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cell free transcription translation protein synthesis reactions  (New England Biolabs)


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    New England Biolabs cell free transcription translation protein synthesis reactions
    T65 is important, but not essential, for NTase and phosphorylation activity. ( A ) In <t>vitro</t> <t>transcription/translation</t> reactions assessing levels of DHFR control protein produced in the presence of either 5 µM AbiEii WT or AbiEii-p. ( B ) In vitro transcription/translation reactions assessing levels of DHFR control protein produced in the absence and presence of 5 µM AbiEii T65A, AbiEii D192A, or AbiEii-p. Reactions were loaded onto 4-20% gradient gels ran at 180 V for 1 h. ( C ) Densitometric analysis of SDS-PAGE bands from ( B ) corresponding to DHFR. ( D ) Dose-response curves of increasing concentrations of either AbiEii WT or AbiEii T65A plotted against DHFR expression, normalized against DHFR expressed in the absence of toxin (100% expression). Data points were generated following densitometric analysis of bands corresponding to DHFR from Supplementary Figs. S4A and B . ( E ) Logarithmic dose-response curves of either 5 µM AbiEii WT or AbiEii T65A incubated with increasing concentrations of AbiEi plotted against DHFR expression, normalized against DHFR expressed in the absence of toxin (100% expression). Data points were generated following densitometric analysis of bands corresponding to DHFR from Supplementary Figs. S4C and D . Logarithmic curves were generated using non-linear regression in Prism (GraphPad). ( F ) Endpoint viable count antitoxicity assays of E. coli DH5α transformed with either pBAD30 empty vector, pTRB482 (AbiEii WT), or pTRB736 (AbiEii T65A), and either pTA100 empty vector or pTRB481 (AbiEi). Overnight cultures were re-seeded into fresh LB supplemented with Ap, Sp and D-glu and grown to mid-log phase. Samples were serially diluted 10 −2 -10 −9 and spotted onto M9A plates containing Ap and Sp, with or without D-glu, L-ara, and IPTG for repression of toxin expression, induction of toxin expression, and induction of antitoxin expression, respectively. Plates were incubated at 37 °C for 48 h, after which they were imaged and colonies counted to determine CFU/ml (one-way ANOVA, P < 0.05). “T” = toxin induced, “TA” = antitoxin + toxin induced. Assays shown are representative of three independent biological replicates and plotted data represent the mean ± SEM.
    Cell Free Transcription Translation Protein Synthesis Reactions, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 820 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cell free transcription translation protein synthesis reactions/product/New England Biolabs
    Average 97 stars, based on 820 article reviews
    cell free transcription translation protein synthesis reactions - by Bioz Stars, 2026-06
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    1) Product Images from "Antitoxin-induced auto-phosphorylation neutralizes the nucleotidyltransferase toxin AbiEii from Streptococcus agalactiae to safeguard global translation"

    Article Title: Antitoxin-induced auto-phosphorylation neutralizes the nucleotidyltransferase toxin AbiEii from Streptococcus agalactiae to safeguard global translation

