltransferase-Catalyzed Reactions

    doi: 10.1038/nchembio.638

    Figure Lengend Snippet: Utilizing a colorimetric screen for glycosyl transfer. ( a ) Scheme for colorimetric screen using the single enzyme (TDP-16) coupled format. ( b ) Evaluation of the colorimetric assay with 58 as the final acceptor. The reactions contained 0.5 mM 9 as donor, 0.5 mM 58 as acceptor, 5 μM UDP, and 11μM TDP-16 in a final total volume of 100 μl with Tris-HCl buffer (50 mM, pH 8.5) in a 96-well plate incubated at 25°C for one hour. ( i ) Qualitative color change after one hour for the full reaction (yellow square), a control lacking the final acceptor 58 (white circle), and a control lacking UDP (red triangle). ( ii ) Δ410 nm over one hour for the full reaction (yellow squares), a control lacking the final acceptor 58 (white circles), and a control reaction lacking UDP (red triangles). ( iii ) HPLC chromatograms of full reaction at 1, 5, and 60 min where 1 is desired product, 9 is the donor, 58 is the target aglycon and ⋄ represents 2-chloro-4-nitrophenolate. (c) The absorbance data and HPLC chromatograms of three representative hits [( i ) 62 (genistein), ( ii ) 79 (tyrphostin), or ( iii ) 92 (ciprofloxacin)] from the broad 50 compound panel screen using the single enzyme (TDP-16) coupled format. In HPLC chromatograms 9 indicates donor; 62 , 79 or 92 represent target aglycon; ⋄ indicates 2-chloro-4-nitrophenolate; and ● depicts glucosylated product(s). For the overall results of the 50 compound screen, additional representative absorbance plots and chromatograms, and combined HPLC and LC/MS characterization, see Supplementary Fig. 19–21 and Supplementary Table 6 .

    Article Snippet: Following, samples were thawed at 4°C and filtered through a MultiScreen Filter Plate (Millipore, Billerica, MA, USA) according to manufacturer’s instructions and evaluated for formation of UDP- ( 33a ) or TDP-α-D-glucose ( 33b ) by analytical reverse-phase HPLC with a 250 mm × 4.6 mm Gemini-NX 5μ C18 column (Phenomenex, Torrance, CA, USA) using a linear gradient of 0% to 15% CH3 CN (solvent B) over 15 minutes (solvent A = aqueous 50 mM triethylammonium acetate buffer [Sigma-Aldrich, St. Louis, MO, USA], flow rate = 1 ml min−1 , with detection monitored at 254 nm).

    Techniques: Colorimetric Assay, Incubation, High Performance Liquid Chromatography, Liquid Chromatography with Mass Spectroscopy

    Serotonin (5-HT) and 5-hydroxyindole-3-acetic acid (5-HIAA) levels in neostriatum. n-3 PUFA supplementation modulates 5-HT turnover in both male and female SHR rats. Levels of ( a ) 5-HT and ( b ) 5-HIAA were used to determine ( c ) 5-HIAA/5-HT ratios. The data are presented as means ± SEM (n = 4–8) and the level of significance is symbolized with * (p-value ≤ 0.05), ** (p-value ≤ 0.01) or *** (p-value ≤ 0.001). The p-values are calculated by comparison with the mid bar, which represents control-fed SHRs. The analyses were performed by HPLC using electrochemical detection.

    Journal: Behavioral and Brain Functions : BBF

    Article Title: Marine omega-3 polyunsaturated fatty acids induce sex-specific changes in reinforcer-controlled behaviour and neurotransmitter metabolism in a spontaneously hypertensive rat model of ADHD

    doi: 10.1186/1744-9081-8-56

    Figure Lengend Snippet: Serotonin (5-HT) and 5-hydroxyindole-3-acetic acid (5-HIAA) levels in neostriatum. n-3 PUFA supplementation modulates 5-HT turnover in both male and female SHR rats. Levels of ( a ) 5-HT and ( b ) 5-HIAA were used to determine ( c ) 5-HIAA/5-HT ratios. The data are presented as means ± SEM (n = 4–8) and the level of significance is symbolized with * (p-value ≤ 0.05), ** (p-value ≤ 0.01) or *** (p-value ≤ 0.001). The p-values are calculated by comparison with the mid bar, which represents control-fed SHRs. The analyses were performed by HPLC using electrochemical detection.

