purexpress in vitro transcription translation system  (New England Biolabs)


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    New England Biolabs purexpress in vitro transcription translation system
    Determination of k R for each construct. (a) Scaled overlay of the individual plots in panels b-h. (b)-(h) Plot of the decrease in fraction arrested protein ( f A ) over time after chasing a 5 min <t>PURExpress</t> translation of the indicated construct. The data were fit to the first order equation in the main text to determine the rate of release ( k R ) of arrested protein from the ribosome. (i) Summary of the fitness of the equation for each construct: degrees of freedom ( d.f. ), R 2 , and goodness-of-fit calculation ( Sy.x ). (j) The goodness-of-fit calculation provided by Prism 8 (Graphpad software) where n is the number of data points and K is the degrees of freedom.
    Purexpress In Vitro Transcription Translation System, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/purexpress in vitro transcription translation system/product/New England Biolabs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    purexpress in vitro transcription translation system - by Bioz Stars, 2022-07
    86/100 stars

    Images

    1) Product Images from "Cotranslational folding cooperativity of contiguous domains of α-spectrin"

    Article Title: Cotranslational folding cooperativity of contiguous domains of α-spectrin

    Journal: bioRxiv

    doi: 10.1101/653360

    Determination of k R for each construct. (a) Scaled overlay of the individual plots in panels b-h. (b)-(h) Plot of the decrease in fraction arrested protein ( f A ) over time after chasing a 5 min PURExpress translation of the indicated construct. The data were fit to the first order equation in the main text to determine the rate of release ( k R ) of arrested protein from the ribosome. (i) Summary of the fitness of the equation for each construct: degrees of freedom ( d.f. ), R 2 , and goodness-of-fit calculation ( Sy.x ). (j) The goodness-of-fit calculation provided by Prism 8 (Graphpad software) where n is the number of data points and K is the degrees of freedom.
    Figure Legend Snippet: Determination of k R for each construct. (a) Scaled overlay of the individual plots in panels b-h. (b)-(h) Plot of the decrease in fraction arrested protein ( f A ) over time after chasing a 5 min PURExpress translation of the indicated construct. The data were fit to the first order equation in the main text to determine the rate of release ( k R ) of arrested protein from the ribosome. (i) Summary of the fitness of the equation for each construct: degrees of freedom ( d.f. ), R 2 , and goodness-of-fit calculation ( Sy.x ). (j) The goodness-of-fit calculation provided by Prism 8 (Graphpad software) where n is the number of data points and K is the degrees of freedom.

    Techniques Used: Construct, Software

    Release rates and estimation of pulling forces. (a) The rate of release ( k R ) obtained from pulse-chase experiments (see Supplementary Fig. S4 and Supplementary Table S2), the fraction full-length protein ( f FL ) measured under standard experimental conditions (20 min. incubation in PURExpress in the continuous presence of [ 35 S] Met), and the pulling force F calculated using Eq. [1] ( k 0 = 3.0 × 10 −4 s −1 , Δ x ‡ = 0.65 nm). The constructs are from Kudva et al. ( 12 ), and are colored to match those in panel b and Supplementary Figs. S4 and S5. (b) F values calculated from Eq. [1] plotted against the standard f FL values, with constructs colored as in panel a . The least-squares fit line is indicated by the blue line, and the analytic relation Eq. [3] between F and f FL , assuming an average delay time Δ t = 550 s (approximately equal to half the standard incubation time), is shown as a red curve.
    Figure Legend Snippet: Release rates and estimation of pulling forces. (a) The rate of release ( k R ) obtained from pulse-chase experiments (see Supplementary Fig. S4 and Supplementary Table S2), the fraction full-length protein ( f FL ) measured under standard experimental conditions (20 min. incubation in PURExpress in the continuous presence of [ 35 S] Met), and the pulling force F calculated using Eq. [1] ( k 0 = 3.0 × 10 −4 s −1 , Δ x ‡ = 0.65 nm). The constructs are from Kudva et al. ( 12 ), and are colored to match those in panel b and Supplementary Figs. S4 and S5. (b) F values calculated from Eq. [1] plotted against the standard f FL values, with constructs colored as in panel a . The least-squares fit line is indicated by the blue line, and the analytic relation Eq. [3] between F and f FL , assuming an average delay time Δ t = 550 s (approximately equal to half the standard incubation time), is shown as a red curve.

