Structured Review

Phenomenex reverse phase hplc
( a ) High-pressure liquid chromatography with UV detector <t>(HPLC-UV)</t> chromatogram of <t>(±)-renzapride</t> N-oxide standard solution (1 mg/mL in acetonitrile). ( b ) HPLC-UV chromatogram of (±)-renzapride and (±)-renzapride N-oxide standard solutions (1 mg/mL in acetonitrile). ( c ) HPLC-UV chromatogram of sample from human liver microsome incubation (60 min) with (±)-renzapride and N-oxide reference standard (1 mg/mL in acetonitrile). ± signifies racemate of (+) and (−) enantiomers present in equal proportions.
Reverse Phase Hplc, supplied by Phenomenex, used in various techniques. Bioz Stars score: 93/100, based on 129 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Images

1) Product Images from "Pharmacology and Metabolism of Renzapride: A Novel Therapeutic Agent for the Potential Treatment of Irritable Bowel Syndrome"

Article Title: Pharmacology and Metabolism of Renzapride: A Novel Therapeutic Agent for the Potential Treatment of Irritable Bowel Syndrome

Journal: Drugs in R & D

doi: 10.2165/00126839-200809010-00004

( a ) High-pressure liquid chromatography with UV detector (HPLC-UV) chromatogram of (±)-renzapride N-oxide standard solution (1 mg/mL in acetonitrile). ( b ) HPLC-UV chromatogram of (±)-renzapride and (±)-renzapride N-oxide standard solutions (1 mg/mL in acetonitrile). ( c ) HPLC-UV chromatogram of sample from human liver microsome incubation (60 min) with (±)-renzapride and N-oxide reference standard (1 mg/mL in acetonitrile). ± signifies racemate of (+) and (−) enantiomers present in equal proportions.
Figure Legend Snippet: ( a ) High-pressure liquid chromatography with UV detector (HPLC-UV) chromatogram of (±)-renzapride N-oxide standard solution (1 mg/mL in acetonitrile). ( b ) HPLC-UV chromatogram of (±)-renzapride and (±)-renzapride N-oxide standard solutions (1 mg/mL in acetonitrile). ( c ) HPLC-UV chromatogram of sample from human liver microsome incubation (60 min) with (±)-renzapride and N-oxide reference standard (1 mg/mL in acetonitrile). ± signifies racemate of (+) and (−) enantiomers present in equal proportions.

Techniques Used: High Performance Liquid Chromatography, Incubation

2) Product Images from "T-1032, a cyclic GMP phosphodiesterase-5 inhibitor, acutely blocks physiologic insulin-mediated muscle haemodynamic effects and glucose uptake in vivo"

Article Title: T-1032, a cyclic GMP phosphodiesterase-5 inhibitor, acutely blocks physiologic insulin-mediated muscle haemodynamic effects and glucose uptake in vivo

Journal: British Journal of Pharmacology

doi: 10.1038/sj.bjp.0705548

Study design. The protocol involved the euglycaemic clamp at 3 mU min −1 kg −1 insulin, commencing at time=0 min, and either saline or T-1032, which was commenced at time=−60 min, or jointly with insulin. Duplicate arterial and femoral venous plasma samples were collected at 120 min, for HPLC analysis, plasma glucose, T-1032, and insulin determinations. Venous infusions are indicated by the bars. Bolus injections of allopurinol or 2-DG were made as indicated. The arterial samples were taken for glucose determinations as indicated. Venous infusions are indicated by the bars. Muscle samples were taken at 120 min for 2-DG and cGMP analyses.
Figure Legend Snippet: Study design. The protocol involved the euglycaemic clamp at 3 mU min −1 kg −1 insulin, commencing at time=0 min, and either saline or T-1032, which was commenced at time=−60 min, or jointly with insulin. Duplicate arterial and femoral venous plasma samples were collected at 120 min, for HPLC analysis, plasma glucose, T-1032, and insulin determinations. Venous infusions are indicated by the bars. Bolus injections of allopurinol or 2-DG were made as indicated. The arterial samples were taken for glucose determinations as indicated. Venous infusions are indicated by the bars. Muscle samples were taken at 120 min for 2-DG and cGMP analyses.

Techniques Used: High Performance Liquid Chromatography

3) Product Images from "Requirements for superoxide-dependent tyrosine hydroperoxide formation in peptides"

Article Title: Requirements for superoxide-dependent tyrosine hydroperoxide formation in peptides

Journal: Biochemical Journal

doi: 10.1042/BJ20040259

Loss of substrate on γ-irradiation of tyrosine, tyramine, N -acetyltyrosine (NAT) and p -hydroxyphenylacetic acid (pOH-PA) γ-Irradiation was performed at pH 7.4 under the conditions described in the Experimental section. □, Parent compound measured by HPLC; ▪, dimer measured by fluorescence. Results represent means and S.E.M. from four independent experiments.
Figure Legend Snippet: Loss of substrate on γ-irradiation of tyrosine, tyramine, N -acetyltyrosine (NAT) and p -hydroxyphenylacetic acid (pOH-PA) γ-Irradiation was performed at pH 7.4 under the conditions described in the Experimental section. □, Parent compound measured by HPLC; ▪, dimer measured by fluorescence. Results represent means and S.E.M. from four independent experiments.

Techniques Used: Irradiation, High Performance Liquid Chromatography, Fluorescence

4) Product Images from "Mapping the Trimethoprim-Induced Secondary Metabolome of Burkholderia thailandensis"

Article Title: Mapping the Trimethoprim-Induced Secondary Metabolome of Burkholderia thailandensis

