shuffle system  (New England Biolabs)


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    New England Biolabs shuffle system
    Shuffle System, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    shuffle system  (New England Biolabs)


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    New England Biolabs shuffle system
    Shuffle System, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    shuffle t7 competent e coli  (New England Biolabs)


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    New England Biolabs shuffle t7 competent e coli
    Shuffle T7 Competent E Coli, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    shuffle competent e coli cells  (New England Biolabs)


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    New England Biolabs shuffle competent e coli cells
    Shuffle Competent E Coli Cells, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    e coli cells  (New England Biolabs)


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    New England Biolabs e coli cells
    E Coli Cells, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    shuffle t7  (New England Biolabs)


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    New England Biolabs shuffle t7
    Shuffle T7, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    e coli shuffle t7 competent cells  (New England Biolabs)


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    New England Biolabs e coli shuffle t7 competent cells
    (A) Schematic showing Them1-mediated de-activation of myristoyl (C14)-CoA into myristic acid plus CoA. Free CoA liberated by Them1 activity covalently binds to a non-fluorescent detection reagent, generating a fluorescent product. (B) Recombinant His-tagged human Them1 (Them1) purified from Shuffle <t>T7</t> competent cells on a HisTrap HP column (Black line). Upper right panel , SDS-PAGE of Them1 (67 kD). (C) Recombinant Them1 and C14-CoA (25 μM) reactions (black dots). (D) Reactions consisting of Them1 and C14-CoA (white dots, 0 μM; black dots, 25 μM) were incubated for the indicated timepoints. (E) Saturation curve of Them1 and C14-CoA reactions (black dots) were used to determined steady state kinetic constants. (F) A pilot small-molecule screen was performed using the library of pharmacologically active compounds (LOPAC, 12.5 μM, 1,056 compounds) with Them1 and C14-CoA as substrate. Compounds were considered as potential inhibitors when Them1 activity was inhibited based on a normalized percent inhibition (NPI) β 30. A representative plate with Z’ factor and 2 compounds fulfilling the inhibitor criteria are shown. Negative values are attributable to the absorbance of a minority of compounds. (G) Reproducibility of the pilot small molecule screen was assessed by NPI values of compounds over 2 independent days. Correlation between d 1 and 2 is indicated by the purple line; R 2 = 0.93. Data represent the mean (s.e.m.) of triplicate determinations. Where not visible, standard error bars are contained within the symbol sizes.
    E Coli Shuffle T7 Competent Cells, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "High-throughput screening identifies small molecule inhibitors of thioesterase superfamily member 1: Implications for the management of non-alcoholic fatty liver disease"

    Article Title: High-throughput screening identifies small molecule inhibitors of thioesterase superfamily member 1: Implications for the management of non-alcoholic fatty liver disease

    Journal: bioRxiv

    doi: 10.1101/2022.10.15.512369

    (A) Schematic showing Them1-mediated de-activation of myristoyl (C14)-CoA into myristic acid plus CoA. Free CoA liberated by Them1 activity covalently binds to a non-fluorescent detection reagent, generating a fluorescent product. (B) Recombinant His-tagged human Them1 (Them1) purified from Shuffle T7 competent cells on a HisTrap HP column (Black line). Upper right panel , SDS-PAGE of Them1 (67 kD). (C) Recombinant Them1 and C14-CoA (25 μM) reactions (black dots). (D) Reactions consisting of Them1 and C14-CoA (white dots, 0 μM; black dots, 25 μM) were incubated for the indicated timepoints. (E) Saturation curve of Them1 and C14-CoA reactions (black dots) were used to determined steady state kinetic constants. (F) A pilot small-molecule screen was performed using the library of pharmacologically active compounds (LOPAC, 12.5 μM, 1,056 compounds) with Them1 and C14-CoA as substrate. Compounds were considered as potential inhibitors when Them1 activity was inhibited based on a normalized percent inhibition (NPI) β 30. A representative plate with Z’ factor and 2 compounds fulfilling the inhibitor criteria are shown. Negative values are attributable to the absorbance of a minority of compounds. (G) Reproducibility of the pilot small molecule screen was assessed by NPI values of compounds over 2 independent days. Correlation between d 1 and 2 is indicated by the purple line; R 2 = 0.93. Data represent the mean (s.e.m.) of triplicate determinations. Where not visible, standard error bars are contained within the symbol sizes.
    Figure Legend Snippet: (A) Schematic showing Them1-mediated de-activation of myristoyl (C14)-CoA into myristic acid plus CoA. Free CoA liberated by Them1 activity covalently binds to a non-fluorescent detection reagent, generating a fluorescent product. (B) Recombinant His-tagged human Them1 (Them1) purified from Shuffle T7 competent cells on a HisTrap HP column (Black line). Upper right panel , SDS-PAGE of Them1 (67 kD). (C) Recombinant Them1 and C14-CoA (25 μM) reactions (black dots). (D) Reactions consisting of Them1 and C14-CoA (white dots, 0 μM; black dots, 25 μM) were incubated for the indicated timepoints. (E) Saturation curve of Them1 and C14-CoA reactions (black dots) were used to determined steady state kinetic constants. (F) A pilot small-molecule screen was performed using the library of pharmacologically active compounds (LOPAC, 12.5 μM, 1,056 compounds) with Them1 and C14-CoA as substrate. Compounds were considered as potential inhibitors when Them1 activity was inhibited based on a normalized percent inhibition (NPI) β 30. A representative plate with Z’ factor and 2 compounds fulfilling the inhibitor criteria are shown. Negative values are attributable to the absorbance of a minority of compounds. (G) Reproducibility of the pilot small molecule screen was assessed by NPI values of compounds over 2 independent days. Correlation between d 1 and 2 is indicated by the purple line; R 2 = 0.93. Data represent the mean (s.e.m.) of triplicate determinations. Where not visible, standard error bars are contained within the symbol sizes.

