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    Name:
    PURExpress In Vitro Protein Synthesis Kit
    Description:
    PURExpress In Vitro Protein Synthesis Kit 100 rxns
    Catalog Number:
    e6800l
    Price:
    2292
    Size:
    100 rxns
    Category:
    Transcription Translation Systems
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    New England Biolabs new england biolabs purexpress
    PURExpress In Vitro Protein Synthesis Kit
    PURExpress In Vitro Protein Synthesis Kit 100 rxns
    https://www.bioz.com/result/new england biolabs purexpress/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    Related Articles

    Transfection:

    Article Title: Identification of functional cis-acting RNA elements in the hepatitis E virus genome required for viral replication
    Article Snippet: .. In vitro transcription assay and viral RNA transfection HEV Kernow-C1/p6, HEV Kernow-C1/p6-Gluc, and the truncated mutant plasmids were linearized by MluI. pSAR55-Gluc was linearized by BglII, pGEM-9Zf-pSHEV3-Gluc was linearized by XbaI, and pGEM-7Zf(-)-TW6196E and pGEM-7Zf(-)-TW6196E/Gluc were linearized by SpeI. .. Viral RNAs were transcribed in vitro from linearized plasmid using HiScribe T7 antireverse cap analog (ARCA) mRNA kit (New England Biolabs, Ipswich, MA) according to the manufacturer’s instructions.

    In Vitro:

    Article Title: Using Group II Introns for Attenuating the In Vitro and In Vivo Expression of a Homing Endonuclease
    Article Snippet: .. The PURExpress In Vitro Protein Synthesis Kit (New England Biolab, MA, USA) which is a cell-free transcription/translation system was utilized to assess if the proteins can be expressed in an “E . coli ” environment. .. Although the PURExpress kit is designed for coupled transcription and translation from an expression construct, direct translation from an mRNA template is also possible provided purified RNA (1–5 μg) is added to the reaction mixture, albeit a proper ribosome binding site must be present for efficient translation.

    Article Title: Protein Synthesis Using A Reconstituted Cell-Free System
    Article Snippet: .. A PURExpress In vitro Protein Synthesis kit (New England Biolabs, ) containing: Solution A (yellow tube) Solution B (red tube) DHFR control template PURExpress Disulfide Bond Enhancer (New England Biolabs, ) containing: Disulfide Bond Enhancer Solution 1 Disulfide Bond Enhancer Solution 2 microcentrifuge tubes or microtiter plate Nuclease-free H2 O (Integrated DNA technologies) Template DNA (See Support Protocol 1 and 2) or mRNA (See Support Protocol 3) Murine RNase inhibitor (40 U/µl, New England Biolabs) or RNasin Ribonuclease inhibitor (20–40 U/µl, Promega) Microcentrifuge Air incubator set at 37°C 3x SDS-PAGE loading buffer (New England Biolabs, CPMB Chapter X) SDS-PAGE gel (4–20% Tris-glycine, Life Technologies, CPMB Chapter X) ..

    Article Title: Protein Synthesis Using A Reconstituted Cell-Free System
    Article Snippet: .. PURExpress In vitro Protein Synthesis kit (New England Biolabs) containing: Solution A (yellow tube) Solution B (red tube) DHFR control template Nuclease free microcentrifuge tubes or microtiter plates Nuclease free H2 O Template DNA (See Support Protocol 1 and 2) or mRNA (See Support Protocol 3) Murine RNase inhibitor (40 U/µl, New England Biolabs) [35 S]-L-Methionine (15 mCi/ml, 1000 Ci/mmol) (PerkinElmer) Microcentrifuge Air incubator set at 37°C 3x SDS-PAGE loading buffer (New England Biolabs) SDS-PAGE gel (4–20 % Tris-glycine, Life Technologies) Filter paper (Whatman) Vacuum gel dryer X-ray film or phosphorimager ..