    Article Snippet: The frozen extracts were used directly for analyses of total DA and 5-HT, as well as their metabolites HVA and 5-HIAA, using a reversed-phase HPLC (Shimadzu, Kyoto, Japan) with electrochemical detection (ECD; Decade II with Sencell flow electrode, Antec Leyden) set to a working potential of 0.6 V. Each sample was eluted for 40 min with a flow rate at 1.3 mL/min and a representative chromatogram is shown in Figure .

    Techniques: Significance Assay, High Performance Liquid Chromatography

    Dopamine (DA) and homovanillic acid (HVA) levels in neostriatum. n-3 PUFA supplementation modulated DA turnover in male SHR: ( a ) DA and ( b ) HVA levels were used to determine ( c ) HVA/DA ratios. Data are presented as means ± SEM (n = 4–8) and the level of significance is symbolized by * (p-value ≤ 0.05), ** (p-value ≤ 0.01) or *** (p-value ≤ 0.001). The p-values are calculated by comparison with the mid bar representing control-fed SHRs. The analyses were performed by HPLC using electrochemical detection.

    Journal: Behavioral and Brain Functions : BBF

    Article Title: Marine omega-3 polyunsaturated fatty acids induce sex-specific changes in reinforcer-controlled behaviour and neurotransmitter metabolism in a spontaneously hypertensive rat model of ADHD

    doi: 10.1186/1744-9081-8-56

    Figure Lengend Snippet: Dopamine (DA) and homovanillic acid (HVA) levels in neostriatum. n-3 PUFA supplementation modulated DA turnover in male SHR: ( a ) DA and ( b ) HVA levels were used to determine ( c ) HVA/DA ratios. Data are presented as means ± SEM (n = 4–8) and the level of significance is symbolized by * (p-value ≤ 0.05), ** (p-value ≤ 0.01) or *** (p-value ≤ 0.001). The p-values are calculated by comparison with the mid bar representing control-fed SHRs. The analyses were performed by HPLC using electrochemical detection.

    Article Snippet: The frozen extracts were used directly for analyses of total DA and 5-HT, as well as their metabolites HVA and 5-HIAA, using a reversed-phase HPLC (Shimadzu, Kyoto, Japan) with electrochemical detection (ECD; Decade II with Sencell flow electrode, Antec Leyden) set to a working potential of 0.6 V. Each sample was eluted for 40 min with a flow rate at 1.3 mL/min and a representative chromatogram is shown in Figure .

    Techniques: Significance Assay, High Performance Liquid Chromatography

    The radioactive signal from oligopeptides being modified with radioactive acetyl CoA . The oligopeptide 1 MLGTE-RRR 24 (200 μM) was incubated with [1- 14 C] acetyl CoA (final 300 μM with specific activity 11.2 mCi/mmol) and purified MBP-hNaa30p (80 nM) in acetylation buffer for 60 minutes at 37°C. Samples were collected at indicated time points and analysed with reverse phase HPLC that had been connected to a radioactivity flow detector after the absorbance detector. A clear time dependent increase in the radioactivity signal is observed, verifying that the eluted oligopeptides are labelled with radioactive acetyl groups.

    Journal: BMC Proceedings

    Article Title: Application of reverse-phase HPLC to quantify oligopeptide acetylation eliminates interference from unspecific acetyl CoA hydrolysis

    doi: 10.1186/1753-6561-3-S6-S5

    Figure Lengend Snippet: The radioactive signal from oligopeptides being modified with radioactive acetyl CoA . The oligopeptide 1 MLGTE-RRR 24 (200 μM) was incubated with [1- 14 C] acetyl CoA (final 300 μM with specific activity 11.2 mCi/mmol) and purified MBP-hNaa30p (80 nM) in acetylation buffer for 60 minutes at 37°C. Samples were collected at indicated time points and analysed with reverse phase HPLC that had been connected to a radioactivity flow detector after the absorbance detector. A clear time dependent increase in the radioactivity signal is observed, verifying that the eluted oligopeptides are labelled with radioactive acetyl groups.