    Techniques Used: Activation Assay, Activity Assay, Recombinant, Purification, SDS Page, Incubation, Inhibition

    (A-G) Acot isoform panels : Left panels , recombinant Acot isoform enzymes including type I [Acot1, Acot2] and type II [Acot9, Acot12 and Acot13], as well as a truncated Them1 either containing only the 2 thioesterase domains but lacking the START domain (Them1-ΔSTART) or the START domain but lacking the 2 thioesterase domains (START) were purified from Shuffle T7 or BL21 (DE3) E. coli competent cells on a HisTrap HP column unless otherwise specified. Recombinant START was further purified using a Superdex 75 column. Recombinant Acot9 was purified on a MBPTrap HP column. Upper right panel , SDS-PAGE of recombinant Acot isoform enzymes. Right panel , saturation curves of recombinant Acot isoform enzymes and myristoyl (C14)-CoA reactions (black dots) were used to determine steady state kinetic constants. Where not visible, standard error bars are contained within the symbol sizes. ( H ) Table , steady state kinetic constants (K m , V max , K cat and K cat /K m ) were determined as described in the text. Data represent the mean (s.e.m.) of triplicate determinations.
    Figure Legend Snippet: (A-G) Acot isoform panels : Left panels , recombinant Acot isoform enzymes including type I [Acot1, Acot2] and type II [Acot9, Acot12 and Acot13], as well as a truncated Them1 either containing only the 2 thioesterase domains but lacking the START domain (Them1-ΔSTART) or the START domain but lacking the 2 thioesterase domains (START) were purified from Shuffle T7 or BL21 (DE3) E. coli competent cells on a HisTrap HP column unless otherwise specified. Recombinant START was further purified using a Superdex 75 column. Recombinant Acot9 was purified on a MBPTrap HP column. Upper right panel , SDS-PAGE of recombinant Acot isoform enzymes. Right panel , saturation curves of recombinant Acot isoform enzymes and myristoyl (C14)-CoA reactions (black dots) were used to determine steady state kinetic constants. Where not visible, standard error bars are contained within the symbol sizes. ( H ) Table , steady state kinetic constants (K m , V max , K cat and K cat /K m ) were determined as described in the text. Data represent the mean (s.e.m.) of triplicate determinations.

    Techniques Used: Recombinant, Purification, SDS Page

    Pre-dialysis reactions were performed with recombinant His-tagged human Them1 (Them1) and myristoyl (C14)-CoA. Reactions subjected to an overnight dialysis were incubated with C14-CoA. ( A ) Time course of Pre- and Post- dialysis reactions for the indicated timepoints. ( B ) Pre- and Post-reactions consisting of compounds U1 , W1 or Ebselen [(-) control]. Each graph is representative of four independent experiments. ( C ) Left panels , recombinant His-tagged mouse Them1 was purified from BL21 (DE3) E. coli competent cells on a HisTrap HP column. Upper right panel , SDS-PAGE of recombinant mouse Them1. Right panel , saturation curve of recombinant mouse Them1 (125 nM) and C14-CoA reactions (black dots) was used to determine steady state kinetic constants. ( D ) All reactions were performed with either recombinant mouse Them1 or recombinant human Them1. Acot activities quantified as IC 50 values are shown. Data represent the mean ± s.e.m. of triplicate determinations. Where not visible, standard error bars are contained within the symbol sizes. ***, P<0.001; Pre- ( U1 ) vs. Post- ( U1 ); ###, P<0.001; Pre- ( W1 ) vs. Post- ( W1 ).
    Figure Legend Snippet: Pre-dialysis reactions were performed with recombinant His-tagged human Them1 (Them1) and myristoyl (C14)-CoA. Reactions subjected to an overnight dialysis were incubated with C14-CoA. ( A ) Time course of Pre- and Post- dialysis reactions for the indicated timepoints. ( B ) Pre- and Post-reactions consisting of compounds U1 , W1 or Ebselen [(-) control]. Each graph is representative of four independent experiments. ( C ) Left panels , recombinant His-tagged mouse Them1 was purified from BL21 (DE3) E. coli competent cells on a HisTrap HP column. Upper right panel , SDS-PAGE of recombinant mouse Them1. Right panel , saturation curve of recombinant mouse Them1 (125 nM) and C14-CoA reactions (black dots) was used to determine steady state kinetic constants. ( D ) All reactions were performed with either recombinant mouse Them1 or recombinant human Them1. Acot activities quantified as IC 50 values are shown. Data represent the mean ± s.e.m. of triplicate determinations. Where not visible, standard error bars are contained within the symbol sizes. ***, P<0.001; Pre- ( U1 ) vs. Post- ( U1 ); ###, P<0.001; Pre- ( W1 ) vs. Post- ( W1 ).