Journal: ACS chemical biology

doi: 10.1021/acschembio.6b00447

Modulation of the products of the bta cluster by small molecules. (A) Products of the three AHL synthases in E264 generated from S -adenosylmethionine and varying acyl groups. Acybolin contains the same acyl chain as the product of BtaI2, which is encoded in the bactobolin gene cluster. (B) The bta gene cluster on chromosome II of E264, as previously annotated. BtaQ (green), btaI2 (red) and its cognate btaR2 (yellow) are outlined. BtaK and BtaN are multi-domain NRPS enzymes with the domain organizations shown. (C) HPLC-MS comparison of wt E264 (red and blue traces) with btaK::Tn (black and gray traces) in the presence of Tmp. Extracted are peaks corresponding to 29 (gray and blue) or 30 (black and red). The btaK mutant fails to produce any of the acybolins. (D) HPLC-Qtof-MS analysis of ΔbtaI1/I2/I3 triple mutant in the presence of only Tmp (black trace), only 3-OH-C 10 -HSL (gray), both Tmp and 3-OH-C 10 -HSL (red), both Tmp and 3-OH-C 8 -HSL (blue), or Tmp, 3-OH-C 10 -HSL and 3-OH-C 8 -HSL (green). Each trace is extracted for acybolin A ( 29 ). Both Tmp and AHL are required for production of any acybolins in this mutant. The traces in (C) and (D) are offset in both axes for clarity.
Figure Legend Snippet: Modulation of the products of the bta cluster by small molecules. (A) Products of the three AHL synthases in E264 generated from S -adenosylmethionine and varying acyl groups. Acybolin contains the same acyl chain as the product of BtaI2, which is encoded in the bactobolin gene cluster. (B) The bta gene cluster on chromosome II of E264, as previously annotated. BtaQ (green), btaI2 (red) and its cognate btaR2 (yellow) are outlined. BtaK and BtaN are multi-domain NRPS enzymes with the domain organizations shown. (C) HPLC-MS comparison of wt E264 (red and blue traces) with btaK::Tn (black and gray traces) in the presence of Tmp. Extracted are peaks corresponding to 29 (gray and blue) or 30 (black and red). The btaK mutant fails to produce any of the acybolins. (D) HPLC-Qtof-MS analysis of ΔbtaI1/I2/I3 triple mutant in the presence of only Tmp (black trace), only 3-OH-C 10 -HSL (gray), both Tmp and 3-OH-C 10 -HSL (red), both Tmp and 3-OH-C 8 -HSL (blue), or Tmp, 3-OH-C 10 -HSL and 3-OH-C 8 -HSL (green). Each trace is extracted for acybolin A ( 29 ). Both Tmp and AHL are required for production of any acybolins in this mutant. The traces in (C) and (D) are offset in both axes for clarity.

Techniques Used: Generated, High Performance Liquid Chromatography, Mass Spectrometry, Mutagenesis

5) Product Images from "The Influence of Double Bond Geometry in the Inhibition of Cyclooxygenases by Sulindac Derivatives"

Article Title: The Influence of Double Bond Geometry in the Inhibition of Cyclooxygenases by Sulindac Derivatives

Journal: Bioorganic & medicinal chemistry letters

doi: 10.1016/j.bmcl.2009.04.078

HPLC separation of the E - and Z -isomers of 2′- des -methyl sulindac sulfide.
Figure Legend Snippet: HPLC separation of the E - and Z -isomers of 2′- des -methyl sulindac sulfide.

Techniques Used: High Performance Liquid Chromatography

6) Product Images from "A novel enediyne‐integrated antibody–drug conjugate shows promising antitumor efficacy against CD30+ lymphomas"

Article Title: A novel enediyne‐integrated antibody–drug conjugate shows promising antitumor efficacy against CD30+ lymphomas

Journal: Molecular Oncology

doi: 10.1002/1878-0261.12166

The characterization and in vitro activity of anti‐CD30‐LDM. (A) Reverse‐phase HPLC analysis of enediyne‐integrated anti‐CD30‐LDM using a Vydac C4 300A column at 340 nm. (B) Binding affinity of anti‐CD30‐LDM and anti‐CD30‐LDP to Karpas299 cells by FACS. (C) Cell viability assay. Karpas299, SU‐DHL‐1, L540, and L428 cell lines were treated with anti‐CD30‐LDM and LDM in a series of concentrations (0.001–1 n m ) for 48 h. Cell viability was tested by Cell Counting Kit‐8 (CCK‐8). Results are the mean values ± SD of three replicates. (D) Flow cytometry analysis of apoptosis of L540 or Karpas299 cells treated with increasing concentrations of anti‐CD30‐LDM for 24 h, respectively. (E) Cell cycle arrest assay of Karpas299 or L540 cells by flow cytometry. Cells were treated with indicated concentrations of anti‐CD30‐LDM for 24 h.
Figure Legend Snippet: The characterization and in vitro activity of anti‐CD30‐LDM. (A) Reverse‐phase HPLC analysis of enediyne‐integrated anti‐CD30‐LDM using a Vydac C4 300A column at 340 nm. (B) Binding affinity of anti‐CD30‐LDM and anti‐CD30‐LDP to Karpas299 cells by FACS. (C) Cell viability assay. Karpas299, SU‐DHL‐1, L540, and L428 cell lines were treated with anti‐CD30‐LDM and LDM in a series of concentrations (0.001–1 n m ) for 48 h. Cell viability was tested by Cell Counting Kit‐8 (CCK‐8). Results are the mean values ± SD of three replicates. (D) Flow cytometry analysis of apoptosis of L540 or Karpas299 cells treated with increasing concentrations of anti‐CD30‐LDM for 24 h, respectively. (E) Cell cycle arrest assay of Karpas299 or L540 cells by flow cytometry. Cells were treated with indicated concentrations of anti‐CD30‐LDM for 24 h.

Techniques Used: In Vitro, Activity Assay, High Performance Liquid Chromatography, Binding Assay, FACS, Viability Assay, Cell Counting, CCK-8 Assay, Flow Cytometry, Cytometry

7) Product Images from "Metabolic Activation of the Anti-Hepatitis C Virus Nucleotide Prodrug PSI-352938"

Article Title: Metabolic Activation of the Anti-Hepatitis C Virus Nucleotide Prodrug PSI-352938

Journal: Antimicrobial Agents and Chemotherapy

doi: 10.1128/AAC.00530-12

Metabolism of PSI-352938 in clone A and primary human hepatocytes. (A and B) HPLC chromatogram of the cell extract collected from clone A cells (A) or primary human hepatocytes (B) after 8 h of incubation with 5 μM radiolabeled PSI-352938. (C
Figure Legend Snippet: Metabolism of PSI-352938 in clone A and primary human hepatocytes. (A and B) HPLC chromatogram of the cell extract collected from clone A cells (A) or primary human hepatocytes (B) after 8 h of incubation with 5 μM radiolabeled PSI-352938. (C

Techniques Used: High Performance Liquid Chromatography, Incubation

Conversion of PSI-352938 to M1. PSI-352938 was incubated with 0.5 μM CYP1A2, 1.5 μM CYP2C8, 1 μM CYP2C9, 1 μM CYP2C19, 1 μM CYP2D6, or 0.5 μM CYP3A4, and the product was separated by HPLC. All reaction mixtures
Figure Legend Snippet: Conversion of PSI-352938 to M1. PSI-352938 was incubated with 0.5 μM CYP1A2, 1.5 μM CYP2C8, 1 μM CYP2C9, 1 μM CYP2C19, 1 μM CYP2D6, or 0.5 μM CYP3A4, and the product was separated by HPLC. All reaction mixtures