    Article Snippet: Separation of non acetylated and acetylated oligopeptides using reverse phase HPLC The acetylation activity was analysed by a reverse phase HPLC system, consisting of a LC-20AB solvent delivery module, an SPD-M20A photodiode array detector and a SIL-20AC autosampler (Shimadzu Prominence), and a 250 mm × 3 mm Nucleosil C18 HD column (Macherey-Nagel) reverse phase HPLC column.

    Techniques: Modification, Incubation, Activity Assay, Purification, High Performance Liquid Chromatography, Radioactivity, Flow Cytometry

    MBP-hNaa30p specificity constants (V/K) for selected oligopeptides based on detection of acetylated oligopeptides (215 nm) . Purified MBP-hNaa30p (80 nM) was incubated with selected oligopeptides and acetyl CoA (300 μM) in acetylation buffer for 30 minutes at 37°C. The acetylation kinetics was analysed by reverse phase HPLC. V/K is the V max /K m (oligopeptides) . Error bars indicate S.D. Experiments are performed in triplicates.

    Journal: BMC Proceedings

    Article Title: Application of reverse-phase HPLC to quantify oligopeptide acetylation eliminates interference from unspecific acetyl CoA hydrolysis

    doi: 10.1186/1753-6561-3-S6-S5

    Figure Lengend Snippet: MBP-hNaa30p specificity constants (V/K) for selected oligopeptides based on detection of acetylated oligopeptides (215 nm) . Purified MBP-hNaa30p (80 nM) was incubated with selected oligopeptides and acetyl CoA (300 μM) in acetylation buffer for 30 minutes at 37°C. The acetylation kinetics was analysed by reverse phase HPLC. V/K is the V max /K m (oligopeptides) . Error bars indicate S.D. Experiments are performed in triplicates.

    Article Snippet: Separation of non acetylated and acetylated oligopeptides using reverse phase HPLC The acetylation activity was analysed by a reverse phase HPLC system, consisting of a LC-20AB solvent delivery module, an SPD-M20A photodiode array detector and a SIL-20AC autosampler (Shimadzu Prominence), and a 250 mm × 3 mm Nucleosil C18 HD column (Macherey-Nagel) reverse phase HPLC column.

    Techniques: Purification, Incubation, High Performance Liquid Chromatography

    Reverse phase HPLC absorbance profile at 215 nm for the separation of acetylated and non-acetylated peptides . A ; The oligopeptide 1 MLGTE-RRR 24 (200 μM) was incubated with acetyl CoA (300 μM) and purified MBP-hNaa30p (80 nM) in acetylation buffer for 60 minutes at 37°C. Samples were collected at indicated time points and analysed with reverse phase HPLC. The resulting absorbance profile at 215 nm indicate good separation of unacetylated ('a') and acetylated oligopeptides ('b'). B ; An expanded version of the absorbance profile for the formation of acetylated oligopeptide. A clear time dependent increase in the absorption signal is observed.

    Journal: BMC Proceedings

    Article Title: Application of reverse-phase HPLC to quantify oligopeptide acetylation eliminates interference from unspecific acetyl CoA hydrolysis

    doi: 10.1186/1753-6561-3-S6-S5

    Figure Lengend Snippet: Reverse phase HPLC absorbance profile at 215 nm for the separation of acetylated and non-acetylated peptides . A ; The oligopeptide 1 MLGTE-RRR 24 (200 μM) was incubated with acetyl CoA (300 μM) and purified MBP-hNaa30p (80 nM) in acetylation buffer for 60 minutes at 37°C. Samples were collected at indicated time points and analysed with reverse phase HPLC. The resulting absorbance profile at 215 nm indicate good separation of unacetylated ('a') and acetylated oligopeptides ('b'). B ; An expanded version of the absorbance profile for the formation of acetylated oligopeptide. A clear time dependent increase in the absorption signal is observed.