    Techniques Used: Recombinant, Incubation, Purification, SDS Page

    t7 competent cells  (New England Biolabs)


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    New England Biolabs t7 competent cells
    (A) Schematic showing Them1-mediated de-activation of myristoyl (C14)-CoA into myristic acid plus CoA. Free CoA liberated by Them1 activity covalently binds to a non-fluorescent detection reagent, generating a fluorescent product. (B) Recombinant His-tagged human Them1 (Them1) purified from Shuffle <t>T7</t> <t>competent</t> cells on a HisTrap HP column (Black line). Upper right panel , SDS-PAGE of Them1 (67 kD). (C) Recombinant Them1 and C14-CoA (25 μM) reactions (black dots). (D) Reactions consisting of Them1 and C14-CoA (white dots, 0 μM; black dots, 25 μM) were incubated for the indicated timepoints. (E) Saturation curve of Them1 and C14-CoA reactions (black dots) were used to determined steady state kinetic constants. (F) A pilot small-molecule screen was performed using the library of pharmacologically active compounds (LOPAC, 12.5 μM, 1,056 compounds) with Them1 and C14-CoA as substrate. Compounds were considered as potential inhibitors when Them1 activity was inhibited based on a normalized percent inhibition (NPI) β 30. A representative plate with Z’ factor and 2 compounds fulfilling the inhibitor criteria are shown. Negative values are attributable to the absorbance of a minority of compounds. (G) Reproducibility of the pilot small molecule screen was assessed by NPI values of compounds over 2 independent days. Correlation between d 1 and 2 is indicated by the purple line; R 2 = 0.93. Data represent the mean (s.e.m.) of triplicate determinations. Where not visible, standard error bars are contained within the symbol sizes.
    T7 Competent Cells, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    t7 competent cells - by Bioz Stars, 2023-02
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    Images

    1) Product Images from "High-throughput screening identifies small molecule inhibitors of thioesterase superfamily member 1: Implications for the management of non-alcoholic fatty liver disease"

    Article Title: High-throughput screening identifies small molecule inhibitors of thioesterase superfamily member 1: Implications for the management of non-alcoholic fatty liver disease

    Journal: bioRxiv

    doi: 10.1101/2022.10.15.512369

    (A) Schematic showing Them1-mediated de-activation of myristoyl (C14)-CoA into myristic acid plus CoA. Free CoA liberated by Them1 activity covalently binds to a non-fluorescent detection reagent, generating a fluorescent product. (B) Recombinant His-tagged human Them1 (Them1) purified from Shuffle T7 competent cells on a HisTrap HP column (Black line). Upper right panel , SDS-PAGE of Them1 (67 kD). (C) Recombinant Them1 and C14-CoA (25 μM) reactions (black dots). (D) Reactions consisting of Them1 and C14-CoA (white dots, 0 μM; black dots, 25 μM) were incubated for the indicated timepoints. (E) Saturation curve of Them1 and C14-CoA reactions (black dots) were used to determined steady state kinetic constants. (F) A pilot small-molecule screen was performed using the library of pharmacologically active compounds (LOPAC, 12.5 μM, 1,056 compounds) with Them1 and C14-CoA as substrate. Compounds were considered as potential inhibitors when Them1 activity was inhibited based on a normalized percent inhibition (NPI) β 30. A representative plate with Z’ factor and 2 compounds fulfilling the inhibitor criteria are shown. Negative values are attributable to the absorbance of a minority of compounds. (G) Reproducibility of the pilot small molecule screen was assessed by NPI values of compounds over 2 independent days. Correlation between d 1 and 2 is indicated by the purple line; R 2 = 0.93. Data represent the mean (s.e.m.) of triplicate determinations. Where not visible, standard error bars are contained within the symbol sizes.
    Figure Legend Snippet: (A) Schematic showing Them1-mediated de-activation of myristoyl (C14)-CoA into myristic acid plus CoA. Free CoA liberated by Them1 activity covalently binds to a non-fluorescent detection reagent, generating a fluorescent product. (B) Recombinant His-tagged human Them1 (Them1) purified from Shuffle T7 competent cells on a HisTrap HP column (Black line). Upper right panel , SDS-PAGE of Them1 (67 kD). (C) Recombinant Them1 and C14-CoA (25 μM) reactions (black dots). (D) Reactions consisting of Them1 and C14-CoA (white dots, 0 μM; black dots, 25 μM) were incubated for the indicated timepoints. (E) Saturation curve of Them1 and C14-CoA reactions (black dots) were used to determined steady state kinetic constants. (F) A pilot small-molecule screen was performed using the library of pharmacologically active compounds (LOPAC, 12.5 μM, 1,056 compounds) with Them1 and C14-CoA as substrate. Compounds were considered as potential inhibitors when Them1 activity was inhibited based on a normalized percent inhibition (NPI) β 30. A representative plate with Z’ factor and 2 compounds fulfilling the inhibitor criteria are shown. Negative values are attributable to the absorbance of a minority of compounds. (G) Reproducibility of the pilot small molecule screen was assessed by NPI values of compounds over 2 independent days. Correlation between d 1 and 2 is indicated by the purple line; R 2 = 0.93. Data represent the mean (s.e.m.) of triplicate determinations. Where not visible, standard error bars are contained within the symbol sizes.