Techniques Used: Incubation, High Performance Liquid Chromatography

8) Product Images from "Human platelets generate phospholipid-esterified prostaglandins via cyclooxygenase-1 that are inhibited by low dose aspirin supplementation [S]"

Article Title: Human platelets generate phospholipid-esterified prostaglandins via cyclooxygenase-1 that are inhibited by low dose aspirin supplementation [S]

Journal: Journal of Lipid Research

doi: 10.1194/jlr.M041533

Identification of esterified PGs in human platelets and analysis of PGE 2 /D 2 -PE using LC/MS/MS . A: Precursor scanning demonstrates lipids with m/z 351.2 eluting during LC/MS/MS. Total lipid extracts from washed human platelets activated with 0.2 U/ml of thrombin for 30 min at 37°C were separated on the Q-Trap platform using LC/MS/MS as described in Materials and Methods, with online negative precursor scanning for m/z 351.2. *, region of LC trace where ions appear that are elevated by thrombin stimulation. Control, broken line. B: Identification of ions that generate m/z 351.2 daughter ions. Shown is a negative MS scan of region marked * in A. Scan shows ions eluting between 19 and 24 min. C: Characterizing phospholipid headgroups of esterified PL. Lipid extracts from thrombin-activated platelets were separated on normal-phase HPLC, as described in Materials and Methods, with fractions collected at 30 sec intervals. Twenty microliters of each fraction was analyzed specific parent → m/z 351.2 MRM transitions. PL class elution was determined using commercial phospholipid standards. Panels D–G: LC/MS/MS of PGE 2 /D 2 -PEs. Platelet lipid extracts were separated using LC/MS/MS as described in Materials and Methods and detected on the Q-Trap platform by parent → m/z 271.2.
Figure Legend Snippet: Identification of esterified PGs in human platelets and analysis of PGE 2 /D 2 -PE using LC/MS/MS . A: Precursor scanning demonstrates lipids with m/z 351.2 eluting during LC/MS/MS. Total lipid extracts from washed human platelets activated with 0.2 U/ml of thrombin for 30 min at 37°C were separated on the Q-Trap platform using LC/MS/MS as described in Materials and Methods, with online negative precursor scanning for m/z 351.2. *, region of LC trace where ions appear that are elevated by thrombin stimulation. Control, broken line. B: Identification of ions that generate m/z 351.2 daughter ions. Shown is a negative MS scan of region marked * in A. Scan shows ions eluting between 19 and 24 min. C: Characterizing phospholipid headgroups of esterified PL. Lipid extracts from thrombin-activated platelets were separated on normal-phase HPLC, as described in Materials and Methods, with fractions collected at 30 sec intervals. Twenty microliters of each fraction was analyzed specific parent → m/z 351.2 MRM transitions. PL class elution was determined using commercial phospholipid standards. Panels D–G: LC/MS/MS of PGE 2 /D 2 -PEs. Platelet lipid extracts were separated using LC/MS/MS as described in Materials and Methods and detected on the Q-Trap platform by parent → m/z 271.2.

Techniques Used: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, High Performance Liquid Chromatography, Size-exclusion Chromatography

9) Product Images from "Design, Synthesis, and Ex Vivo Evaluation of a Selective Inhibitor for Retinaldehyde Dehydrogenase Enzymes."

Article Title: Design, Synthesis, and Ex Vivo Evaluation of a Selective Inhibitor for Retinaldehyde Dehydrogenase Enzymes.

Journal: Bioorganic & medicinal chemistry

doi: 10.1016/j.bmc.2018.10.009

DAR Inhibits ATRA Synthesis in a RALDH2 Expressing Cell Line. (A) Induction of chicken RALDH2-eGFP expression in a stably transfected, doxycycline (DOX)-inducible HEK 293 cell line ( (Dox) RALDH2-eGFP). RALDH2-eGFP expression is not detected in cell lysates not treated with DOX (-DOX). (Scale bar =100 pixels) (B) Toxicity curves measuring DNA content and ATP production in cells treated with DAR (0.01 – 50 μM) for 24 hrs. Data represent results of two experiments (n = 5 for each concentration/exp). (C) Inhibition of ATRA synthesis in (Dox) RALDH2-eGFP cells treated with DAR (0.05 – 2 μM) or vehicle (DMSO) for 24 hrs. Following isolation of cell lysates, the enzyme reaction was initiated by addition of NAD + and all- trans -retinaldehyde (25 μM). ATRA was quantified by HPLC. No ATRA synthesis was detected in cells not induced with DOX (-DOX). (D) Results in ( C ) normalized to RALDH2 protein expression. Relative RALDH2 expression was quantified from western blots of (Dox) RALDH2-eGFP cell lysates ( inset ). ** p
Figure Legend Snippet: DAR Inhibits ATRA Synthesis in a RALDH2 Expressing Cell Line. (A) Induction of chicken RALDH2-eGFP expression in a stably transfected, doxycycline (DOX)-inducible HEK 293 cell line ( (Dox) RALDH2-eGFP). RALDH2-eGFP expression is not detected in cell lysates not treated with DOX (-DOX). (Scale bar =100 pixels) (B) Toxicity curves measuring DNA content and ATP production in cells treated with DAR (0.01 – 50 μM) for 24 hrs. Data represent results of two experiments (n = 5 for each concentration/exp). (C) Inhibition of ATRA synthesis in (Dox) RALDH2-eGFP cells treated with DAR (0.05 – 2 μM) or vehicle (DMSO) for 24 hrs. Following isolation of cell lysates, the enzyme reaction was initiated by addition of NAD + and all- trans -retinaldehyde (25 μM). ATRA was quantified by HPLC. No ATRA synthesis was detected in cells not induced with DOX (-DOX). (D) Results in ( C ) normalized to RALDH2 protein expression. Relative RALDH2 expression was quantified from western blots of (Dox) RALDH2-eGFP cell lysates ( inset ). ** p

Techniques Used: Expressing, Stable Transfection, Transfection, Concentration Assay, Inhibition, Isolation, High Performance Liquid Chromatography, Western Blot