    Techniques Used: Pulse Chase, Incubation, Construct

    2) Product Images from "Plasmid replication-associated single-strand-specific methyltransferases"

    Article Title: Plasmid replication-associated single-strand-specific methyltransferases

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkaa1163

    Polymerase and MTase activities copurify when domains are fused. Panel ( A ): Size and purity of fusion proteins. For each MTase, both of the immunoreactive components of the MTase-PolI fusion proteins run at the same position, and comigrate with the Coomassie-stained purified proteins. Western blot (lanes 1, 5, 9 and 10) detected 1 μg of MTase-PolI fusion proteins; Coomassie (lanes 2, 3, 6, 7) visualized 1 μg or 20 μg of the same fractions. Western blots were probed separately with anti-Pol1 rabbit polyclonal or anti-6xHis (detecting the MTase) monoclonal antibodies and developed with horseradish peroxidase-labeled antirabbit or antimouse following kit instructions as detailed in Material and Methods. Dots on lane 1 correspond to the position of protein markers after Western blotting. The bands at the side of lane 10 are spillover from the adjacent lane, which were control 6xHis tagged proteins from a PurExpress extract. Panel ( B ): Activity copurification through two columns. Pooled HiTrapHepHP (#22–26) and HiTrapQHP (#15–19) protein fractions were tested for MTase activity on single-stranded M13mp18 DNA in the presence of [H 3 ]SAM and for DNA-polymerase activity on sonicated sperm-whale DNA in the presence of [H 3 ]TTP.
    Figure Legend Snippet: Polymerase and MTase activities copurify when domains are fused. Panel ( A ): Size and purity of fusion proteins. For each MTase, both of the immunoreactive components of the MTase-PolI fusion proteins run at the same position, and comigrate with the Coomassie-stained purified proteins. Western blot (lanes 1, 5, 9 and 10) detected 1 μg of MTase-PolI fusion proteins; Coomassie (lanes 2, 3, 6, 7) visualized 1 μg or 20 μg of the same fractions. Western blots were probed separately with anti-Pol1 rabbit polyclonal or anti-6xHis (detecting the MTase) monoclonal antibodies and developed with horseradish peroxidase-labeled antirabbit or antimouse following kit instructions as detailed in Material and Methods. Dots on lane 1 correspond to the position of protein markers after Western blotting. The bands at the side of lane 10 are spillover from the adjacent lane, which were control 6xHis tagged proteins from a PurExpress extract. Panel ( B ): Activity copurification through two columns. Pooled HiTrapHepHP (#22–26) and HiTrapQHP (#15–19) protein fractions were tested for MTase activity on single-stranded M13mp18 DNA in the presence of [H 3 ]SAM and for DNA-polymerase activity on sonicated sperm-whale DNA in the presence of [H 3 ]TTP.

    Techniques Used: Staining, Purification, Western Blot, Labeling, Activity Assay, Copurification, Sonication

    MTase activity requires single strands. Panels ( A ) and ( C ): M13 substrates stained with ethidium bromide. Panels ( B ) and ( D ): fluorograms of modification reactions using [H 3 ]SAM. M13 SS: virion DNA substrate. M13 RF cut: DS replication intermediate RFI was digested following the labelling reaction for visual simplification; NdeI (Panels A and B) or NdeI+BamHI (Panels C and D). The substrates were treated with MTase proteins obtained with PURExpress in vitro transcription-translation (Panels A and B) or were partially-purified (Ni-NTA purification) proteins synthesized in vivo (Panels C and D). Lanes 1) empty pSAPv6 vector, 2) M.BceJIII WT (pAF9), 3) M.EcoGIX WT (pAF10) and 4) M.EcoGIX APPA variant (pAF11). H 3 radiolabeled markers (M) are HindIII digested lambda DNA modified at A by M.EcoGII.
    Figure Legend Snippet: MTase activity requires single strands. Panels ( A ) and ( C ): M13 substrates stained with ethidium bromide. Panels ( B ) and ( D ): fluorograms of modification reactions using [H 3 ]SAM. M13 SS: virion DNA substrate. M13 RF cut: DS replication intermediate RFI was digested following the labelling reaction for visual simplification; NdeI (Panels A and B) or NdeI+BamHI (Panels C and D). The substrates were treated with MTase proteins obtained with PURExpress in vitro transcription-translation (Panels A and B) or were partially-purified (Ni-NTA purification) proteins synthesized in vivo (Panels C and D). Lanes 1) empty pSAPv6 vector, 2) M.BceJIII WT (pAF9), 3) M.EcoGIX WT (pAF10) and 4) M.EcoGIX APPA variant (pAF11). H 3 radiolabeled markers (M) are HindIII digested lambda DNA modified at A by M.EcoGII.