    Journal: bioRxiv

    doi: 10.64898/2026.01.19.700257

    T65 is important, but not essential, for NTase and phosphorylation activity. ( A ) In vitro transcription/translation reactions assessing levels of DHFR control protein produced in the presence of either 5 µM AbiEii WT or AbiEii-p. ( B ) In vitro transcription/translation reactions assessing levels of DHFR control protein produced in the absence and presence of 5 µM AbiEii T65A, AbiEii D192A, or AbiEii-p. Reactions were loaded onto 4-20% gradient gels ran at 180 V for 1 h. ( C ) Densitometric analysis of SDS-PAGE bands from ( B ) corresponding to DHFR. ( D ) Dose-response curves of increasing concentrations of either AbiEii WT or AbiEii T65A plotted against DHFR expression, normalized against DHFR expressed in the absence of toxin (100% expression). Data points were generated following densitometric analysis of bands corresponding to DHFR from Supplementary Figs. S4A and B . ( E ) Logarithmic dose-response curves of either 5 µM AbiEii WT or AbiEii T65A incubated with increasing concentrations of AbiEi plotted against DHFR expression, normalized against DHFR expressed in the absence of toxin (100% expression). Data points were generated following densitometric analysis of bands corresponding to DHFR from Supplementary Figs. S4C and D . Logarithmic curves were generated using non-linear regression in Prism (GraphPad). ( F ) Endpoint viable count antitoxicity assays of E. coli DH5α transformed with either pBAD30 empty vector, pTRB482 (AbiEii WT), or pTRB736 (AbiEii T65A), and either pTA100 empty vector or pTRB481 (AbiEi). Overnight cultures were re-seeded into fresh LB supplemented with Ap, Sp and D-glu and grown to mid-log phase. Samples were serially diluted 10 −2 -10 −9 and spotted onto M9A plates containing Ap and Sp, with or without D-glu, L-ara, and IPTG for repression of toxin expression, induction of toxin expression, and induction of antitoxin expression, respectively. Plates were incubated at 37 °C for 48 h, after which they were imaged and colonies counted to determine CFU/ml (one-way ANOVA, P < 0.05). “T” = toxin induced, “TA” = antitoxin + toxin induced. Assays shown are representative of three independent biological replicates and plotted data represent the mean ± SEM.
    Figure Legend Snippet: T65 is important, but not essential, for NTase and phosphorylation activity. ( A ) In vitro transcription/translation reactions assessing levels of DHFR control protein produced in the presence of either 5 µM AbiEii WT or AbiEii-p. ( B ) In vitro transcription/translation reactions assessing levels of DHFR control protein produced in the absence and presence of 5 µM AbiEii T65A, AbiEii D192A, or AbiEii-p. Reactions were loaded onto 4-20% gradient gels ran at 180 V for 1 h. ( C ) Densitometric analysis of SDS-PAGE bands from ( B ) corresponding to DHFR. ( D ) Dose-response curves of increasing concentrations of either AbiEii WT or AbiEii T65A plotted against DHFR expression, normalized against DHFR expressed in the absence of toxin (100% expression). Data points were generated following densitometric analysis of bands corresponding to DHFR from Supplementary Figs. S4A and B . ( E ) Logarithmic dose-response curves of either 5 µM AbiEii WT or AbiEii T65A incubated with increasing concentrations of AbiEi plotted against DHFR expression, normalized against DHFR expressed in the absence of toxin (100% expression). Data points were generated following densitometric analysis of bands corresponding to DHFR from Supplementary Figs. S4C and D . Logarithmic curves were generated using non-linear regression in Prism (GraphPad). ( F ) Endpoint viable count antitoxicity assays of E. coli DH5α transformed with either pBAD30 empty vector, pTRB482 (AbiEii WT), or pTRB736 (AbiEii T65A), and either pTA100 empty vector or pTRB481 (AbiEi). Overnight cultures were re-seeded into fresh LB supplemented with Ap, Sp and D-glu and grown to mid-log phase. Samples were serially diluted 10 −2 -10 −9 and spotted onto M9A plates containing Ap and Sp, with or without D-glu, L-ara, and IPTG for repression of toxin expression, induction of toxin expression, and induction of antitoxin expression, respectively. Plates were incubated at 37 °C for 48 h, after which they were imaged and colonies counted to determine CFU/ml (one-way ANOVA, P < 0.05). “T” = toxin induced, “TA” = antitoxin + toxin induced. Assays shown are representative of three independent biological replicates and plotted data represent the mean ± SEM.

    Techniques Used: Phospho-proteomics, Activity Assay, In Vitro, Control, Produced, SDS Page, Expressing, Generated, Incubation, Transformation Assay, Plasmid Preparation