    Techniques Used: Activation Assay, Activity Assay, Recombinant, Purification, SDS Page, Incubation, Inhibition

    (A-G) Acot isoform panels : Left panels , recombinant Acot isoform enzymes including type I [Acot1, Acot2] and type II [Acot9, Acot12 and Acot13], as well as a truncated Them1 either containing only the 2 thioesterase domains but lacking the START domain (Them1-ΔSTART) or the START domain but lacking the 2 thioesterase domains (START) were purified from Shuffle T7 or BL21 (DE3) E. coli competent cells on a HisTrap HP column unless otherwise specified. Recombinant START was further purified using a Superdex 75 column. Recombinant Acot9 was purified on a MBPTrap HP column. Upper right panel , SDS-PAGE of recombinant Acot isoform enzymes. Right panel , saturation curves of recombinant Acot isoform enzymes and myristoyl (C14)-CoA reactions (black dots) were used to determine steady state kinetic constants. Where not visible, standard error bars are contained within the symbol sizes. ( H ) Table , steady state kinetic constants (K m , V max , K cat and K cat /K m ) were determined as described in the text. Data represent the mean (s.e.m.) of triplicate determinations.
    Figure Legend Snippet: (A-G) Acot isoform panels : Left panels , recombinant Acot isoform enzymes including type I [Acot1, Acot2] and type II [Acot9, Acot12 and Acot13], as well as a truncated Them1 either containing only the 2 thioesterase domains but lacking the START domain (Them1-ΔSTART) or the START domain but lacking the 2 thioesterase domains (START) were purified from Shuffle T7 or BL21 (DE3) E. coli competent cells on a HisTrap HP column unless otherwise specified. Recombinant START was further purified using a Superdex 75 column. Recombinant Acot9 was purified on a MBPTrap HP column. Upper right panel , SDS-PAGE of recombinant Acot isoform enzymes. Right panel , saturation curves of recombinant Acot isoform enzymes and myristoyl (C14)-CoA reactions (black dots) were used to determine steady state kinetic constants. Where not visible, standard error bars are contained within the symbol sizes. ( H ) Table , steady state kinetic constants (K m , V max , K cat and K cat /K m ) were determined as described in the text. Data represent the mean (s.e.m.) of triplicate determinations.

    Techniques Used: Recombinant, Purification, SDS Page

    shuffle t7 e coli competent cells  (New England Biolabs)


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    New England Biolabs shuffle t7 e coli competent cells
    Shuffle T7 E Coli Competent Cells, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    escherichia coli shuffle t7  (New England Biolabs)


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    New England Biolabs escherichia coli shuffle t7
    Escherichia Coli Shuffle T7, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    shuffle t7 competent e coli cells  (New England Biolabs)