    Techniques Used: Activity Assay, Staining, Modification, In Vitro, Purification, Synthesized, In Vivo, Plasmid Preparation, Variant Assay, Lambda DNA Preparation

    3) Product Images from "Cotranslational folding cooperativity of contiguous domains of α-spectrin"

    Article Title: Cotranslational folding cooperativity of contiguous domains of α-spectrin

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1909683117

    Release rates and estimation of pulling forces. ( A ) The rate of release ( k R ), the fraction full-length protein ( f FL ) measured under standard experimental conditions (20-min incubation in PURExpress in the continuous presence of [ 35 S]Met), and the pulling force F ( k 0 = 3.0 × 10 − 4 s − 1 , Δ x ‡ = 0.65 nm ) and are colored to match those in B . ( B ) F plotted against the standard f FL values, with constructs colored as in A between F and f FL , assuming an average delay time Δ t = 550 s (approximately equal to half the standard incubation time), is shown as a red curve.
    Figure Legend Snippet: Release rates and estimation of pulling forces. ( A ) The rate of release ( k R ), the fraction full-length protein ( f FL ) measured under standard experimental conditions (20-min incubation in PURExpress in the continuous presence of [ 35 S]Met), and the pulling force F ( k 0 = 3.0 × 10 − 4 s − 1 , Δ x ‡ = 0.65 nm ) and are colored to match those in B . ( B ) F plotted against the standard f FL values, with constructs colored as in A between F and f FL , assuming an average delay time Δ t = 550 s (approximately equal to half the standard incubation time), is shown as a red curve.

    Techniques Used: Incubation, Construct

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    New England Biolabs purexpress in vitro transcription translation system
    Determination of k R for each construct. (a) Scaled overlay of the individual plots in panels b-h. (b)-(h) Plot of the decrease in fraction arrested protein ( f A ) over time after chasing a 5 min <t>PURExpress</t> translation of the indicated construct. The data were fit to the first order equation in the main text to determine the rate of release ( k R ) of arrested protein from the ribosome. (i) Summary of the fitness of the equation for each construct: degrees of freedom ( d.f. ), R 2 , and goodness-of-fit calculation ( Sy.x ). (j) The goodness-of-fit calculation provided by Prism 8 (Graphpad software) where n is the number of data points and K is the degrees of freedom.
    Purexpress In Vitro Transcription Translation System, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/purexpress in vitro transcription translation system/product/New England Biolabs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    purexpress in vitro transcription translation system - by Bioz Stars, 2022-07
    86/100 stars
      Buy from Supplier

    86
    New England Biolabs in vitro transcription translation system
    Reduced tyrosine autophosphorylation of <t>in</t> <t>vitro</t> -translated DYRK1A-L295F. DYRK1A constructs (28-499) were expressed in a cell-free E. coli- derived expression <t>system.</t> Coupled in vitro <t>transcription-translation</t> reactions were incubated for 90 min at 37°C before reaction products were subjected to immunoblot analysis. For quantification, pTyr signals were normalized to the total amounts of recombinant DYRK1A as determined by detection of the N-terminal StrepTag (means and s.d., n =3). The difference in relative tyrosine autophosphorylation was tested for statistical significance by one-sample t -test (* P
    In Vitro Transcription Translation System, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/in vitro transcription translation system/product/New England Biolabs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    in vitro transcription translation system - by Bioz Stars, 2022-07
    86/100 stars
      Buy from Supplier