    Article Snippet: Separation of non acetylated and acetylated oligopeptides using reverse phase HPLC The acetylation activity was analysed by a reverse phase HPLC system, consisting of a LC-20AB solvent delivery module, an SPD-M20A photodiode array detector and a SIL-20AC autosampler (Shimadzu Prominence), and a 250 mm × 3 mm Nucleosil C18 HD column (Macherey-Nagel) reverse phase HPLC column.

    Techniques: High Performance Liquid Chromatography, Incubation, Purification

    Effect of DB3 and DB3DB3 on different Aβ aggregation species. A) Analysis of Aβ(1–42) aggregation species with density gradient centrifugation and followed by analysis using silver-stained Tricine-SDS-PAGE to analyze the influence of DB3 and DB3DB3 on the distribution of Aβ assemblies. B) Quantification of Aβ(1–42) by RP-HPLC. All data were recorded in triplicate.

    Journal: PLoS ONE

    Article Title: Optimization of the All-D Peptide D3 for Aβ Oligomer Elimination

    doi: 10.1371/journal.pone.0153035

    Figure Lengend Snippet: Effect of DB3 and DB3DB3 on different Aβ aggregation species. A) Analysis of Aβ(1–42) aggregation species with density gradient centrifugation and followed by analysis using silver-stained Tricine-SDS-PAGE to analyze the influence of DB3 and DB3DB3 on the distribution of Aβ assemblies. B) Quantification of Aβ(1–42) by RP-HPLC. All data were recorded in triplicate.

    Article Snippet: For quantification of the Aβ(1–42) amount in each fraction, reversed-phase high performance liquid chromatography (RP-HPLC) was performed using a Zorbax SB-300 C8 column (Agilent, Böblingen, Germany) connected to an Agilent 1260 Infinity system using 30% (v/v) acetonitrile with 0.1% (v/v) trifluoroacetic acid (TFA) as the mobile phase with a flow of 1 ml/min and a column temperature of 80°C.

    Techniques: Gradient Centrifugation, Staining, SDS Page, High Performance Liquid Chromatography

    Chromatography elution profiles and calcium binding capacities of calcium-binding peptides. ( A ) Sephadex G-25 gel filtration chromatography of SPH; ( B ) Semi-preparative C18 RP-HPLC of fraction III; ( C ) RP-HPLC of fraction 17 from semi-preparative HPLC.

    Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

    Article Title: A Specific Peptide with Calcium-Binding Capacity from Defatted Schizochytrium sp. Protein Hydrolysates and the Molecular Properties

    doi: 10.3390/molecules22040544

    Figure Lengend Snippet: Chromatography elution profiles and calcium binding capacities of calcium-binding peptides. ( A ) Sephadex G-25 gel filtration chromatography of SPH; ( B ) Semi-preparative C18 RP-HPLC of fraction III; ( C ) RP-HPLC of fraction 17 from semi-preparative HPLC.

    Article Snippet: The fraction with the highest calcium-binding activity from Sephadex G-25 chromatography was pooled and further purified by semi-preparative reversed phase HPLC on a C18 reversed-silica gel column (Gemini 5 μ C18, 250 × 10 mm; Phenomenex Inc., Torrance, CA, USA).

    Techniques: Chromatography, Binding Assay, Filtration, High Performance Liquid Chromatography

    ( A ) Coomassie blue staining of the purified region-1 and region-2 of mPCSK9 proteins. Region-1 (residues 152–351) and region-2 (residues 249–452) of mouse catalytic domain were cloned and expressed by pET-3a plasmids. The proteins were reduced and purified by a reverse-phase high-performance liquid chromatography (HPLC) on an Agilent 1100 HPLC system. The purified proteins were analyzed by SDS/PAGE and stained with Coomassie blue. The positions of expressed proteins are shown; ( B ) MALDI mass spectrometry on Region-1. The molecular mass of purified PCSK9 region-1 in fully reduced form (PCSK9-1R). The molecular mass of PCSK9 region-1 as determined by MALDI was 21,201 Da; ( C ) MALDI mass spectrometry on Region-2. The molecular mass of purified PCSK9 region-2 in fully reduced form (PCSK9-2R). The molecular mass of PCSK9 region-2 as determined by MALDI was 21,355 Da.