Effect of DAR and WIN on RALDH2 Activity in Control and Recovering Choroids. (A) Inhibition of ATRA synthesis in control and recovering choroidal lysates following 20 min pre-incubation with DAR (0.01 – 6 μM) or vehicle (DMSO). After pre-incubation, the enzyme reaction was initiated by addition of NAD + and RAL (25 μM) and allowed to proceed for 30 min at 37°C. Data are representative of results from two experiments (n = 3 – 5/experiment). (C) Inhibition of RALDH2 activity in living choroid tissue, “ ex vivo ” by DAR. Control and recovering choroids were placed in organ culture and treated with DAR (0.01 – 10 μM) or vehicle (DMSO) for 24 hrs at 37°C and 5% CO 2 . Following isolation of cytosol fractions, the enzyme reaction was initiated by addition of NAD + and RAL (25 μM) (n = 9 −14 choroids/concentration). Data represent results of three independent experiments (n = 3 – 5 choroids/experiment) (E) Inhibition of RALDH2 activity in living choroid tissue, “ ex vivo ” by WIN 18,446 (WIN). Control and recovering choroids were placed in organ culture and treated with WIN (0.1 – 10 μM) or vehicle (DMSO) for 24 hrs at 37°C and 5% CO 2 . Following isolation of cytosol fractions, the enzyme reaction was initiated by addition of NAD + and RAL (25 μM) (n = 6 choroids/concentration). ATRA was quantified using HPLC. Data represent results of two independent experiments (n = 3/choroids/experiment). (B, D, F) IC 50 and EC 50 values for DARand WIN were calculated by non-linear regression analyses of data from recovering choroids from figures A, C, and E, respectively. * p
Figure Legend Snippet: Effect of DAR and WIN on RALDH2 Activity in Control and Recovering Choroids. (A) Inhibition of ATRA synthesis in control and recovering choroidal lysates following 20 min pre-incubation with DAR (0.01 – 6 μM) or vehicle (DMSO). After pre-incubation, the enzyme reaction was initiated by addition of NAD + and RAL (25 μM) and allowed to proceed for 30 min at 37°C. Data are representative of results from two experiments (n = 3 – 5/experiment). (C) Inhibition of RALDH2 activity in living choroid tissue, “ ex vivo ” by DAR. Control and recovering choroids were placed in organ culture and treated with DAR (0.01 – 10 μM) or vehicle (DMSO) for 24 hrs at 37°C and 5% CO 2 . Following isolation of cytosol fractions, the enzyme reaction was initiated by addition of NAD + and RAL (25 μM) (n = 9 −14 choroids/concentration). Data represent results of three independent experiments (n = 3 – 5 choroids/experiment) (E) Inhibition of RALDH2 activity in living choroid tissue, “ ex vivo ” by WIN 18,446 (WIN). Control and recovering choroids were placed in organ culture and treated with WIN (0.1 – 10 μM) or vehicle (DMSO) for 24 hrs at 37°C and 5% CO 2 . Following isolation of cytosol fractions, the enzyme reaction was initiated by addition of NAD + and RAL (25 μM) (n = 6 choroids/concentration). ATRA was quantified using HPLC. Data represent results of two independent experiments (n = 3/choroids/experiment). (B, D, F) IC 50 and EC 50 values for DARand WIN were calculated by non-linear regression analyses of data from recovering choroids from figures A, C, and E, respectively. * p

Techniques Used: Activity Assay, Inhibition, Incubation, Ex Vivo, Organ Culture, Isolation, Concentration Assay, High Performance Liquid Chromatography

10) Product Images from "Patterns of crystallin distribution in porcine eye lenses"

Article Title: Patterns of crystallin distribution in porcine eye lenses

Journal: Molecular Vision

doi:

Typical size-exclusion HPLC elution profiles of the water-soluble proteins from selected layers of porcine lens. The layers are numbered consecutively from the lens periphery into the center. Numbers above the chromatogram indicate positions at which various molecular weight standards eluted. From lens periphery to center, HPLC profiles show a decrease in HMW proteins, an increase in LMW proteins and a change in proportion of the MMW proteins.
Figure Legend Snippet: Typical size-exclusion HPLC elution profiles of the water-soluble proteins from selected layers of porcine lens. The layers are numbered consecutively from the lens periphery into the center. Numbers above the chromatogram indicate positions at which various molecular weight standards eluted. From lens periphery to center, HPLC profiles show a decrease in HMW proteins, an increase in LMW proteins and a change in proportion of the MMW proteins.

Techniques Used: High Performance Liquid Chromatography, Molecular Weight

11) Product Images from "Mapping the Trimethoprim-Induced Secondary Metabolome of Burkholderia thailandensis"

Article Title: Mapping the Trimethoprim-Induced Secondary Metabolome of Burkholderia thailandensis

Journal: ACS chemical biology

doi: 10.1021/acschembio.6b00447

Modulation of the products of the bta cluster by small molecules. (A) Products of the three AHL synthases in E264 generated from S -adenosylmethionine and varying acyl groups. Acybolin contains the same acyl chain as the product of BtaI2, which is encoded in the bactobolin gene cluster. (B) The bta gene cluster on chromosome II of E264, as previously annotated. BtaQ (green), btaI2 (red) and its cognate btaR2 (yellow) are outlined. BtaK and BtaN are multi-domain NRPS enzymes with the domain organizations shown. (C) HPLC-MS comparison of wt E264 (red and blue traces) with btaK::Tn (black and gray traces) in the presence of Tmp. Extracted are peaks corresponding to 29 (gray and blue) or 30 (black and red). The btaK mutant fails to produce any of the acybolins. (D) HPLC-Qtof-MS analysis of ΔbtaI1/I2/I3 triple mutant in the presence of only Tmp (black trace), only 3-OH-C 10 -HSL (gray), both Tmp and 3-OH-C 10 -HSL (red), both Tmp and 3-OH-C 8 -HSL (blue), or Tmp, 3-OH-C 10 -HSL and 3-OH-C 8 -HSL (green). Each trace is extracted for acybolin A ( 29 ). Both Tmp and AHL are required for production of any acybolins in this mutant. The traces in (C) and (D) are offset in both axes for clarity.
Figure Legend Snippet: Modulation of the products of the bta cluster by small molecules. (A) Products of the three AHL synthases in E264 generated from S -adenosylmethionine and varying acyl groups. Acybolin contains the same acyl chain as the product of BtaI2, which is encoded in the bactobolin gene cluster. (B) The bta gene cluster on chromosome II of E264, as previously annotated. BtaQ (green), btaI2 (red) and its cognate btaR2 (yellow) are outlined. BtaK and BtaN are multi-domain NRPS enzymes with the domain organizations shown. (C) HPLC-MS comparison of wt E264 (red and blue traces) with btaK::Tn (black and gray traces) in the presence of Tmp. Extracted are peaks corresponding to 29 (gray and blue) or 30 (black and red). The btaK mutant fails to produce any of the acybolins. (D) HPLC-Qtof-MS analysis of ΔbtaI1/I2/I3 triple mutant in the presence of only Tmp (black trace), only 3-OH-C 10 -HSL (gray), both Tmp and 3-OH-C 10 -HSL (red), both Tmp and 3-OH-C 8 -HSL (blue), or Tmp, 3-OH-C 10 -HSL and 3-OH-C 8 -HSL (green). Each trace is extracted for acybolin A ( 29 ). Both Tmp and AHL are required for production of any acybolins in this mutant. The traces in (C) and (D) are offset in both axes for clarity.