    Article Title: Intramolecular chaperone-mediated secretion of an Rhs effector toxin by a type VI secretion system
    Article Snippet: .. In vitro protein expression was performed with a PURExpress® In Vitro Protein Synthesis Kit (NEB) following the instruction of the manufacturer. .. For TseI purification under denaturing conditions, His-tagged proteins were first purified with Ni-NTA resin, eluted with elution buffer A (50 mM NaH2 PO4 pH 8.0, 300 mM NaCl, 250 mM imidazole), and dialyzed with dialysis buffer (50 mM NaH2 PO4 pH 8.0, 300 mM NaCl) three times to remove imidazole.

    Article Title: Riboneogenesis in yeast
    Article Snippet: .. Initial enzymatic screens for enzymatic activity were performed using in vitro synthesized, untagged Shb17 (New England Biolabs Inc. PURExpress® In Vitro Protein Synthesis Kit). .. Subsequent studies were performed using N-terminal His-tagged recombinant protein purified from E. coli .

    Article Title: Protein Synthesis Using A Reconstituted Cell-Free System
    Article Snippet: .. A PURExpress In vitro Protein Synthesis kit (New England Biolabs, ) 10 mM magnesium acetate Amicon Ultracel −0.5 ml-100 K MW cut off spin concentrator (Millipore) Microcentrifuge at 4°C Ni-NTA agarose (Qiagen) Microcentrifuge tubes Bio-Rad micro-spin column (Bio-Rad) ..

    Article Title: High Affinity Binding of N2-Modified Guanine Derivatives Significantly Disrupts the Ligand Binding Pocket of the Guanine Riboswitch
    Article Snippet: .. Single-Turnover In Vitro Transcription Assays Transcription assays were performed using both wild-type B. subtilis xpt and chimeric xpt /yxjA riboswitches [ , , ]. ..

    Article Title: Identification of functional cis-acting RNA elements in the hepatitis E virus genome required for viral replication
    Article Snippet: .. In vitro transcription assay and viral RNA transfection HEV Kernow-C1/p6, HEV Kernow-C1/p6-Gluc, and the truncated mutant plasmids were linearized by MluI. pSAR55-Gluc was linearized by BglII, pGEM-9Zf-pSHEV3-Gluc was linearized by XbaI, and pGEM-7Zf(-)-TW6196E and pGEM-7Zf(-)-TW6196E/Gluc were linearized by SpeI. .. Viral RNAs were transcribed in vitro from linearized plasmid using HiScribe T7 antireverse cap analog (ARCA) mRNA kit (New England Biolabs, Ipswich, MA) according to the manufacturer’s instructions.

    Synthesized:

    Article Title: Riboneogenesis in yeast
    Article Snippet: .. Initial enzymatic screens for enzymatic activity were performed using in vitro synthesized, untagged Shb17 (New England Biolabs Inc. PURExpress® In Vitro Protein Synthesis Kit). .. Subsequent studies were performed using N-terminal His-tagged recombinant protein purified from E. coli .

    Mutagenesis:

    Article Title: Identification of functional cis-acting RNA elements in the hepatitis E virus genome required for viral replication
    Article Snippet: .. In vitro transcription assay and viral RNA transfection HEV Kernow-C1/p6, HEV Kernow-C1/p6-Gluc, and the truncated mutant plasmids were linearized by MluI. pSAR55-Gluc was linearized by BglII, pGEM-9Zf-pSHEV3-Gluc was linearized by XbaI, and pGEM-7Zf(-)-TW6196E and pGEM-7Zf(-)-TW6196E/Gluc were linearized by SpeI. .. Viral RNAs were transcribed in vitro from linearized plasmid using HiScribe T7 antireverse cap analog (ARCA) mRNA kit (New England Biolabs, Ipswich, MA) according to the manufacturer’s instructions.

    Activity Assay:

    Article Title: Riboneogenesis in yeast
    Article Snippet: .. Initial enzymatic screens for enzymatic activity were performed using in vitro synthesized, untagged Shb17 (New England Biolabs Inc. PURExpress® In Vitro Protein Synthesis Kit). .. Subsequent studies were performed using N-terminal His-tagged recombinant protein purified from E. coli .