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    New England Biolabs shuffle t7 competent e coli cells
    Shuffle T7 Competent E Coli Cells, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs e coli shuffle t7 competent cells
    (A) Schematic showing Them1-mediated de-activation of myristoyl (C14)-CoA into myristic acid plus CoA. Free CoA liberated by Them1 activity covalently binds to a non-fluorescent detection reagent, generating a fluorescent product. (B) Recombinant His-tagged human Them1 (Them1) purified from Shuffle <t>T7</t> competent cells on a HisTrap HP column (Black line). Upper right panel , SDS-PAGE of Them1 (67 kD). (C) Recombinant Them1 and C14-CoA (25 μM) reactions (black dots). (D) Reactions consisting of Them1 and C14-CoA (white dots, 0 μM; black dots, 25 μM) were incubated for the indicated timepoints. (E) Saturation curve of Them1 and C14-CoA reactions (black dots) were used to determined steady state kinetic constants. (F) A pilot small-molecule screen was performed using the library of pharmacologically active compounds (LOPAC, 12.5 μM, 1,056 compounds) with Them1 and C14-CoA as substrate. Compounds were considered as potential inhibitors when Them1 activity was inhibited based on a normalized percent inhibition (NPI) β 30. A representative plate with Z’ factor and 2 compounds fulfilling the inhibitor criteria are shown. Negative values are attributable to the absorbance of a minority of compounds. (G) Reproducibility of the pilot small molecule screen was assessed by NPI values of compounds over 2 independent days. Correlation between d 1 and 2 is indicated by the purple line; R 2 = 0.93. Data represent the mean (s.e.m.) of triplicate determinations. Where not visible, standard error bars are contained within the symbol sizes.
    E Coli Shuffle T7 Competent Cells, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs t7 competent cells
    (A) Schematic showing Them1-mediated de-activation of myristoyl (C14)-CoA into myristic acid plus CoA. Free CoA liberated by Them1 activity covalently binds to a non-fluorescent detection reagent, generating a fluorescent product. (B) Recombinant His-tagged human Them1 (Them1) purified from Shuffle <t>T7</t> <t>competent</t> cells on a HisTrap HP column (Black line). Upper right panel , SDS-PAGE of Them1 (67 kD). (C) Recombinant Them1 and C14-CoA (25 μM) reactions (black dots). (D) Reactions consisting of Them1 and C14-CoA (white dots, 0 μM; black dots, 25 μM) were incubated for the indicated timepoints. (E) Saturation curve of Them1 and C14-CoA reactions (black dots) were used to determined steady state kinetic constants. (F) A pilot small-molecule screen was performed using the library of pharmacologically active compounds (LOPAC, 12.5 μM, 1,056 compounds) with Them1 and C14-CoA as substrate. Compounds were considered as potential inhibitors when Them1 activity was inhibited based on a normalized percent inhibition (NPI) β 30. A representative plate with Z’ factor and 2 compounds fulfilling the inhibitor criteria are shown. Negative values are attributable to the absorbance of a minority of compounds. (G) Reproducibility of the pilot small molecule screen was assessed by NPI values of compounds over 2 independent days. Correlation between d 1 and 2 is indicated by the purple line; R 2 = 0.93. Data represent the mean (s.e.m.) of triplicate determinations. Where not visible, standard error bars are contained within the symbol sizes.
    T7 Competent Cells, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs shuffle t7 e coli competent cells
    (A) Schematic showing Them1-mediated de-activation of myristoyl (C14)-CoA into myristic acid plus CoA. Free CoA liberated by Them1 activity covalently binds to a non-fluorescent detection reagent, generating a fluorescent product. (B) Recombinant His-tagged human Them1 (Them1) purified from Shuffle <t>T7</t> <t>competent</t> cells on a HisTrap HP column (Black line). Upper right panel , SDS-PAGE of Them1 (67 kD). (C) Recombinant Them1 and C14-CoA (25 μM) reactions (black dots). (D) Reactions consisting of Them1 and C14-CoA (white dots, 0 μM; black dots, 25 μM) were incubated for the indicated timepoints. (E) Saturation curve of Them1 and C14-CoA reactions (black dots) were used to determined steady state kinetic constants. (F) A pilot small-molecule screen was performed using the library of pharmacologically active compounds (LOPAC, 12.5 μM, 1,056 compounds) with Them1 and C14-CoA as substrate. Compounds were considered as potential inhibitors when Them1 activity was inhibited based on a normalized percent inhibition (NPI) β 30. A representative plate with Z’ factor and 2 compounds fulfilling the inhibitor criteria are shown. Negative values are attributable to the absorbance of a minority of compounds. (G) Reproducibility of the pilot small molecule screen was assessed by NPI values of compounds over 2 independent days. Correlation between d 1 and 2 is indicated by the purple line; R 2 = 0.93. Data represent the mean (s.e.m.) of triplicate determinations. Where not visible, standard error bars are contained within the symbol sizes.
    Shuffle T7 E Coli Competent Cells, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs escherichia coli shuffle t7
    (A) Schematic showing Them1-mediated de-activation of myristoyl (C14)-CoA into myristic acid plus CoA. Free CoA liberated by Them1 activity covalently binds to a non-fluorescent detection reagent, generating a fluorescent product. (B) Recombinant His-tagged human Them1 (Them1) purified from Shuffle <t>T7</t> <t>competent</t> cells on a HisTrap HP column (Black line). Upper right panel , SDS-PAGE of Them1 (67 kD). (C) Recombinant Them1 and C14-CoA (25 μM) reactions (black dots). (D) Reactions consisting of Them1 and C14-CoA (white dots, 0 μM; black dots, 25 μM) were incubated for the indicated timepoints. (E) Saturation curve of Them1 and C14-CoA reactions (black dots) were used to determined steady state kinetic constants. (F) A pilot small-molecule screen was performed using the library of pharmacologically active compounds (LOPAC, 12.5 μM, 1,056 compounds) with Them1 and C14-CoA as substrate. Compounds were considered as potential inhibitors when Them1 activity was inhibited based on a normalized percent inhibition (NPI) β 30. A representative plate with Z’ factor and 2 compounds fulfilling the inhibitor criteria are shown. Negative values are attributable to the absorbance of a minority of compounds. (G) Reproducibility of the pilot small molecule screen was assessed by NPI values of compounds over 2 independent days. Correlation between d 1 and 2 is indicated by the purple line; R 2 = 0.93. Data represent the mean (s.e.m.) of triplicate determinations. Where not visible, standard error bars are contained within the symbol sizes.
    Escherichia Coli Shuffle T7, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs shuffle t7 competent e coli cells
    (A) Schematic showing Them1-mediated de-activation of myristoyl (C14)-CoA into myristic acid plus CoA. Free CoA liberated by Them1 activity covalently binds to a non-fluorescent detection reagent, generating a fluorescent product. (B) Recombinant His-tagged human Them1 (Them1) purified from Shuffle <t>T7</t> <t>competent</t> cells on a HisTrap HP column (Black line). Upper right panel , SDS-PAGE of Them1 (67 kD). (C) Recombinant Them1 and C14-CoA (25 μM) reactions (black dots). (D) Reactions consisting of Them1 and C14-CoA (white dots, 0 μM; black dots, 25 μM) were incubated for the indicated timepoints. (E) Saturation curve of Them1 and C14-CoA reactions (black dots) were used to determined steady state kinetic constants. (F) A pilot small-molecule screen was performed using the library of pharmacologically active compounds (LOPAC, 12.5 μM, 1,056 compounds) with Them1 and C14-CoA as substrate. Compounds were considered as potential inhibitors when Them1 activity was inhibited based on a normalized percent inhibition (NPI) β 30. A representative plate with Z’ factor and 2 compounds fulfilling the inhibitor criteria are shown. Negative values are attributable to the absorbance of a minority of compounds. (G) Reproducibility of the pilot small molecule screen was assessed by NPI values of compounds over 2 independent days. Correlation between d 1 and 2 is indicated by the purple line; R 2 = 0.93. Data represent the mean (s.e.m.) of triplicate determinations. Where not visible, standard error bars are contained within the symbol sizes.
    Shuffle T7 Competent E Coli Cells, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    (A) Schematic showing Them1-mediated de-activation of myristoyl (C14)-CoA into myristic acid plus CoA. Free CoA liberated by Them1 activity covalently binds to a non-fluorescent detection reagent, generating a fluorescent product. (B) Recombinant His-tagged human Them1 (Them1) purified from Shuffle T7 competent cells on a HisTrap HP column (Black line). Upper right panel , SDS-PAGE of Them1 (67 kD). (C) Recombinant Them1 and C14-CoA (25 μM) reactions (black dots). (D) Reactions consisting of Them1 and C14-CoA (white dots, 0 μM; black dots, 25 μM) were incubated for the indicated timepoints. (E) Saturation curve of Them1 and C14-CoA reactions (black dots) were used to determined steady state kinetic constants. (F) A pilot small-molecule screen was performed using the library of pharmacologically active compounds (LOPAC, 12.5 μM, 1,056 compounds) with Them1 and C14-CoA as substrate. Compounds were considered as potential inhibitors when Them1 activity was inhibited based on a normalized percent inhibition (NPI) β 30. A representative plate with Z’ factor and 2 compounds fulfilling the inhibitor criteria are shown. Negative values are attributable to the absorbance of a minority of compounds. (G) Reproducibility of the pilot small molecule screen was assessed by NPI values of compounds over 2 independent days. Correlation between d 1 and 2 is indicated by the purple line; R 2 = 0.93. Data represent the mean (s.e.m.) of triplicate determinations. Where not visible, standard error bars are contained within the symbol sizes.