Techniques Used: Generated, High Performance Liquid Chromatography, Mass Spectrometry, Mutagenesis

12) Product Images from "Complexity Generation in Fungal Peptidyl Alkaloid Biosynthesis: Oxidation of Fumiquinazoline A to the Heptacyclic Hemiaminal Fumiquinazoline C by the Flavoenzyme Af12070 from Aspergillus fumigatus"

Article Title: Complexity Generation in Fungal Peptidyl Alkaloid Biosynthesis: Oxidation of Fumiquinazoline A to the Heptacyclic Hemiaminal Fumiquinazoline C by the Flavoenzyme Af12070 from Aspergillus fumigatus

Journal: Biochemistry

doi: 10.1021/bi201302w

HPLC-based timecourse data (with detection at 254 nm) for 2 µM Af12070 Δ1–24 with 200 µM FQA as substrate. (A) HPLC chromatogram overlay of reaction aliquots quenched at the indicated time by addition of MeCN prior to injection
Figure Legend Snippet: HPLC-based timecourse data (with detection at 254 nm) for 2 µM Af12070 Δ1–24 with 200 µM FQA as substrate. (A) HPLC chromatogram overlay of reaction aliquots quenched at the indicated time by addition of MeCN prior to injection

Techniques Used: High Performance Liquid Chromatography, Injection

13) Product Images from "Human platelets generate phospholipid-esterified prostaglandins via cyclooxygenase-1 that are inhibited by low dose aspirin supplementation [S]"

Article Title: Human platelets generate phospholipid-esterified prostaglandins via cyclooxygenase-1 that are inhibited by low dose aspirin supplementation [S]

Journal: Journal of Lipid Research

doi: 10.1194/jlr.M041533

Identification of esterified PGs in human platelets and analysis of PGE 2 /D 2 -PE using LC/MS/MS . A: Precursor scanning demonstrates lipids with m/z 351.2 eluting during LC/MS/MS. Total lipid extracts from washed human platelets activated with 0.2 U/ml of thrombin for 30 min at 37°C were separated on the Q-Trap platform using LC/MS/MS as described in Materials and Methods, with online negative precursor scanning for m/z 351.2. *, region of LC trace where ions appear that are elevated by thrombin stimulation. Control, broken line. B: Identification of ions that generate m/z 351.2 daughter ions. Shown is a negative MS scan of region marked * in A. Scan shows ions eluting between 19 and 24 min. C: Characterizing phospholipid headgroups of esterified PL. Lipid extracts from thrombin-activated platelets were separated on normal-phase HPLC, as described in Materials and Methods, with fractions collected at 30 sec intervals. Twenty microliters of each fraction was analyzed specific parent → m/z 351.2 MRM transitions. PL class elution was determined using commercial phospholipid standards. Panels D–G: LC/MS/MS of PGE 2 /D 2 -PEs. Platelet lipid extracts were separated using LC/MS/MS as described in Materials and Methods and detected on the Q-Trap platform by parent → m/z 271.2.
Figure Legend Snippet: Identification of esterified PGs in human platelets and analysis of PGE 2 /D 2 -PE using LC/MS/MS . A: Precursor scanning demonstrates lipids with m/z 351.2 eluting during LC/MS/MS. Total lipid extracts from washed human platelets activated with 0.2 U/ml of thrombin for 30 min at 37°C were separated on the Q-Trap platform using LC/MS/MS as described in Materials and Methods, with online negative precursor scanning for m/z 351.2. *, region of LC trace where ions appear that are elevated by thrombin stimulation. Control, broken line. B: Identification of ions that generate m/z 351.2 daughter ions. Shown is a negative MS scan of region marked * in A. Scan shows ions eluting between 19 and 24 min. C: Characterizing phospholipid headgroups of esterified PL. Lipid extracts from thrombin-activated platelets were separated on normal-phase HPLC, as described in Materials and Methods, with fractions collected at 30 sec intervals. Twenty microliters of each fraction was analyzed specific parent → m/z 351.2 MRM transitions. PL class elution was determined using commercial phospholipid standards. Panels D–G: LC/MS/MS of PGE 2 /D 2 -PEs. Platelet lipid extracts were separated using LC/MS/MS as described in Materials and Methods and detected on the Q-Trap platform by parent → m/z 271.2.

Techniques Used: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, High Performance Liquid Chromatography, Size-exclusion Chromatography

14) Product Images from "Sirt6 regulates TNF? secretion via hydrolysis of long chain fatty acyl lysine"

Article Title: Sirt6 regulates TNF? secretion via hydrolysis of long chain fatty acyl lysine

Journal: Nature

doi: 10.1038/nature12038

Sirt6 prefers to hydrolyze long chain fatty acyl lysine in vitro. (A) HPLC traces showing Sirt6-catalyzed hydrolysis of different acyl peptides based on the H3K9 sequence. (B) H2B K12 myristoyl peptide can be hydrolyzed by Sirt6 while the corresponding acetyl peptide cannot. Reactions were carried out with 50 μM peptide, 1 μM Sirt6, 20 mM Tris pH 8.0, 0.5 mM NAD, and 1 mM DTT at 37°C for 30 min.
Figure Legend Snippet: Sirt6 prefers to hydrolyze long chain fatty acyl lysine in vitro. (A) HPLC traces showing Sirt6-catalyzed hydrolysis of different acyl peptides based on the H3K9 sequence. (B) H2B K12 myristoyl peptide can be hydrolyzed by Sirt6 while the corresponding acetyl peptide cannot. Reactions were carried out with 50 μM peptide, 1 μM Sirt6, 20 mM Tris pH 8.0, 0.5 mM NAD, and 1 mM DTT at 37°C for 30 min.

Techniques Used: In Vitro, High Performance Liquid Chromatography, Sequencing

15) Product Images from "Using Simple Donors to Drive the Equilibria of Glycosyltransferase-Catalyzed Reactions"

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

Journal: Nature chemical biology

doi: 10.1038/nchembio.638

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
Figure Legend 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

Techniques Used: 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 ) .
Figure Legend 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 ) .