    Expressing:

    Article Title: Intramolecular chaperone-mediated secretion of an Rhs effector toxin by a type VI secretion system
    Article Snippet: .. In vitro protein expression was performed with a PURExpress® In Vitro Protein Synthesis Kit (NEB) following the instruction of the manufacturer. .. For TseI purification under denaturing conditions, His-tagged proteins were first purified with Ni-NTA resin, eluted with elution buffer A (50 mM NaH2 PO4 pH 8.0, 300 mM NaCl, 250 mM imidazole), and dialyzed with dialysis buffer (50 mM NaH2 PO4 pH 8.0, 300 mM NaCl) three times to remove imidazole.

    SDS Page:

    Article Title: Protein Synthesis Using A Reconstituted Cell-Free System
    Article Snippet: .. A PURExpress In vitro Protein Synthesis kit (New England Biolabs, ) containing: Solution A (yellow tube) Solution B (red tube) DHFR control template PURExpress Disulfide Bond Enhancer (New England Biolabs, ) containing: Disulfide Bond Enhancer Solution 1 Disulfide Bond Enhancer Solution 2 microcentrifuge tubes or microtiter plate Nuclease-free H2 O (Integrated DNA technologies) Template DNA (See Support Protocol 1 and 2) or mRNA (See Support Protocol 3) Murine RNase inhibitor (40 U/µl, New England Biolabs) or RNasin Ribonuclease inhibitor (20–40 U/µl, Promega) Microcentrifuge Air incubator set at 37°C 3x SDS-PAGE loading buffer (New England Biolabs, CPMB Chapter X) SDS-PAGE gel (4–20% Tris-glycine, Life Technologies, CPMB Chapter X) ..

    Article Title: Protein Synthesis Using A Reconstituted Cell-Free System
    Article Snippet: .. PURExpress In vitro Protein Synthesis kit (New England Biolabs) containing: Solution A (yellow tube) Solution B (red tube) DHFR control template Nuclease free microcentrifuge tubes or microtiter plates Nuclease free H2 O Template DNA (See Support Protocol 1 and 2) or mRNA (See Support Protocol 3) Murine RNase inhibitor (40 U/µl, New England Biolabs) [35 S]-L-Methionine (15 mCi/ml, 1000 Ci/mmol) (PerkinElmer) Microcentrifuge Air incubator set at 37°C 3x SDS-PAGE loading buffer (New England Biolabs) SDS-PAGE gel (4–20 % Tris-glycine, Life Technologies) Filter paper (Whatman) Vacuum gel dryer X-ray film or phosphorimager ..

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  • 99
    New England Biolabs in vitro transcription assay
    Full-length HEV genome with synonymous mutations <t>in</t> the cis -acting RNA elements is significantly impaired in its ability to produce infectious virus. (A) Schematic diagrams of the protocol to determine the production of infectious virus. For the cis -acting element mutants, synonymous mutations were introduced in the ORF1 coding region (G113C or G113T) or ORF2 coding region (G7335A). (B-C) Transfection of in <t>vitro</t> -transcribed WT, synonymous mutants or GAD (Pol-) RNA of full-length Kernow C1/p6 (GT 3) into S10-3 cells. Cell lysate supernatant was collected 7 days after transfection, and virus was titrated by infecting HepG2C3A cells. Cells were stained with anti-HEV ORF2 mAbs at 5 days post of infection. (B) to quantify infectious viral particles by foci-forming <t>assay</t> (C). Values are means plus SD (n = 3). IF, immunofluorescence; FFU, foci-forming units; LOD, limit of detection.
    In Vitro Transcription Assay, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 87 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/in vitro transcription assay/product/New England Biolabs
    Average 99 stars, based on 87 article reviews
    Price from $9.99 to $1999.99
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    90
    New England Biolabs purexpress
    Testing agsA thermometer function in a cell-free protein synthesis system using purified pre-transcribed mRNA. (a) Fluorescence trajectories over time for translation of SFGFP from agsA constructs and control mRNA in the <t>PURExpress</t> protein synthesis system at 30 °C and 42 °C. Shading represents standard deviation over three replicates. (b) SFGFP production rates, calculated from the trajectories in (a), during the linear synthesis regime for agsA constructs and control at 30 °C (45–50 minutes) and 42 °C (30–35 minutes), with error bars representing standard deviation.
    Purexpress, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 90/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/purexpress/product/New England Biolabs
    Average 90 stars, based on 18 article reviews
    Price from $9.99 to $1999.99
    purexpress - by Bioz Stars, 2020-09
    90/100 stars
      Buy from Supplier