    Journal: International Journal of Molecular Sciences

    Article Title: Non-Native Conformational Isomers of the Catalytic Domain of PCSK9 Induce an Immune Response, Reduce Lipids and Increase LDL Receptor Levels

    doi: 10.3390/ijms19020640

    Figure Lengend Snippet: ( A ) Coomassie blue staining of the purified region-1 and region-2 of mPCSK9 proteins. Region-1 (residues 152–351) and region-2 (residues 249–452) of mouse catalytic domain were cloned and expressed by pET-3a plasmids. The proteins were reduced and purified by a reverse-phase high-performance liquid chromatography (HPLC) on an Agilent 1100 HPLC system. The purified proteins were analyzed by SDS/PAGE and stained with Coomassie blue. The positions of expressed proteins are shown; ( B ) MALDI mass spectrometry on Region-1. The molecular mass of purified PCSK9 region-1 in fully reduced form (PCSK9-1R). The molecular mass of PCSK9 region-1 as determined by MALDI was 21,201 Da; ( C ) MALDI mass spectrometry on Region-2. The molecular mass of purified PCSK9 region-2 in fully reduced form (PCSK9-2R). The molecular mass of PCSK9 region-2 as determined by MALDI was 21,355 Da.

    Article Snippet: The authors used a reverse-phase high-performance liquid chromatography (HPLC) on an Agilent 1100 HPLC system (Column ZORBAX 3000 SB-C18, 9.4 mm × 25 cm) to purify the proteins.

    Techniques: Staining, Purification, Clone Assay, Positron Emission Tomography, High Performance Liquid Chromatography, SDS Page, Mass Spectrometry

    Determination of the purity of [ 18 F]FDG-6-P. By (a) iTLC at 0 h and (b) RP-HPLC after incubation of the radiopharmaceutical in water for 3 h. The retention profiles of [ 18 F]FDG-6-P were easily resolved from that of [ 18 F]FDG, the most likely breakdown product of [ 18 F]FDG-6-P. No changes in the [ 18 F]FDG-6-P peak or retention time were observed.

    Journal: EJNMMI Research

    Article Title: [18F]FDG-6-P as a novel in vivo tool for imaging staphylococcal infections

    doi: 10.1186/s13550-015-0095-1

    Figure Lengend Snippet: Determination of the purity of [ 18 F]FDG-6-P. By (a) iTLC at 0 h and (b) RP-HPLC after incubation of the radiopharmaceutical in water for 3 h. The retention profiles of [ 18 F]FDG-6-P were easily resolved from that of [ 18 F]FDG, the most likely breakdown product of [ 18 F]FDG-6-P. No changes in the [ 18 F]FDG-6-P peak or retention time were observed.

    Article Snippet: The purity of the [18 F]FDG-6-P product was determined by reverse-phase high-performance liquid chromatography (RP-HPLC) on an Agilent 1260 Series LC (Agilent Technologies, Sta.

    Techniques: High Performance Liquid Chromatography, Incubation

    Representative HPLC chromatogram of ceftriaxone sodium

    Journal: Progress in Biomaterials

    Article Title: Development and evaluation of a calcium alginate based oral ceftriaxone sodium formulation

    doi: 10.1007/s40204-016-0051-9

    Figure Lengend Snippet: Representative HPLC chromatogram of ceftriaxone sodium

    Article Snippet: Analysis of ceftriaxone sodium by RP-HPLC Ceftriaxone sodium was analyzed using reversed phase-high-performance liquid chromatography (RP-HPLC) (Waters Alliance e2695 separation module, Milford, MA) equipped with a Phenomenex C18 column (Gemini 5u, 110A, 250 × 4.6 mm) and photodiode array detector (Waters Alliance 2998).