    Journal: bioRxiv

    Article Title: High-throughput screening identifies small molecule inhibitors of thioesterase superfamily member 1: Implications for the management of non-alcoholic fatty liver disease

    doi: 10.1101/2022.10.15.512369

    Figure Lengend Snippet: (A) Schematic showing Them1-mediated de-activation of myristoyl (C14)-CoA into myristic acid plus CoA. Free CoA liberated by Them1 activity covalently binds to a non-fluorescent detection reagent, generating a fluorescent product. (B) Recombinant His-tagged human Them1 (Them1) purified from Shuffle T7 competent cells on a HisTrap HP column (Black line). Upper right panel , SDS-PAGE of Them1 (67 kD). (C) Recombinant Them1 and C14-CoA (25 μM) reactions (black dots). (D) Reactions consisting of Them1 and C14-CoA (white dots, 0 μM; black dots, 25 μM) were incubated for the indicated timepoints. (E) Saturation curve of Them1 and C14-CoA reactions (black dots) were used to determined steady state kinetic constants. (F) A pilot small-molecule screen was performed using the library of pharmacologically active compounds (LOPAC, 12.5 μM, 1,056 compounds) with Them1 and C14-CoA as substrate. Compounds were considered as potential inhibitors when Them1 activity was inhibited based on a normalized percent inhibition (NPI) β 30. A representative plate with Z’ factor and 2 compounds fulfilling the inhibitor criteria are shown. Negative values are attributable to the absorbance of a minority of compounds. (G) Reproducibility of the pilot small molecule screen was assessed by NPI values of compounds over 2 independent days. Correlation between d 1 and 2 is indicated by the purple line; R 2 = 0.93. Data represent the mean (s.e.m.) of triplicate determinations. Where not visible, standard error bars are contained within the symbol sizes.

    Article Snippet: Cultures of E. coli Shuffle T7 competent cells (New England Biolabs, Ipswich, MA) transformed with pET19b-Them1 plasmid were grown to an A 600 of ∼ 0.5 – 0.7 in terrific broth followed by induction of recombinant Them1 by the addition of 0.5 mM isoproyl β-D-thiogalactoside with 16 h of shaking (250 rpm) at 18 °C.

    Techniques: Activation Assay, Activity Assay, Recombinant, Purification, SDS Page, Incubation, Inhibition

    (A-G) Acot isoform panels : Left panels , recombinant Acot isoform enzymes including type I [Acot1, Acot2] and type II [Acot9, Acot12 and Acot13], as well as a truncated Them1 either containing only the 2 thioesterase domains but lacking the START domain (Them1-ΔSTART) or the START domain but lacking the 2 thioesterase domains (START) were purified from Shuffle T7 or BL21 (DE3) E. coli competent cells on a HisTrap HP column unless otherwise specified. Recombinant START was further purified using a Superdex 75 column. Recombinant Acot9 was purified on a MBPTrap HP column. Upper right panel , SDS-PAGE of recombinant Acot isoform enzymes. Right panel , saturation curves of recombinant Acot isoform enzymes and myristoyl (C14)-CoA reactions (black dots) were used to determine steady state kinetic constants. Where not visible, standard error bars are contained within the symbol sizes. ( H ) Table , steady state kinetic constants (K m , V max , K cat and K cat /K m ) were determined as described in the text. Data represent the mean (s.e.m.) of triplicate determinations.

    Journal: bioRxiv

    Article Title: High-throughput screening identifies small molecule inhibitors of thioesterase superfamily member 1: Implications for the management of non-alcoholic fatty liver disease

    doi: 10.1101/2022.10.15.512369

    Figure Lengend Snippet: (A-G) Acot isoform panels : Left panels , recombinant Acot isoform enzymes including type I [Acot1, Acot2] and type II [Acot9, Acot12 and Acot13], as well as a truncated Them1 either containing only the 2 thioesterase domains but lacking the START domain (Them1-ΔSTART) or the START domain but lacking the 2 thioesterase domains (START) were purified from Shuffle T7 or BL21 (DE3) E. coli competent cells on a HisTrap HP column unless otherwise specified. Recombinant START was further purified using a Superdex 75 column. Recombinant Acot9 was purified on a MBPTrap HP column. Upper right panel , SDS-PAGE of recombinant Acot isoform enzymes. Right panel , saturation curves of recombinant Acot isoform enzymes and myristoyl (C14)-CoA reactions (black dots) were used to determine steady state kinetic constants. Where not visible, standard error bars are contained within the symbol sizes. ( H ) Table , steady state kinetic constants (K m , V max , K cat and K cat /K m ) were determined as described in the text. Data represent the mean (s.e.m.) of triplicate determinations.