    97
    New England Biolabs in vitro transcription translation reaction
    Tre23 inhibits protein synthesis. ( A ) Phase contrast and fluorescence microscopy recordings of E. coli cells constitutively producing short-lived green fluorescent protein (GFP-LVA) and the Tre23 toxin from the low-copy number pNDM220 vector, before and 100 min post toxin induction. Cells bearing the empty pNDM220 vector (Ø) were used as control. ( B ) Replication, <t>transcription</t> and <t>translation</t> rates before (time 0) and 60 min after the induction of Tre23 toxin from pNDM220 vector (red line) as compared to control carrying empty vector (black line) were measured by the incorporation <t>(in</t> count per minute, cpm) of [ 3 H] thymidine, [ 3 H] uridine and [ 35 S] methionine, respectively. ( C ) Coupled in <t>vitro</t> transcription-translation <t>reaction.</t> In vitro transcription/translation of the Strep-tagged GFP reporter protein was estimated by immunodetection. Reactions were supplemented with NAD + , Tre23 and Tri23, as indicated. In the last reaction, Tri23 was added 1 h after incubating the reaction with Tre23 (aft) and reaction proceeded for 2 h. ( D ) Tre23-Tri23 co-purification. Total cell lysates of E. coli BL21(DE3) cells producing FLAG-tagged Tri23 (Tri23 FL ) alone or co-producing Strep-tagged Tre23 (Tre23 ST ) (T) were subjected to purification on streptactin agarose beads followed by immunoprecipitation on anti-FLAG affinity gel (IP). Proteins were separated by SDS-PAGE and stained by Coomassie (upper panel) or immunodetected using anti-Strep and anti-FLAG antibodies (middle and bottom panels, respectively).
    In Vitro Transcription Translation Reaction, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/in vitro transcription translation reaction/product/New England Biolabs
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    Determination of k R for each construct. (a) Scaled overlay of the individual plots in panels b-h. (b)-(h) Plot of the decrease in fraction arrested protein ( f A ) over time after chasing a 5 min PURExpress translation of the indicated construct. The data were fit to the first order equation in the main text to determine the rate of release ( k R ) of arrested protein from the ribosome. (i) Summary of the fitness of the equation for each construct: degrees of freedom ( d.f. ), R 2 , and goodness-of-fit calculation ( Sy.x ). (j) The goodness-of-fit calculation provided by Prism 8 (Graphpad software) where n is the number of data points and K is the degrees of freedom.

    Journal: bioRxiv

    Article Title: Cotranslational folding cooperativity of contiguous domains of α-spectrin

    doi: 10.1101/653360

    Figure Lengend Snippet: Determination of k R for each construct. (a) Scaled overlay of the individual plots in panels b-h. (b)-(h) Plot of the decrease in fraction arrested protein ( f A ) over time after chasing a 5 min PURExpress translation of the indicated construct. The data were fit to the first order equation in the main text to determine the rate of release ( k R ) of arrested protein from the ribosome. (i) Summary of the fitness of the equation for each construct: degrees of freedom ( d.f. ), R 2 , and goodness-of-fit calculation ( Sy.x ). (j) The goodness-of-fit calculation provided by Prism 8 (Graphpad software) where n is the number of data points and K is the degrees of freedom.

    Article Snippet: The PURExpress in vitro transcription-translation system was purchased from NEB.

    Techniques: Construct, Software

    Release rates and estimation of pulling forces. (a) The rate of release ( k R ) obtained from pulse-chase experiments (see Supplementary Fig. S4 and Supplementary Table S2), the fraction full-length protein ( f FL ) measured under standard experimental conditions (20 min. incubation in PURExpress in the continuous presence of [ 35 S] Met), and the pulling force F calculated using Eq. [1] ( k 0 = 3.0 × 10 −4 s −1 , Δ x ‡ = 0.65 nm). The constructs are from Kudva et al. ( 12 ), and are colored to match those in panel b and Supplementary Figs. S4 and S5. (b) F values calculated from Eq. [1] plotted against the standard f FL values, with constructs colored as in panel a . The least-squares fit line is indicated by the blue line, and the analytic relation Eq. [3] between F and f FL , assuming an average delay time Δ t = 550 s (approximately equal to half the standard incubation time), is shown as a red curve.