    Image Search Results


    Full-length HEV genome with synonymous mutations in the cis -acting RNA elements is significantly impaired in its ability to produce infectious virus. (A) Schematic diagrams of the protocol to determine the production of infectious virus. For the cis -acting element mutants, synonymous mutations were introduced in the ORF1 coding region (G113C or G113T) or ORF2 coding region (G7335A). (B-C) Transfection of in vitro -transcribed WT, synonymous mutants or GAD (Pol-) RNA of full-length Kernow C1/p6 (GT 3) into S10-3 cells. Cell lysate supernatant was collected 7 days after transfection, and virus was titrated by infecting HepG2C3A cells. Cells were stained with anti-HEV ORF2 mAbs at 5 days post of infection. (B) to quantify infectious viral particles by foci-forming assay (C). Values are means plus SD (n = 3). IF, immunofluorescence; FFU, foci-forming units; LOD, limit of detection.

    Journal: PLoS Pathogens

    Article Title: Identification of functional cis-acting RNA elements in the hepatitis E virus genome required for viral replication

    doi: 10.1371/journal.ppat.1008488

    Figure Lengend Snippet: Full-length HEV genome with synonymous mutations in the cis -acting RNA elements is significantly impaired in its ability to produce infectious virus. (A) Schematic diagrams of the protocol to determine the production of infectious virus. For the cis -acting element mutants, synonymous mutations were introduced in the ORF1 coding region (G113C or G113T) or ORF2 coding region (G7335A). (B-C) Transfection of in vitro -transcribed WT, synonymous mutants or GAD (Pol-) RNA of full-length Kernow C1/p6 (GT 3) into S10-3 cells. Cell lysate supernatant was collected 7 days after transfection, and virus was titrated by infecting HepG2C3A cells. Cells were stained with anti-HEV ORF2 mAbs at 5 days post of infection. (B) to quantify infectious viral particles by foci-forming assay (C). Values are means plus SD (n = 3). IF, immunofluorescence; FFU, foci-forming units; LOD, limit of detection.

    Article Snippet: In vitro transcription assay and viral RNA transfection HEV Kernow-C1/p6, HEV Kernow-C1/p6-Gluc, and the truncated mutant plasmids were linearized by MluI. pSAR55-Gluc was linearized by BglII, pGEM-9Zf-pSHEV3-Gluc was linearized by XbaI, and pGEM-7Zf(-)-TW6196E and pGEM-7Zf(-)-TW6196E/Gluc were linearized by SpeI.

    Techniques: Transfection, In Vitro, Staining, Infection, Immunofluorescence

    Shb17 feeds carbon into the non-oxidative pentose phosphate pathway. (A) Flux through Shb17 into S7P can be measured using [6- 13 C 1 ]-glucose. [6- 13 C 1 ]-glucose leads to [7- 13 C 1 ]-S7P when S7P is made via the oxidative PPP or the non-oxidative PPP. However, when S7P is produced from SBP via Shb17, a fraction of the S7P pool is doubly labeled: [1,7- 13 C 2 ]-S7P. Flux is calculated based on the measured isotopic distribution of SBP and S7P. (B) Flux through Shb17 is increased by supplementation with nutrients whose endogenous production requires NADPH, and thus drives oxidative PPP flux. All measurements are performed in wild type yeast. YNB is yeast nitrogen base without amino acids plus 2% glucose. Supplementation with amino acids includes 17 amino acids. Data shown is the arithmetic mean ± SE of N=3 technical replicates. (C) Effects of PPP gene deletions on Shb17 flux. Deletions are: glucose 6-phosphate dehydrogenase zwf1 Δ; transketolase tkl1 Δ/ tkl2 Δ; transaldolase is tal1 Δ/ nqm1 Δ. Less than 1% doubly labeled S7P was observed in any shb17 Δ strain in all measured conditions. All strains were grown in YNB + 2% glucose and supplements as required: methionine for zwf1 Δ; synthetic complete media including aromatic amino acids for tkl1 Δ/ tkl2 Δ. (C) Triple deletion of the sedoheptulose bisphosphatase SHB17 , the glucose-6-phosphate dehydrogenase ZWF1 , and the transaldolase TAL1 .