    Techniques: High Performance Liquid Chromatography

    Effects of purified enamel matrix derivative (EMD) fractions on epithelial cell proliferation. (A) Results of reversed-phase high-performance liquid chromatography (HPLC) using C18 hydrophobic support. Lyophilized EMD was dissolved in 0.1% TFA, applied to reversed-phase HPLC, and eluted at 0.5 mL/minute. The arrow indicates the bioactive peak; (B) GE-1 cells were stimulated with each fraction (50 μg/mL) for 48 hours. Cell viability was determined using WST-1 analysis. The data show the percentage inhibition of cell proliferation from independent samples (n=3). The bars represent mean±standard deviation. Data was analyzed by Dunnett’s test after one-way ANOVA (*P

    Journal: Journal of Applied Oral Science

    Article Title: Novel biological activity of ameloblastin in enamel matrix derivative

    doi: 10.1590/1678-775720140291

    Figure Lengend Snippet: Effects of purified enamel matrix derivative (EMD) fractions on epithelial cell proliferation. (A) Results of reversed-phase high-performance liquid chromatography (HPLC) using C18 hydrophobic support. Lyophilized EMD was dissolved in 0.1% TFA, applied to reversed-phase HPLC, and eluted at 0.5 mL/minute. The arrow indicates the bioactive peak; (B) GE-1 cells were stimulated with each fraction (50 μg/mL) for 48 hours. Cell viability was determined using WST-1 analysis. The data show the percentage inhibition of cell proliferation from independent samples (n=3). The bars represent mean±standard deviation. Data was analyzed by Dunnett’s test after one-way ANOVA (*P

    Article Snippet: Lyophilized material was dissolved in 0.1% trifluoroacetic acid (TFA) (30 mg/mL), after which reversed-phase high-performance liquid chromatography (HPLC) was performed using a Waters system (Midford, MA, USA) and C18 column (4.6×150 mm; Vydac, Hesperia, CA, USA) equilibrated with 0.1% TFA.

    Techniques: Purification, High Performance Liquid Chromatography, Inhibition, Standard Deviation

    RP-HPLC analysis of fibrinopeptides. Standard fibrinopeptides A ( a ) and B ( b ), used for comparative purposes ( A ) and fibrinopeptides formed by incubating human fibrinogen (3 mg/mL) with thrombin (5 µg/mL); ( B ) or Moojase (20 µg/mL); ( C ) at 37 °C for 120 min were analyzed by reversed phase HPLC at 214 nm.

    Journal: Toxins

    Article Title: New Insights on Moojase, a Thrombin-Like Serine Protease from Bothrops moojeni Snake Venom

    doi: 10.3390/toxins10120500

    Figure Lengend Snippet: RP-HPLC analysis of fibrinopeptides. Standard fibrinopeptides A ( a ) and B ( b ), used for comparative purposes ( A ) and fibrinopeptides formed by incubating human fibrinogen (3 mg/mL) with thrombin (5 µg/mL); ( B ) or Moojase (20 µg/mL); ( C ) at 37 °C for 120 min were analyzed by reversed phase HPLC at 214 nm.

    Article Snippet: Fibrinopeptides were analyzed by reversed-phase high performance liquid chromatography (RP-HPLC) at 214 nm, using a C18 column (0.46 × 25 cm, CLC-ODS, Shimadzu, Japan) and a gradient of solutions A (0.1% TFA) and B (70% acetonitrile in 0.1% TFA).

    Techniques: High Performance Liquid Chromatography

    Translocation. A. Colicin E3-cleaved (closed circles) or control (open circles) pre-translocation complexes (0.1 μM) were mixed in a quench-flow apparatus with EF-G·GTP (3 μM) and Pmn (10 mM) and translocation analysed by formation of fMetPhe-Pmn. The amount of pre-translocation complex (PTC) was determined by HPLC analysis of fMetPhe. B. Translocation was monitored by the fluorescence change of fMetPhe-tRNA Phe (Prf16/17). Curve 1 – control ribosomes; curve 2 – colicin E3-cleaved ribosomes; curve 3 – control ribosomes in the presence of viomycin (200 μM); curve 4 – colicin E3-cleaved ribosomes with viomycin (200 μM). Normalized ΔF, fluorescence change normalized by the maximum extent of translocation determined by the Pmn reaction. C. Pi release upon GTP hydrolysis by EF-G (curves 1 and 2) or buffer control (curves 3 and 4) with control ribosomes (curves 1 and 3) or colicin E3-cleaved (curves 2 and 4). Pi release was measured by the fluorescence change of MDCC-PBP upon Pi binding ( Experimental procedures ). D. Multiple-turnover GTP hydrolysis by EF-G. Velocity of GTP hydrolysis was measured with catalytic concentrations of EF-G (10 nM) and increasing concentrations of colicin E3-cleaved ribosomes (closed circles) or control ribosomes (open circles) in the presence of excess [γ- 32 P]-GTP (200 μM).