Techniques Used: 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 .
Figure Legend 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 .

Techniques Used: 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 .
Figure Legend 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 .

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

16) Product Images from "Design, Synthesis, and Ex Vivo Evaluation of a Selective Inhibitor for Retinaldehyde Dehydrogenase Enzymes."

Article Title: Design, Synthesis, and Ex Vivo Evaluation of a Selective Inhibitor for Retinaldehyde Dehydrogenase Enzymes.

Journal: Bioorganic & medicinal chemistry

doi: 10.1016/j.bmc.2018.10.009

DAR Inhibits ATRA Synthesis in a RALDH2 Expressing Cell Line. (A) Induction of chicken RALDH2-eGFP expression in a stably transfected, doxycycline (DOX)-inducible HEK 293 cell line ( (Dox) RALDH2-eGFP). RALDH2-eGFP expression is not detected in cell lysates not treated with DOX (-DOX). (Scale bar =100 pixels) (B) Toxicity curves measuring DNA content and ATP production in cells treated with DAR (0.01 – 50 μM) for 24 hrs. Data represent results of two experiments (n = 5 for each concentration/exp). (C) Inhibition of ATRA synthesis in (Dox) RALDH2-eGFP cells treated with DAR (0.05 – 2 μM) or vehicle (DMSO) for 24 hrs. Following isolation of cell lysates, the enzyme reaction was initiated by addition of NAD + and all- trans -retinaldehyde (25 μM). ATRA was quantified by HPLC. No ATRA synthesis was detected in cells not induced with DOX (-DOX). (D) Results in ( C ) normalized to RALDH2 protein expression. Relative RALDH2 expression was quantified from western blots of (Dox) RALDH2-eGFP cell lysates ( inset ). ** p
Figure Legend Snippet: DAR Inhibits ATRA Synthesis in a RALDH2 Expressing Cell Line. (A) Induction of chicken RALDH2-eGFP expression in a stably transfected, doxycycline (DOX)-inducible HEK 293 cell line ( (Dox) RALDH2-eGFP). RALDH2-eGFP expression is not detected in cell lysates not treated with DOX (-DOX). (Scale bar =100 pixels) (B) Toxicity curves measuring DNA content and ATP production in cells treated with DAR (0.01 – 50 μM) for 24 hrs. Data represent results of two experiments (n = 5 for each concentration/exp). (C) Inhibition of ATRA synthesis in (Dox) RALDH2-eGFP cells treated with DAR (0.05 – 2 μM) or vehicle (DMSO) for 24 hrs. Following isolation of cell lysates, the enzyme reaction was initiated by addition of NAD + and all- trans -retinaldehyde (25 μM). ATRA was quantified by HPLC. No ATRA synthesis was detected in cells not induced with DOX (-DOX). (D) Results in ( C ) normalized to RALDH2 protein expression. Relative RALDH2 expression was quantified from western blots of (Dox) RALDH2-eGFP cell lysates ( inset ). ** p

Techniques Used: Expressing, Stable Transfection, Transfection, Concentration Assay, Inhibition, Isolation, High Performance Liquid Chromatography, Western Blot

Effect of DAR and WIN on RALDH2 Activity in Control and Recovering Choroids. (A) Inhibition of ATRA synthesis in control and recovering choroidal lysates following 20 min pre-incubation with DAR (0.01 – 6 μM) or vehicle (DMSO). After pre-incubation, the enzyme reaction was initiated by addition of NAD + and RAL (25 μM) and allowed to proceed for 30 min at 37°C. Data are representative of results from two experiments (n = 3 – 5/experiment). (C) Inhibition of RALDH2 activity in living choroid tissue, “ ex vivo ” by DAR. Control and recovering choroids were placed in organ culture and treated with DAR (0.01 – 10 μM) or vehicle (DMSO) for 24 hrs at 37°C and 5% CO 2 . Following isolation of cytosol fractions, the enzyme reaction was initiated by addition of NAD + and RAL (25 μM) (n = 9 −14 choroids/concentration). Data represent results of three independent experiments (n = 3 – 5 choroids/experiment) (E) Inhibition of RALDH2 activity in living choroid tissue, “ ex vivo ” by WIN 18,446 (WIN). Control and recovering choroids were placed in organ culture and treated with WIN (0.1 – 10 μM) or vehicle (DMSO) for 24 hrs at 37°C and 5% CO 2 . Following isolation of cytosol fractions, the enzyme reaction was initiated by addition of NAD + and RAL (25 μM) (n = 6 choroids/concentration). ATRA was quantified using HPLC. Data represent results of two independent experiments (n = 3/choroids/experiment). (B, D, F) IC 50 and EC 50 values for DARand WIN were calculated by non-linear regression analyses of data from recovering choroids from figures A, C, and E, respectively. * p
Figure Legend Snippet: Effect of DAR and WIN on RALDH2 Activity in Control and Recovering Choroids. (A) Inhibition of ATRA synthesis in control and recovering choroidal lysates following 20 min pre-incubation with DAR (0.01 – 6 μM) or vehicle (DMSO). After pre-incubation, the enzyme reaction was initiated by addition of NAD + and RAL (25 μM) and allowed to proceed for 30 min at 37°C. Data are representative of results from two experiments (n = 3 – 5/experiment). (C) Inhibition of RALDH2 activity in living choroid tissue, “ ex vivo ” by DAR. Control and recovering choroids were placed in organ culture and treated with DAR (0.01 – 10 μM) or vehicle (DMSO) for 24 hrs at 37°C and 5% CO 2 . Following isolation of cytosol fractions, the enzyme reaction was initiated by addition of NAD + and RAL (25 μM) (n = 9 −14 choroids/concentration). Data represent results of three independent experiments (n = 3 – 5 choroids/experiment) (E) Inhibition of RALDH2 activity in living choroid tissue, “ ex vivo ” by WIN 18,446 (WIN). Control and recovering choroids were placed in organ culture and treated with WIN (0.1 – 10 μM) or vehicle (DMSO) for 24 hrs at 37°C and 5% CO 2 . Following isolation of cytosol fractions, the enzyme reaction was initiated by addition of NAD + and RAL (25 μM) (n = 6 choroids/concentration). ATRA was quantified using HPLC. Data represent results of two independent experiments (n = 3/choroids/experiment). (B, D, F) IC 50 and EC 50 values for DARand WIN were calculated by non-linear regression analyses of data from recovering choroids from figures A, C, and E, respectively. * p