    Journal: bioRxiv

    Article Title: Cotranslational folding cooperativity of contiguous domains of α-spectrin

    doi: 10.1101/653360

    Figure Lengend Snippet: Release rates and estimation of pulling forces. (a) The rate of release ( k R ) obtained from pulse-chase experiments (see Supplementary Fig. S4 and Supplementary Table S2), the fraction full-length protein ( f FL ) measured under standard experimental conditions (20 min. incubation in PURExpress in the continuous presence of [ 35 S] Met), and the pulling force F calculated using Eq. [1] ( k 0 = 3.0 × 10 −4 s −1 , Δ x ‡ = 0.65 nm). The constructs are from Kudva et al. ( 12 ), and are colored to match those in panel b and Supplementary Figs. S4 and S5. (b) F values calculated from Eq. [1] plotted against the standard f FL values, with constructs colored as in panel a . The least-squares fit line is indicated by the blue line, and the analytic relation Eq. [3] between F and f FL , assuming an average delay time Δ t = 550 s (approximately equal to half the standard incubation time), is shown as a red curve.

    Article Snippet: The PURExpress in vitro transcription-translation system was purchased from NEB.

    Techniques: Pulse Chase, Incubation, Construct

    Reduced tyrosine autophosphorylation of in vitro -translated DYRK1A-L295F. DYRK1A constructs (28-499) were expressed in a cell-free E. coli- derived expression system. Coupled in vitro transcription-translation reactions were incubated for 90 min at 37°C before reaction products were subjected to immunoblot analysis. For quantification, pTyr signals were normalized to the total amounts of recombinant DYRK1A as determined by detection of the N-terminal StrepTag (means and s.d., n =3). The difference in relative tyrosine autophosphorylation was tested for statistical significance by one-sample t -test (* P

    Journal: Biology Open

    Article Title: Functional characterization of DYRK1A missense variants associated with a syndromic form of intellectual deficiency and autism

    doi: 10.1242/bio.032862

    Figure Lengend Snippet: Reduced tyrosine autophosphorylation of in vitro -translated DYRK1A-L295F. DYRK1A constructs (28-499) were expressed in a cell-free E. coli- derived expression system. Coupled in vitro transcription-translation reactions were incubated for 90 min at 37°C before reaction products were subjected to immunoblot analysis. For quantification, pTyr signals were normalized to the total amounts of recombinant DYRK1A as determined by detection of the N-terminal StrepTag (means and s.d., n =3). The difference in relative tyrosine autophosphorylation was tested for statistical significance by one-sample t -test (* P

    Article Snippet: A reconstituted E. coli -based in vitro transcription-translation system (PURExpress, New England Biolabs, Beverley, MA, USA) was used to express a DYRK1A(28-499) construct fused to an N-terminal Strep-tag 2 sequence.

    Techniques: In Vitro, Construct, Derivative Assay, Expressing, Incubation, Recombinant

    Reduced tyrosine autophosphorylation of in vitro -translated DYRK1A-L295F. DYRK1A constructs (28-499) were expressed in a cell-free E. coli- derived expression system. Coupled in vitro transcription-translation reactions were incubated for 90 min at 37°C before reaction products were subjected to immunoblot analysis. For quantification, pTyr signals were normalized to the total amounts of recombinant DYRK1A as determined by detection of the N-terminal StrepTag (means and s.d., n =3). The difference in relative tyrosine autophosphorylation was tested for statistical significance by one-sample t -test (* P

    Journal: Biology Open

    Article Title: Functional characterization of DYRK1A missense variants associated with a syndromic form of intellectual deficiency and autism

    doi: 10.1242/bio.032862

    Figure Lengend Snippet: Reduced tyrosine autophosphorylation of in vitro -translated DYRK1A-L295F. DYRK1A constructs (28-499) were expressed in a cell-free E. coli- derived expression system. Coupled in vitro transcription-translation reactions were incubated for 90 min at 37°C before reaction products were subjected to immunoblot analysis. For quantification, pTyr signals were normalized to the total amounts of recombinant DYRK1A as determined by detection of the N-terminal StrepTag (means and s.d., n =3). The difference in relative tyrosine autophosphorylation was tested for statistical significance by one-sample t -test (* P

    Article Snippet: In vitro translation A reconstituted E. coli -based in vitro transcription-translation system (PURExpress, New England Biolabs, Beverley, MA, USA) was used to express a DYRK1A(28-499) construct fused to an N-terminal Strep-tag 2 sequence.