    Journal: Molecular Microbiology

    Article Title: Colicin E3 cleavage of 16S rRNA impairs decoding and accelerates tRNA translocation on Escherichia coli ribosomes

    doi: 10.1111/j.1365-2958.2008.06283.x

    Figure Lengend Snippet: Translocation. A. Colicin E3-cleaved (closed circles) or control (open circles) pre-translocation complexes (0.1 μM) were mixed in a quench-flow apparatus with EF-G·GTP (3 μM) and Pmn (10 mM) and translocation analysed by formation of fMetPhe-Pmn. The amount of pre-translocation complex (PTC) was determined by HPLC analysis of fMetPhe. B. Translocation was monitored by the fluorescence change of fMetPhe-tRNA Phe (Prf16/17). Curve 1 – control ribosomes; curve 2 – colicin E3-cleaved ribosomes; curve 3 – control ribosomes in the presence of viomycin (200 μM); curve 4 – colicin E3-cleaved ribosomes with viomycin (200 μM). Normalized ΔF, fluorescence change normalized by the maximum extent of translocation determined by the Pmn reaction. C. Pi release upon GTP hydrolysis by EF-G (curves 1 and 2) or buffer control (curves 3 and 4) with control ribosomes (curves 1 and 3) or colicin E3-cleaved (curves 2 and 4). Pi release was measured by the fluorescence change of MDCC-PBP upon Pi binding ( Experimental procedures ). D. Multiple-turnover GTP hydrolysis by EF-G. Velocity of GTP hydrolysis was measured with catalytic concentrations of EF-G (10 nM) and increasing concentrations of colicin E3-cleaved ribosomes (closed circles) or control ribosomes (open circles) in the presence of excess [γ- 32 P]-GTP (200 μM).

    Article Snippet: The amount of fMetPhe formed was determined by reversed-phase HPLC (Lichrospher 100 RP-8, Merck).

    Techniques: Translocation Assay, Flow Cytometry, High Performance Liquid Chromatography, Fluorescence, Binding Assay

    ESI/LC/MS analysis of CYP2B1 apoprotein inactivated by BPA or BMP. The incubation conditions, HPLC, and MS analysis conditions were as described under Materials and Methods. A, representative deconvoluted mass spectrum from a control incubation of CYP2B1 with either BPA or BMP in the absence of NADPH. B, representative deconvoluted mass spectrum of P450 2B1 incubated with BPA in the presence of NADPH. C, representative deconvoluted mass spectrum of CYP2B1 incubated with BMP in the presence of NADPH.

    Journal: The Journal of Pharmacology and Experimental Therapeutics

    Article Title: Mechanism-Based Inactivation of CYP2B1 and Its F-Helix Mutant by Two tert-Butyl Acetylenic Compounds: Covalent Modification of Prosthetic Heme Versus Apoprotein

    doi: 10.1124/jpet.109.158782

    Figure Lengend Snippet: ESI/LC/MS analysis of CYP2B1 apoprotein inactivated by BPA or BMP. The incubation conditions, HPLC, and MS analysis conditions were as described under Materials and Methods. A, representative deconvoluted mass spectrum from a control incubation of CYP2B1 with either BPA or BMP in the absence of NADPH. B, representative deconvoluted mass spectrum of P450 2B1 incubated with BPA in the presence of NADPH. C, representative deconvoluted mass spectrum of CYP2B1 incubated with BMP in the presence of NADPH.

    Article Snippet: An aliquot (25 pmol) of the primary reaction mixtures inactivated by incubation with BMP was injected onto a reverse-phase HPLC column (XTerra MS C18, 2.1 × 150 mm; Waters) equilibrated with 65% solvent A (0.05% TFA/0.05% formic acid) and 35% solvent B (0.05% TFA/0.05% formic acid in acetonitrile) at a flow rate of 0.3 ml/min.