Techniques Used: Activity Assay, Inhibition, Incubation, Ex Vivo, Organ Culture, Isolation, Concentration Assay, High Performance Liquid Chromatography

17) Product Images from "Azemiopsin from Azemiops feae Viper Venom, a Novel Polypeptide Ligand of Nicotinic Acetylcholine Receptor *"

Article Title: Azemiopsin from Azemiops feae Viper Venom, a Novel Polypeptide Ligand of Nicotinic Acetylcholine Receptor *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M112.363051

Isolation of azemiopsin. A , separation of crude A. feae venom by gel filtration on Superdex HR75 column (10 × 300 mm) in 0.1 m ammonium acetate buffer, pH 6.2, at a flow rate of 30 ml/h. B , isolation of azemiopsin by reverse-phase HPLC on a Jupiter C18 (4.6 × 250 mm) column in a linear gradient of acetonitrile in water (0.1% TFA). Flow rate is 1 ml/min.
Figure Legend Snippet: Isolation of azemiopsin. A , separation of crude A. feae venom by gel filtration on Superdex HR75 column (10 × 300 mm) in 0.1 m ammonium acetate buffer, pH 6.2, at a flow rate of 30 ml/h. B , isolation of azemiopsin by reverse-phase HPLC on a Jupiter C18 (4.6 × 250 mm) column in a linear gradient of acetonitrile in water (0.1% TFA). Flow rate is 1 ml/min.

Techniques Used: Isolation, Filtration, Flow Cytometry, High Performance Liquid Chromatography

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    Phenomenex rp hplc
    Production of recombinant Ct1a (A) Schematic representation of the pLicC–Ct1a vector used for periplasmic expression of recombinant Ct1a. The coding region includes a MalE signal sequence (MalE SS ) for periplasmic export, a His 6 affinity tag, an MBP fusion tag and a codon-optimized gene encoding Ct1a, with a <t>TEV</t> protease recognition site inserted between the MBP and toxin-coding regions. The locations of key elements of the vector are shown, including the ribosome-binding site (RBS), T7 promoter and lac operator. (B) <t>RP-HPLC</t> chromatogram showing purification of Ct1a following removal of the His 6 -MBP tag by TEV protease. The dotted line shows the gradient of solvent B (right ordinate axis). Asterisk denotes the peak corresponding to correctly folded Ct1a. Inset is a MALDI-TOF MS spectrum showing the [M+H] + ion for the purified recombinant toxin (observed = 4413.2 Da; calculated = 4413.0 Da). Note that the recombinant Ct1a contains a non-native N-terminal serine residue and hence has a slightly higher mass than native Ct1a.
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    Phenomenex reverse phased liquid chromatography rp hplc
    Purification of Crotalus adamanteus toxin-II (CaTx-II) from Eastern Diamondback Rattlesnake venom. (A) High Performance Liquid Chromatography <t>(HPLC)</t> profiles of C. adamanteus crude venom from a Superdex G-75 column, (B–D) Reverse-phase (RP)-HPLC chromatograms from Sepharose <t>C18</t> and C8 columns, (E) Molecular mass of pure CaTx-II, (F) Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) profile of RP-HPLC fractions, lanes indicate: CA-CV C. adamanteus crude venom (1–2), lane (4) homogeneity of CaTx-II confirmed by SDS-PAGE as 15 kDa of CA-F1 - reverse-phase fraction and (5) marker, 25 µg of protein loaded per lane, respectively.
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    Phenomenex reverse phase c18 hplc column
    Demonstration of oxidized PS species in apoptotic membranes using negative ion LC/MS/MS. The presence of oxidized PAPS or PLPS species in lipid extracts of apoptotic cells was analyzed by <t>HPLC</t> with on-line negative ion electrospray ionization tandem mass spectrometry. Separation was done on a reverse phase <t>C18</t> column (2.1 × 250 mm, 5μm) using methanol/water as mobile phase at a flow rate of 0.2 ml/min with gradient and oxidized PLPS (a) and PAPS (b) molecular species identified by their characteristic retention time and daughter ions specific for each analyte, as described in Materials and methods.
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    Phenomenex reversed phase hplc procedure
    Reversed-phase <t>HPLC</t> analysis of 5′-deoxyadenosine produced by BciD during reaction with BChlide c . Shown is a reversed-phase HPLC elution profile of <t>SAM</t> standard ( dashed line ), 5′-deoxyadenosine standard ( long-dashed line ), 1-h control reaction with BChlide c ( dotted line ), and 1-h complete reaction with BciD ( solid line ) using BChlide c as the substrate. SAM (4.5 min) is consumed, and 5′-deoxyadenosine (17.8 min) is produced during the reaction.
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    Production of recombinant Ct1a (A) Schematic representation of the pLicC–Ct1a vector used for periplasmic expression of recombinant Ct1a. The coding region includes a MalE signal sequence (MalE SS ) for periplasmic export, a His 6 affinity tag, an MBP fusion tag and a codon-optimized gene encoding Ct1a, with a TEV protease recognition site inserted between the MBP and toxin-coding regions. The locations of key elements of the vector are shown, including the ribosome-binding site (RBS), T7 promoter and lac operator. (B) RP-HPLC chromatogram showing purification of Ct1a following removal of the His 6 -MBP tag by TEV protease. The dotted line shows the gradient of solvent B (right ordinate axis). Asterisk denotes the peak corresponding to correctly folded Ct1a. Inset is a MALDI-TOF MS spectrum showing the [M+H] + ion for the purified recombinant toxin (observed = 4413.2 Da; calculated = 4413.0 Da). Note that the recombinant Ct1a contains a non-native N-terminal serine residue and hence has a slightly higher mass than native Ct1a.

    Journal: Toxicon : official journal of the International Society on Toxinology

    Article Title: Isolation of two insecticidal toxins from venom of the Australian theraphosid spider Coremiocnemis tropix

    doi: 10.1016/j.toxicon.2016.10.013

    Figure Lengend Snippet: Production of recombinant Ct1a (A) Schematic representation of the pLicC–Ct1a vector used for periplasmic expression of recombinant Ct1a. The coding region includes a MalE signal sequence (MalE SS ) for periplasmic export, a His 6 affinity tag, an MBP fusion tag and a codon-optimized gene encoding Ct1a, with a TEV protease recognition site inserted between the MBP and toxin-coding regions. The locations of key elements of the vector are shown, including the ribosome-binding site (RBS), T7 promoter and lac operator. (B) RP-HPLC chromatogram showing purification of Ct1a following removal of the His 6 -MBP tag by TEV protease. The dotted line shows the gradient of solvent B (right ordinate axis). Asterisk denotes the peak corresponding to correctly folded Ct1a. Inset is a MALDI-TOF MS spectrum showing the [M+H] + ion for the purified recombinant toxin (observed = 4413.2 Da; calculated = 4413.0 Da). Note that the recombinant Ct1a contains a non-native N-terminal serine residue and hence has a slightly higher mass than native Ct1a.