    Journal: Cell

    Article Title: Riboneogenesis in yeast

    doi: 10.1016/j.cell.2011.05.022

    Figure Lengend Snippet: Shb17 feeds carbon into the non-oxidative pentose phosphate pathway. (A) Flux through Shb17 into S7P can be measured using [6- 13 C 1 ]-glucose. [6- 13 C 1 ]-glucose leads to [7- 13 C 1 ]-S7P when S7P is made via the oxidative PPP or the non-oxidative PPP. However, when S7P is produced from SBP via Shb17, a fraction of the S7P pool is doubly labeled: [1,7- 13 C 2 ]-S7P. Flux is calculated based on the measured isotopic distribution of SBP and S7P. (B) Flux through Shb17 is increased by supplementation with nutrients whose endogenous production requires NADPH, and thus drives oxidative PPP flux. All measurements are performed in wild type yeast. YNB is yeast nitrogen base without amino acids plus 2% glucose. Supplementation with amino acids includes 17 amino acids. Data shown is the arithmetic mean ± SE of N=3 technical replicates. (C) Effects of PPP gene deletions on Shb17 flux. Deletions are: glucose 6-phosphate dehydrogenase zwf1 Δ; transketolase tkl1 Δ/ tkl2 Δ; transaldolase is tal1 Δ/ nqm1 Δ. Less than 1% doubly labeled S7P was observed in any shb17 Δ strain in all measured conditions. All strains were grown in YNB + 2% glucose and supplements as required: methionine for zwf1 Δ; synthetic complete media including aromatic amino acids for tkl1 Δ/ tkl2 Δ. (C) Triple deletion of the sedoheptulose bisphosphatase SHB17 , the glucose-6-phosphate dehydrogenase ZWF1 , and the transaldolase TAL1 .

    Article Snippet: Initial enzymatic screens for enzymatic activity were performed using in vitro synthesized, untagged Shb17 (New England Biolabs Inc. PURExpress® In Vitro Protein Synthesis Kit).

    Techniques: Produced, Labeling

    Structure of the Shb17/SBP complex. (A) Overall fold of the Shb17 (H13A) in complex with SBP (PDB 3OI7, grey ribbon) shown in two orientations with secondary structural elements being labeled. The SBP molecule (magenta carbon atoms) is shown in a stick representation. (B) Close-up view of the active site of Shb17 in complex with SBP. The side chains of residues in contact with SBP are displayed in a stick representation (green carbon atoms) and labeled. SBP is shown in a stick representation (magenta carbon atoms) and labeled, whereas the Mg 2+ ion is shown as a purple sphere and labeled. (C) Active site of Shb17 in complex with FBP, a similar view as (B). The red sphere denotes a water molecule. Y102 makes two hydrogen bonds with SBP, whereas only one hydrogen bond can be formed between this residue and FBP. These hydrogen bonds are shown by dashed lines in parts B and C.

    Journal: Cell

    Article Title: Riboneogenesis in yeast

    doi: 10.1016/j.cell.2011.05.022

    Figure Lengend Snippet: Structure of the Shb17/SBP complex. (A) Overall fold of the Shb17 (H13A) in complex with SBP (PDB 3OI7, grey ribbon) shown in two orientations with secondary structural elements being labeled. The SBP molecule (magenta carbon atoms) is shown in a stick representation. (B) Close-up view of the active site of Shb17 in complex with SBP. The side chains of residues in contact with SBP are displayed in a stick representation (green carbon atoms) and labeled. SBP is shown in a stick representation (magenta carbon atoms) and labeled, whereas the Mg 2+ ion is shown as a purple sphere and labeled. (C) Active site of Shb17 in complex with FBP, a similar view as (B). The red sphere denotes a water molecule. Y102 makes two hydrogen bonds with SBP, whereas only one hydrogen bond can be formed between this residue and FBP. These hydrogen bonds are shown by dashed lines in parts B and C.