    Article Snippet: Cultures of E. coli Shuffle T7 competent cells (New England Biolabs, Ipswich, MA) transformed with pET19b-Them1 plasmid were grown to an A 600 of ∼ 0.5 – 0.7 in terrific broth followed by induction of recombinant Them1 by the addition of 0.5 mM isoproyl β-D-thiogalactoside with 16 h of shaking (250 rpm) at 18 °C.

    Techniques: Recombinant, Purification, SDS Page

    Pre-dialysis reactions were performed with recombinant His-tagged human Them1 (Them1) and myristoyl (C14)-CoA. Reactions subjected to an overnight dialysis were incubated with C14-CoA. ( A ) Time course of Pre- and Post- dialysis reactions for the indicated timepoints. ( B ) Pre- and Post-reactions consisting of compounds U1 , W1 or Ebselen [(-) control]. Each graph is representative of four independent experiments. ( C ) Left panels , recombinant His-tagged mouse Them1 was purified from BL21 (DE3) E. coli competent cells on a HisTrap HP column. Upper right panel , SDS-PAGE of recombinant mouse Them1. Right panel , saturation curve of recombinant mouse Them1 (125 nM) and C14-CoA reactions (black dots) was used to determine steady state kinetic constants. ( D ) All reactions were performed with either recombinant mouse Them1 or recombinant human Them1. Acot activities quantified as IC 50 values are shown. Data represent the mean ± s.e.m. of triplicate determinations. Where not visible, standard error bars are contained within the symbol sizes. ***, P<0.001; Pre- ( U1 ) vs. Post- ( U1 ); ###, P<0.001; Pre- ( W1 ) vs. Post- ( W1 ).

    Journal: bioRxiv

    Article Title: High-throughput screening identifies small molecule inhibitors of thioesterase superfamily member 1: Implications for the management of non-alcoholic fatty liver disease

    doi: 10.1101/2022.10.15.512369

    Figure Lengend Snippet: Pre-dialysis reactions were performed with recombinant His-tagged human Them1 (Them1) and myristoyl (C14)-CoA. Reactions subjected to an overnight dialysis were incubated with C14-CoA. ( A ) Time course of Pre- and Post- dialysis reactions for the indicated timepoints. ( B ) Pre- and Post-reactions consisting of compounds U1 , W1 or Ebselen [(-) control]. Each graph is representative of four independent experiments. ( C ) Left panels , recombinant His-tagged mouse Them1 was purified from BL21 (DE3) E. coli competent cells on a HisTrap HP column. Upper right panel , SDS-PAGE of recombinant mouse Them1. Right panel , saturation curve of recombinant mouse Them1 (125 nM) and C14-CoA reactions (black dots) was used to determine steady state kinetic constants. ( D ) All reactions were performed with either recombinant mouse Them1 or recombinant human Them1. Acot activities quantified as IC 50 values are shown. Data represent the mean ± s.e.m. of triplicate determinations. Where not visible, standard error bars are contained within the symbol sizes. ***, P<0.001; Pre- ( U1 ) vs. Post- ( U1 ); ###, P<0.001; Pre- ( W1 ) vs. Post- ( W1 ).

    Article Snippet: Cultures of E. coli Shuffle T7 competent cells (New England Biolabs, Ipswich, MA) transformed with pET19b-Them1 plasmid were grown to an A 600 of ∼ 0.5 – 0.7 in terrific broth followed by induction of recombinant Them1 by the addition of 0.5 mM isoproyl β-D-thiogalactoside with 16 h of shaking (250 rpm) at 18 °C.

    Techniques: Recombinant, Incubation, Purification, SDS Page

    (A) Schematic showing Them1-mediated de-activation of myristoyl (C14)-CoA into myristic acid plus CoA. Free CoA liberated by Them1 activity covalently binds to a non-fluorescent detection reagent, generating a fluorescent product. (B) Recombinant His-tagged human Them1 (Them1) purified from Shuffle T7 competent cells on a HisTrap HP column (Black line). Upper right panel , SDS-PAGE of Them1 (67 kD). (C) Recombinant Them1 and C14-CoA (25 μM) reactions (black dots). (D) Reactions consisting of Them1 and C14-CoA (white dots, 0 μM; black dots, 25 μM) were incubated for the indicated timepoints. (E) Saturation curve of Them1 and C14-CoA reactions (black dots) were used to determined steady state kinetic constants. (F) A pilot small-molecule screen was performed using the library of pharmacologically active compounds (LOPAC, 12.5 μM, 1,056 compounds) with Them1 and C14-CoA as substrate. Compounds were considered as potential inhibitors when Them1 activity was inhibited based on a normalized percent inhibition (NPI) β 30. A representative plate with Z’ factor and 2 compounds fulfilling the inhibitor criteria are shown. Negative values are attributable to the absorbance of a minority of compounds. (G) Reproducibility of the pilot small molecule screen was assessed by NPI values of compounds over 2 independent days. Correlation between d 1 and 2 is indicated by the purple line; R 2 = 0.93. Data represent the mean (s.e.m.) of triplicate determinations. Where not visible, standard error bars are contained within the symbol sizes.