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    cell free transcription translation protein synthesis reactions  (New England Biolabs)
    97
    New England Biolabs cell free transcription translation protein synthesis reactions
    T65 is important, but not essential, for NTase and phosphorylation activity. ( A ) In <t>vitro</t> <t>transcription/translation</t> reactions assessing levels of DHFR control protein produced in the presence of either 5 µM AbiEii WT or AbiEii-p. ( B ) In vitro transcription/translation reactions assessing levels of DHFR control protein produced in the absence and presence of 5 µM AbiEii T65A, AbiEii D192A, or AbiEii-p. Reactions were loaded onto 4-20% gradient gels ran at 180 V for 1 h. ( C ) Densitometric analysis of SDS-PAGE bands from ( B ) corresponding to DHFR. ( D ) Dose-response curves of increasing concentrations of either AbiEii WT or AbiEii T65A plotted against DHFR expression, normalized against DHFR expressed in the absence of toxin (100% expression). Data points were generated following densitometric analysis of bands corresponding to DHFR from Supplementary Figs. S4A and B . ( E ) Logarithmic dose-response curves of either 5 µM AbiEii WT or AbiEii T65A incubated with increasing concentrations of AbiEi plotted against DHFR expression, normalized against DHFR expressed in the absence of toxin (100% expression). Data points were generated following densitometric analysis of bands corresponding to DHFR from Supplementary Figs. S4C and D . Logarithmic curves were generated using non-linear regression in Prism (GraphPad). ( F ) Endpoint viable count antitoxicity assays of E. coli DH5α transformed with either pBAD30 empty vector, pTRB482 (AbiEii WT), or pTRB736 (AbiEii T65A), and either pTA100 empty vector or pTRB481 (AbiEi). Overnight cultures were re-seeded into fresh LB supplemented with Ap, Sp and D-glu and grown to mid-log phase. Samples were serially diluted 10 −2 -10 −9 and spotted onto M9A plates containing Ap and Sp, with or without D-glu, L-ara, and IPTG for repression of toxin expression, induction of toxin expression, and induction of antitoxin expression, respectively. Plates were incubated at 37 °C for 48 h, after which they were imaged and colonies counted to determine CFU/ml (one-way ANOVA, P < 0.05). “T” = toxin induced, “TA” = antitoxin + toxin induced. Assays shown are representative of three independent biological replicates and plotted data represent the mean ± SEM.
    Cell Free Transcription Translation Protein Synthesis Reactions, 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/cell free transcription translation protein synthesis reactions/product/New England Biolabs
    Average 97 stars, based on 1 article reviews
    cell free transcription translation protein synthesis reactions - by Bioz Stars, 2026-06
    97/100 stars
    		  Buy from Supplier
    cell free transcription translation reactions  (New England Biolabs)
    97
    New England Biolabs cell free transcription translation reactions
    T65 is important, but not essential, for NTase and phosphorylation activity. ( A ) In <t>vitro</t> <t>transcription/translation</t> reactions assessing levels of DHFR control protein produced in the presence of either 5 µM AbiEii WT or AbiEii-p. ( B ) In vitro transcription/translation reactions assessing levels of DHFR control protein produced in the absence and presence of 5 µM AbiEii T65A, AbiEii D192A, or AbiEii-p. Reactions were loaded onto 4-20% gradient gels ran at 180 V for 1 h. ( C ) Densitometric analysis of SDS-PAGE bands from ( B ) corresponding to DHFR. ( D ) Dose-response curves of increasing concentrations of either AbiEii WT or AbiEii T65A plotted against DHFR expression, normalized against DHFR expressed in the absence of toxin (100% expression). Data points were generated following densitometric analysis of bands corresponding to DHFR from Supplementary Figs. S4A and B . ( E ) Logarithmic dose-response curves of either 5 µM AbiEii WT or AbiEii T65A incubated with increasing concentrations of AbiEi plotted against DHFR expression, normalized against DHFR expressed in the absence of toxin (100% expression). Data points were generated following densitometric analysis of bands corresponding to DHFR from Supplementary Figs. S4C and D . Logarithmic curves were generated using non-linear regression in Prism (GraphPad). ( F ) Endpoint viable count antitoxicity assays of E. coli DH5α transformed with either pBAD30 empty vector, pTRB482 (AbiEii WT), or pTRB736 (AbiEii T65A), and either pTA100 empty vector or pTRB481 (AbiEi). Overnight cultures were re-seeded into fresh LB supplemented with Ap, Sp and D-glu and grown to mid-log phase. Samples were serially diluted 10 −2 -10 −9 and spotted onto M9A plates containing Ap and Sp, with or without D-glu, L-ara, and IPTG for repression of toxin expression, induction of toxin expression, and induction of antitoxin expression, respectively. Plates were incubated at 37 °C for 48 h, after which they were imaged and colonies counted to determine CFU/ml (one-way ANOVA, P < 0.05). “T” = toxin induced, “TA” = antitoxin + toxin induced. Assays shown are representative of three independent biological replicates and plotted data represent the mean ± SEM.
    Cell Free Transcription Translation Reactions, 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/cell free transcription translation reactions/product/New England Biolabs
    Average 97 stars, based on 1 article reviews
    cell free transcription translation reactions - by Bioz Stars, 2026-06
    97/100 stars
    		  Buy from Supplier