    Techniques: In Vitro, Construct, Derivative Assay, Expressing, Incubation, Recombinant

    Tre23 inhibits protein synthesis. ( A ) Phase contrast and fluorescence microscopy recordings of E. coli cells constitutively producing short-lived green fluorescent protein (GFP-LVA) and the Tre23 toxin from the low-copy number pNDM220 vector, before and 100 min post toxin induction. Cells bearing the empty pNDM220 vector (Ø) were used as control. ( B ) Replication, transcription and translation rates before (time 0) and 60 min after the induction of Tre23 toxin from pNDM220 vector (red line) as compared to control carrying empty vector (black line) were measured by the incorporation (in count per minute, cpm) of [ 3 H] thymidine, [ 3 H] uridine and [ 35 S] methionine, respectively. ( C ) Coupled in vitro transcription-translation reaction. In vitro transcription/translation of the Strep-tagged GFP reporter protein was estimated by immunodetection. Reactions were supplemented with NAD + , Tre23 and Tri23, as indicated. In the last reaction, Tri23 was added 1 h after incubating the reaction with Tre23 (aft) and reaction proceeded for 2 h. ( D ) Tre23-Tri23 co-purification. Total cell lysates of E. coli BL21(DE3) cells producing FLAG-tagged Tri23 (Tri23 FL ) alone or co-producing Strep-tagged Tre23 (Tre23 ST ) (T) were subjected to purification on streptactin agarose beads followed by immunoprecipitation on anti-FLAG affinity gel (IP). Proteins were separated by SDS-PAGE and stained by Coomassie (upper panel) or immunodetected using anti-Strep and anti-FLAG antibodies (middle and bottom panels, respectively).

    Journal: Nucleic Acids Research

    Article Title: Photorhabdus antibacterial Rhs polymorphic toxin inhibits translation through ADP-ribosylation of 23S ribosomal RNA

    doi: 10.1093/nar/gkab608

    Figure Lengend Snippet: Tre23 inhibits protein synthesis. ( A ) Phase contrast and fluorescence microscopy recordings of E. coli cells constitutively producing short-lived green fluorescent protein (GFP-LVA) and the Tre23 toxin from the low-copy number pNDM220 vector, before and 100 min post toxin induction. Cells bearing the empty pNDM220 vector (Ø) were used as control. ( B ) Replication, transcription and translation rates before (time 0) and 60 min after the induction of Tre23 toxin from pNDM220 vector (red line) as compared to control carrying empty vector (black line) were measured by the incorporation (in count per minute, cpm) of [ 3 H] thymidine, [ 3 H] uridine and [ 35 S] methionine, respectively. ( C ) Coupled in vitro transcription-translation reaction. In vitro transcription/translation of the Strep-tagged GFP reporter protein was estimated by immunodetection. Reactions were supplemented with NAD + , Tre23 and Tri23, as indicated. In the last reaction, Tri23 was added 1 h after incubating the reaction with Tre23 (aft) and reaction proceeded for 2 h. ( D ) Tre23-Tri23 co-purification. Total cell lysates of E. coli BL21(DE3) cells producing FLAG-tagged Tri23 (Tri23 FL ) alone or co-producing Strep-tagged Tre23 (Tre23 ST ) (T) were subjected to purification on streptactin agarose beads followed by immunoprecipitation on anti-FLAG affinity gel (IP). Proteins were separated by SDS-PAGE and stained by Coomassie (upper panel) or immunodetected using anti-Strep and anti-FLAG antibodies (middle and bottom panels, respectively).

    Article Snippet: C-terminally Strep-tagged Tre23 (Tre23ST ) was synthesized in a coupled in vitro transcription-translation reaction (PURExpress Kit, NEB).