    Journal: bioRxiv

    Article Title: High-throughput screening identifies small molecule inhibitors of thioesterase superfamily member 1: Implications for the management of non-alcoholic fatty liver disease

    doi: 10.1101/2022.10.15.512369

    Figure Lengend Snippet: (A) Schematic showing Them1-mediated de-activation of myristoyl (C14)-CoA into myristic acid plus CoA. Free CoA liberated by Them1 activity covalently binds to a non-fluorescent detection reagent, generating a fluorescent product. (B) Recombinant His-tagged human Them1 (Them1) purified from Shuffle T7 competent cells on a HisTrap HP column (Black line). Upper right panel , SDS-PAGE of Them1 (67 kD). (C) Recombinant Them1 and C14-CoA (25 μM) reactions (black dots). (D) Reactions consisting of Them1 and C14-CoA (white dots, 0 μM; black dots, 25 μM) were incubated for the indicated timepoints. (E) Saturation curve of Them1 and C14-CoA reactions (black dots) were used to determined steady state kinetic constants. (F) A pilot small-molecule screen was performed using the library of pharmacologically active compounds (LOPAC, 12.5 μM, 1,056 compounds) with Them1 and C14-CoA as substrate. Compounds were considered as potential inhibitors when Them1 activity was inhibited based on a normalized percent inhibition (NPI) β 30. A representative plate with Z’ factor and 2 compounds fulfilling the inhibitor criteria are shown. Negative values are attributable to the absorbance of a minority of compounds. (G) Reproducibility of the pilot small molecule screen was assessed by NPI values of compounds over 2 independent days. Correlation between d 1 and 2 is indicated by the purple line; R 2 = 0.93. Data represent the mean (s.e.m.) of triplicate determinations. Where not visible, standard error bars are contained within the symbol sizes.

    Article Snippet: These plasmids were transformed into E. coli strains BL21 (DE3) (New England Biolabs; Ipswich, MA) or Shuffle T7 competent cells (New England Biolabs, Ipswich, MA), grown to an A 600 of 0.5 – 0.7 in terrific broth and then induced by the addition of 0.5 mM isoproyl β-D-thiogalactoside with 16 h of shaking (250 rpm) at 18 °C.

    Techniques: Activation Assay, Activity Assay, Recombinant, Purification, SDS Page, Incubation, Inhibition

    (A-G) Acot isoform panels : Left panels , recombinant Acot isoform enzymes including type I [Acot1, Acot2] and type II [Acot9, Acot12 and Acot13], as well as a truncated Them1 either containing only the 2 thioesterase domains but lacking the START domain (Them1-ΔSTART) or the START domain but lacking the 2 thioesterase domains (START) were purified from Shuffle T7 or BL21 (DE3) E. coli competent cells on a HisTrap HP column unless otherwise specified. Recombinant START was further purified using a Superdex 75 column. Recombinant Acot9 was purified on a MBPTrap HP column. Upper right panel , SDS-PAGE of recombinant Acot isoform enzymes. Right panel , saturation curves of recombinant Acot isoform enzymes and myristoyl (C14)-CoA reactions (black dots) were used to determine steady state kinetic constants. Where not visible, standard error bars are contained within the symbol sizes. ( H ) Table , steady state kinetic constants (K m , V max , K cat and K cat /K m ) were determined as described in the text. Data represent the mean (s.e.m.) of triplicate determinations.

    Journal: bioRxiv

    Article Title: High-throughput screening identifies small molecule inhibitors of thioesterase superfamily member 1: Implications for the management of non-alcoholic fatty liver disease

    doi: 10.1101/2022.10.15.512369

    Figure Lengend Snippet: (A-G) Acot isoform panels : Left panels , recombinant Acot isoform enzymes including type I [Acot1, Acot2] and type II [Acot9, Acot12 and Acot13], as well as a truncated Them1 either containing only the 2 thioesterase domains but lacking the START domain (Them1-ΔSTART) or the START domain but lacking the 2 thioesterase domains (START) were purified from Shuffle T7 or BL21 (DE3) E. coli competent cells on a HisTrap HP column unless otherwise specified. Recombinant START was further purified using a Superdex 75 column. Recombinant Acot9 was purified on a MBPTrap HP column. Upper right panel , SDS-PAGE of recombinant Acot isoform enzymes. Right panel , saturation curves of recombinant Acot isoform enzymes and myristoyl (C14)-CoA reactions (black dots) were used to determine steady state kinetic constants. Where not visible, standard error bars are contained within the symbol sizes. ( H ) Table , steady state kinetic constants (K m , V max , K cat and K cat /K m ) were determined as described in the text. Data represent the mean (s.e.m.) of triplicate determinations.

    Article Snippet: These plasmids were transformed into E. coli strains BL21 (DE3) (New England Biolabs; Ipswich, MA) or Shuffle T7 competent cells (New England Biolabs, Ipswich, MA), grown to an A 600 of 0.5 – 0.7 in terrific broth and then induced by the addition of 0.5 mM isoproyl β-D-thiogalactoside with 16 h of shaking (250 rpm) at 18 °C.

    Techniques: Recombinant, Purification, SDS Page