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    T65 is important, but not essential, for NTase and phosphorylation activity. ( A ) In vitro transcription/translation reactions assessing levels of DHFR control protein produced in the presence of either 5 µM AbiEii WT or AbiEii-p. ( B ) In vitro transcription/translation reactions assessing levels of DHFR control protein produced in the absence and presence of 5 µM AbiEii T65A, AbiEii D192A, or AbiEii-p. Reactions were loaded onto 4-20% gradient gels ran at 180 V for 1 h. ( C ) Densitometric analysis of SDS-PAGE bands from ( B ) corresponding to DHFR. ( D ) Dose-response curves of increasing concentrations of either AbiEii WT or AbiEii T65A plotted against DHFR expression, normalized against DHFR expressed in the absence of toxin (100% expression). Data points were generated following densitometric analysis of bands corresponding to DHFR from Supplementary Figs. S4A and B . ( E ) Logarithmic dose-response curves of either 5 µM AbiEii WT or AbiEii T65A incubated with increasing concentrations of AbiEi plotted against DHFR expression, normalized against DHFR expressed in the absence of toxin (100% expression). Data points were generated following densitometric analysis of bands corresponding to DHFR from Supplementary Figs. S4C and D . Logarithmic curves were generated using non-linear regression in Prism (GraphPad). ( F ) Endpoint viable count antitoxicity assays of E. coli DH5α transformed with either pBAD30 empty vector, pTRB482 (AbiEii WT), or pTRB736 (AbiEii T65A), and either pTA100 empty vector or pTRB481 (AbiEi). Overnight cultures were re-seeded into fresh LB supplemented with Ap, Sp and D-glu and grown to mid-log phase. Samples were serially diluted 10 −2 -10 −9 and spotted onto M9A plates containing Ap and Sp, with or without D-glu, L-ara, and IPTG for repression of toxin expression, induction of toxin expression, and induction of antitoxin expression, respectively. Plates were incubated at 37 °C for 48 h, after which they were imaged and colonies counted to determine CFU/ml (one-way ANOVA, P < 0.05). “T” = toxin induced, “TA” = antitoxin + toxin induced. Assays shown are representative of three independent biological replicates and plotted data represent the mean ± SEM.

    Journal: bioRxiv

    Article Title: Antitoxin-induced auto-phosphorylation neutralizes the nucleotidyltransferase toxin AbiEii from Streptococcus agalactiae to safeguard global translation

    doi: 10.64898/2026.01.19.700257

    Figure Lengend Snippet: T65 is important, but not essential, for NTase and phosphorylation activity. ( A ) In vitro transcription/translation reactions assessing levels of DHFR control protein produced in the presence of either 5 µM AbiEii WT or AbiEii-p. ( B ) In vitro transcription/translation reactions assessing levels of DHFR control protein produced in the absence and presence of 5 µM AbiEii T65A, AbiEii D192A, or AbiEii-p. Reactions were loaded onto 4-20% gradient gels ran at 180 V for 1 h. ( C ) Densitometric analysis of SDS-PAGE bands from ( B ) corresponding to DHFR. ( D ) Dose-response curves of increasing concentrations of either AbiEii WT or AbiEii T65A plotted against DHFR expression, normalized against DHFR expressed in the absence of toxin (100% expression). Data points were generated following densitometric analysis of bands corresponding to DHFR from Supplementary Figs. S4A and B . ( E ) Logarithmic dose-response curves of either 5 µM AbiEii WT or AbiEii T65A incubated with increasing concentrations of AbiEi plotted against DHFR expression, normalized against DHFR expressed in the absence of toxin (100% expression). Data points were generated following densitometric analysis of bands corresponding to DHFR from Supplementary Figs. S4C and D . Logarithmic curves were generated using non-linear regression in Prism (GraphPad). ( F ) Endpoint viable count antitoxicity assays of E. coli DH5α transformed with either pBAD30 empty vector, pTRB482 (AbiEii WT), or pTRB736 (AbiEii T65A), and either pTA100 empty vector or pTRB481 (AbiEi). Overnight cultures were re-seeded into fresh LB supplemented with Ap, Sp and D-glu and grown to mid-log phase. Samples were serially diluted 10 −2 -10 −9 and spotted onto M9A plates containing Ap and Sp, with or without D-glu, L-ara, and IPTG for repression of toxin expression, induction of toxin expression, and induction of antitoxin expression, respectively. Plates were incubated at 37 °C for 48 h, after which they were imaged and colonies counted to determine CFU/ml (one-way ANOVA, P < 0.05). “T” = toxin induced, “TA” = antitoxin + toxin induced. Assays shown are representative of three independent biological replicates and plotted data represent the mean ± SEM.

    Article Snippet: Five μM of purified AbiEii WT or AbiEii-p were added to cell-free transcription/translation protein synthesis reactions (PURExpress ® ; NEB) to quantify changes in DHFR control protein expression.

    Techniques: Phospho-proteomics, Activity Assay, In Vitro, Control, Produced, SDS Page, Expressing, Generated, Incubation, Transformation Assay, Plasmid Preparation