    Techniques: Fluorescence, Microscopy, Low Copy Number, Plasmid Preparation, In Vitro, Immunodetection, Copurification, Purification, Immunoprecipitation, SDS Page, Staining

    Tre23 ADP-ribosylates the 23S RNA of actively translating ribosomes. ( A and B ) Native PAGE (A) and SDS-PAGE (B) analyses of in vitro couple translation-transcription reaction supplemented with 6-biotin-17-NAD + ( biot NAD + ) and with Tre23, as indicated. Samples were stained with Coomassie blue (left panels) or detected using streptavidin-AP (right panels). Mass spectrometry analysis of the indicated band identified subunits of 30S and 50S ribosome (Ribos.). ( C ) RNA extracted from of in vitro transcription-translation reactions supplemented with 6-biotin-17-NAD + ( biot NAD + ), Tre23 and Tri23, as indicated were stained with ethidium bromide (EtBr, left panel) or detected using streptavidin-AP conjugate (right panel). The positions of tRNAs, 16S and 23S ribosomal RNAs are indicated. ( D ) Western-blot analyses with anti-Strep antibodies of GFP-strepII in vitro transcription-translation in presence of ribosomes purified from wild-type E. coli cells carrying empty pNDM220 vector (Ø), or cells producing Tre23. Ribosomes (R) included in the coupled transcription-translation kit were used as control. Ribosome profile changes over time after toxin induction are shown in Supplementary Figure S2 . ( E ) RNA analyses of in vitro transcription-translation reactions supplemented with 6-biotin-17-NAD + ( biot NAD + ), and Tre23, as indicated, using ribosomes purified from wild-type E. coli cells bearing empty vector (Ø), or cells producing Tre23. RNAs were stained with EtBr (left panel) or detected using streptavidin-AP (right panel). ( F ) EtBr staining (left panel) and Streptavidin-AP (right panel) RNA analyses of purified ribosomal RNAs (rRNA), or purified ribosomes alone or supplemented with translation factors (F) or translation reagents (tRNAs, amino acids, nucleotides; A), in presence of 6-biotin-17-NAD + ( biot NAD + ) and Tre23, as indicated.

    Journal: Nucleic Acids Research

    Article Title: Photorhabdus antibacterial Rhs polymorphic toxin inhibits translation through ADP-ribosylation of 23S ribosomal RNA

    doi: 10.1093/nar/gkab608

    Figure Lengend Snippet: Tre23 ADP-ribosylates the 23S RNA of actively translating ribosomes. ( A and B ) Native PAGE (A) and SDS-PAGE (B) analyses of in vitro couple translation-transcription reaction supplemented with 6-biotin-17-NAD + ( biot NAD + ) and with Tre23, as indicated. Samples were stained with Coomassie blue (left panels) or detected using streptavidin-AP (right panels). Mass spectrometry analysis of the indicated band identified subunits of 30S and 50S ribosome (Ribos.). ( C ) RNA extracted from of in vitro transcription-translation reactions supplemented with 6-biotin-17-NAD + ( biot NAD + ), Tre23 and Tri23, as indicated were stained with ethidium bromide (EtBr, left panel) or detected using streptavidin-AP conjugate (right panel). The positions of tRNAs, 16S and 23S ribosomal RNAs are indicated. ( D ) Western-blot analyses with anti-Strep antibodies of GFP-strepII in vitro transcription-translation in presence of ribosomes purified from wild-type E. coli cells carrying empty pNDM220 vector (Ø), or cells producing Tre23. Ribosomes (R) included in the coupled transcription-translation kit were used as control. Ribosome profile changes over time after toxin induction are shown in Supplementary Figure S2 . ( E ) RNA analyses of in vitro transcription-translation reactions supplemented with 6-biotin-17-NAD + ( biot NAD + ), and Tre23, as indicated, using ribosomes purified from wild-type E. coli cells bearing empty vector (Ø), or cells producing Tre23. RNAs were stained with EtBr (left panel) or detected using streptavidin-AP (right panel). ( F ) EtBr staining (left panel) and Streptavidin-AP (right panel) RNA analyses of purified ribosomal RNAs (rRNA), or purified ribosomes alone or supplemented with translation factors (F) or translation reagents (tRNAs, amino acids, nucleotides; A), in presence of 6-biotin-17-NAD + ( biot NAD + ) and Tre23, as indicated.

    Article Snippet: C-terminally Strep-tagged Tre23 (Tre23ST ) was synthesized in a coupled in vitro transcription-translation reaction (PURExpress Kit, NEB).

    Techniques: Clear Native PAGE, SDS Page, In Vitro, Staining, Mass Spectrometry, Western Blot, Purification, Plasmid Preparation