    Article Snippet: The peptide was liberated from the fusion protein by addition of TEV protease and purified using RP-HPLC ( ).

    Techniques: Recombinant, Plasmid Preparation, Expressing, Sequencing, Binding Assay, High Performance Liquid Chromatography, Purification, Mass Spectrometry

    Purification of Crotalus adamanteus toxin-II (CaTx-II) from Eastern Diamondback Rattlesnake venom. (A) High Performance Liquid Chromatography (HPLC) profiles of C. adamanteus crude venom from a Superdex G-75 column, (B–D) Reverse-phase (RP)-HPLC chromatograms from Sepharose C18 and C8 columns, (E) Molecular mass of pure CaTx-II, (F) Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) profile of RP-HPLC fractions, lanes indicate: CA-CV C. adamanteus crude venom (1–2), lane (4) homogeneity of CaTx-II confirmed by SDS-PAGE as 15 kDa of CA-F1 - reverse-phase fraction and (5) marker, 25 µg of protein loaded per lane, respectively.

    Journal: PLoS ONE

    Article Title: Wound Healing Activity and Mechanisms of Action of an Antibacterial Protein from the Venom of the Eastern Diamondback Rattlesnake (Crotalus adamanteus)

    doi: 10.1371/journal.pone.0080199

    Figure Lengend Snippet: Purification of Crotalus adamanteus toxin-II (CaTx-II) from Eastern Diamondback Rattlesnake venom. (A) High Performance Liquid Chromatography (HPLC) profiles of C. adamanteus crude venom from a Superdex G-75 column, (B–D) Reverse-phase (RP)-HPLC chromatograms from Sepharose C18 and C8 columns, (E) Molecular mass of pure CaTx-II, (F) Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) profile of RP-HPLC fractions, lanes indicate: CA-CV C. adamanteus crude venom (1–2), lane (4) homogeneity of CaTx-II confirmed by SDS-PAGE as 15 kDa of CA-F1 - reverse-phase fraction and (5) marker, 25 µg of protein loaded per lane, respectively.

    Article Snippet: All the fractions were tested for enzymatic and antibacterial effects, and the fraction that showed the highest enzymatic and antibacterial effects were then subjected to reverse-phased liquid chromatography (RP-HPLC) via a C18 column (4.6×150 mm, Phenomenex, Upsala, Sweden), using a linear gradient of aqueous acetonitrile (80%) in 0.1% trifluoroacetic acid (Sigma Aldrich Co, St Louis, Missouri, USA) at a flow rate of 1.0 ml per min.

    Techniques: Purification, High Performance Liquid Chromatography, Polyacrylamide Gel Electrophoresis, SDS Page, Marker

    Demonstration of oxidized PS species in apoptotic membranes using negative ion LC/MS/MS. The presence of oxidized PAPS or PLPS species in lipid extracts of apoptotic cells was analyzed by HPLC with on-line negative ion electrospray ionization tandem mass spectrometry. Separation was done on a reverse phase C18 column (2.1 × 250 mm, 5μm) using methanol/water as mobile phase at a flow rate of 0.2 ml/min with gradient and oxidized PLPS (a) and PAPS (b) molecular species identified by their characteristic retention time and daughter ions specific for each analyte, as described in Materials and methods.

    Journal: The Journal of Experimental Medicine

    Article Title: Oxidized phosphatidylserine-CD36 interactions play an essential role in macrophage-dependent phagocytosis of apoptotic cells

    doi: 10.1084/jem.20060370

    Figure Lengend Snippet: Demonstration of oxidized PS species in apoptotic membranes using negative ion LC/MS/MS. The presence of oxidized PAPS or PLPS species in lipid extracts of apoptotic cells was analyzed by HPLC with on-line negative ion electrospray ionization tandem mass spectrometry. Separation was done on a reverse phase C18 column (2.1 × 250 mm, 5μm) using methanol/water as mobile phase at a flow rate of 0.2 ml/min with gradient and oxidized PLPS (a) and PAPS (b) molecular species identified by their characteristic retention time and daughter ions specific for each analyte, as described in Materials and methods.

    Article Snippet: Each sample preparation (in 90% methanol) was injected onto a reverse phase C18 HPLC column (2 × 150 mm, 5 μm; ODS; Phenomenex) at a flow rate of 0.2 ml/min. oxPSes were resolved using a gradient from water containing 0.2% ammonium to methanol containing 0.2% ammonium.

    Techniques: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Papanicolaou Stain, High Performance Liquid Chromatography, Flow Cytometry

    Reversed-phase HPLC analysis of 5′-deoxyadenosine produced by BciD during reaction with BChlide c . Shown is a reversed-phase HPLC elution profile of SAM standard ( dashed line ), 5′-deoxyadenosine standard ( long-dashed line ), 1-h control reaction with BChlide c ( dotted line ), and 1-h complete reaction with BciD ( solid line ) using BChlide c as the substrate. SAM (4.5 min) is consumed, and 5′-deoxyadenosine (17.8 min) is produced during the reaction.

    Journal: The Journal of Biological Chemistry

    Article Title: BciD Is a Radical S-Adenosyl-l-methionine (SAM) Enzyme That Completes Bacteriochlorophyllide e Biosynthesis by Oxidizing a Methyl Group into a Formyl Group at C-7 *

    doi: 10.1074/jbc.M116.767665

    Figure Lengend Snippet: Reversed-phase HPLC analysis of 5′-deoxyadenosine produced by BciD during reaction with BChlide c . Shown is a reversed-phase HPLC elution profile of SAM standard ( dashed line ), 5′-deoxyadenosine standard ( long-dashed line ), 1-h control reaction with BChlide c ( dotted line ), and 1-h complete reaction with BciD ( solid line ) using BChlide c as the substrate. SAM (4.5 min) is consumed, and 5′-deoxyadenosine (17.8 min) is produced during the reaction.

    Article Snippet: SAM products were separated by a reversed-phase HPLC procedure modified from Lanz et al. , using a 150 × 4.6-mm Kinetex 5-μm C-18 column (Phenomenex Inc., Torrance, CA) and a Shimadzu UFLC system (Shimadzu Scientific Instruments, Columbia, MD).

    Techniques: High Performance Liquid Chromatography, Produced