    Techniques: Liquid Chromatography with Mass Spectroscopy, Incubation, High Performance Liquid Chromatography, Mass Spectrometry

    Analysis of CYP2B1 heme adducts after incubating the reaction mixture with BMP and NADPH. After inactivation of WT by BMP, as described under Materials and Methods , the reaction mixture was injected directly onto a reverse-phase HPLC column, and the effluent was analyzed using a photodiode-array detector and a Thermo Fisher Scientific LTQ linear ion trap mass spectrometer as described under Materials and Methods . A, HPLC elution profile for peaks a, b, c, d, e, and f monitored at 405 nm. B, the extracted ion chromatograms for each of the peaks observed in the HPLC elution profile. C, the full mass spectra for peak a having m / z 616.2, peaks b, c, and f having m / z values 705.5, and peaks d and e having m / z 758.5.

    Journal: The Journal of Pharmacology and Experimental Therapeutics

    Article Title: Mechanism-Based Inactivation of CYP2B1 and Its F-Helix Mutant by Two tert-Butyl Acetylenic Compounds: Covalent Modification of Prosthetic Heme Versus Apoprotein

    doi: 10.1124/jpet.109.158782

    Figure Lengend Snippet: Analysis of CYP2B1 heme adducts after incubating the reaction mixture with BMP and NADPH. After inactivation of WT by BMP, as described under Materials and Methods , the reaction mixture was injected directly onto a reverse-phase HPLC column, and the effluent was analyzed using a photodiode-array detector and a Thermo Fisher Scientific LTQ linear ion trap mass spectrometer as described under Materials and Methods . A, HPLC elution profile for peaks a, b, c, d, e, and f monitored at 405 nm. B, the extracted ion chromatograms for each of the peaks observed in the HPLC elution profile. C, the full mass spectra for peak a having m / z 616.2, peaks b, c, and f having m / z values 705.5, and peaks d and e having m / z 758.5.

    Article Snippet: An aliquot (25 pmol) of the primary reaction mixtures inactivated by incubation with BMP was injected onto a reverse-phase HPLC column (XTerra MS C18, 2.1 × 150 mm; Waters) equilibrated with 65% solvent A (0.05% TFA/0.05% formic acid) and 35% solvent B (0.05% TFA/0.05% formic acid in acetonitrile) at a flow rate of 0.3 ml/min.

    Techniques: Injection, High Performance Liquid Chromatography, Mass Spectrometry

    HPLC elution profiles for the reconstituted system after incubating the WT (A) or T205A mutant (B) 2B1 with BPA or BMP as described under Materials and Methods . WT/control and T205A/control are the reaction mixtures incubated without NADPH. WT/BMP, WT/BPA, T205A/BMP, and T205A/BPA are the reaction mixtures incubated with inactivator and NADPH. Native heme, peak 1, and peak 2 eluted at 20, 24, and 26 min, respectively.

    Journal: The Journal of Pharmacology and Experimental Therapeutics

    Article Title: Mechanism-Based Inactivation of CYP2B1 and Its F-Helix Mutant by Two tert-Butyl Acetylenic Compounds: Covalent Modification of Prosthetic Heme Versus Apoprotein

    doi: 10.1124/jpet.109.158782

    Figure Lengend Snippet: HPLC elution profiles for the reconstituted system after incubating the WT (A) or T205A mutant (B) 2B1 with BPA or BMP as described under Materials and Methods . WT/control and T205A/control are the reaction mixtures incubated without NADPH. WT/BMP, WT/BPA, T205A/BMP, and T205A/BPA are the reaction mixtures incubated with inactivator and NADPH. Native heme, peak 1, and peak 2 eluted at 20, 24, and 26 min, respectively.

    Article Snippet: An aliquot (25 pmol) of the primary reaction mixtures inactivated by incubation with BMP was injected onto a reverse-phase HPLC column (XTerra MS C18, 2.1 × 150 mm; Waters) equilibrated with 65% solvent A (0.05% TFA/0.05% formic acid) and 35% solvent B (0.05% TFA/0.05% formic acid in acetonitrile) at a flow rate of 0.3 ml/min.

    Techniques: High Performance Liquid Chromatography, Mutagenesis, Incubation