    Article Snippet: Initial enzymatic screens for enzymatic activity were performed using in vitro synthesized, untagged Shb17 (New England Biolabs Inc. PURExpress® In Vitro Protein Synthesis Kit).

    Techniques: Labeling

    SBP and OBP are synthesized in vivo by C 3 + C 4 and C 3 + C 5 subunits via fructose bisphosphate aldolase. (A) Cells were switched from unlabeled to 70:30 unlabeled glucose:[U- 13 C]-glucose. Labeling patterns of erythrose-4-phosphate (E4P), dihydroxyacetone-phosphate (DHAP), ribose-5-phosphate (R5P), SBP and OBP were measured in shb17 Δ, where SBP and OBP accumulate and hence are more readily quantitated. The reaction products sedoheptulose-7-phosphate (S7P) and octulose 8-phosphate (O8P) were measured in wild type (for data on S7P in shb17 ). Labeling is reported 20 minutes after nutrient switch for all compounds except OBP, where data is taken at 120 min due to its slower labeling. (B) Kinetics of labeling of SBP after switching shb17 Δ cells with wild type fructose bisphosphate aldolase ( FBA1-wt ), or the Decreased Abundance by mRNA Perturbation allele ( FBA1-DAmP ) into [U- 13 C 6 . (C) Kinetics of labeling of SBP and S1P after switching shb17 Δ cells into [U- 13 C 6 ]-glucose.

    Journal: Cell

    Article Title: Riboneogenesis in yeast

    doi: 10.1016/j.cell.2011.05.022

    Figure Lengend Snippet: SBP and OBP are synthesized in vivo by C 3 + C 4 and C 3 + C 5 subunits via fructose bisphosphate aldolase. (A) Cells were switched from unlabeled to 70:30 unlabeled glucose:[U- 13 C]-glucose. Labeling patterns of erythrose-4-phosphate (E4P), dihydroxyacetone-phosphate (DHAP), ribose-5-phosphate (R5P), SBP and OBP were measured in shb17 Δ, where SBP and OBP accumulate and hence are more readily quantitated. The reaction products sedoheptulose-7-phosphate (S7P) and octulose 8-phosphate (O8P) were measured in wild type (for data on S7P in shb17 ). Labeling is reported 20 minutes after nutrient switch for all compounds except OBP, where data is taken at 120 min due to its slower labeling. (B) Kinetics of labeling of SBP after switching shb17 Δ cells with wild type fructose bisphosphate aldolase ( FBA1-wt ), or the Decreased Abundance by mRNA Perturbation allele ( FBA1-DAmP ) into [U- 13 C 6 . (C) Kinetics of labeling of SBP and S1P after switching shb17 Δ cells into [U- 13 C 6 ]-glucose.

    Article Snippet: Initial enzymatic screens for enzymatic activity were performed using in vitro synthesized, untagged Shb17 (New England Biolabs Inc. PURExpress® In Vitro Protein Synthesis Kit).

    Techniques: Synthesized, In Vivo, Labeling

    Metabolomic phenotype of shb17 Δ. (A) Metabolite structures associated with metabolic phenotype of shb17Δ . (B) Relative quantitation of metabolites. Data shown is arithmetic mean ± SE of N=4 independent biological replicates. (C)The negative ionization mode extracted ion chromatogram for SBP in shb17 Δ and wild type S. cerevisiae . Inset: Mass spectrum displaying the accurate mass for the parent ion (M) and natural 13 C abundance ion (M+1) observed for SBP in negative ionization mode via LC/Exactive Orbitrap MS. (D) Table of [M-H] ions with altered abundance between shb17 Δ and wild type.

    Journal: Cell

    Article Title: Riboneogenesis in yeast

    doi: 10.1016/j.cell.2011.05.022

    Figure Lengend Snippet: Metabolomic phenotype of shb17 Δ. (A) Metabolite structures associated with metabolic phenotype of shb17Δ . (B) Relative quantitation of metabolites. Data shown is arithmetic mean ± SE of N=4 independent biological replicates. (C)The negative ionization mode extracted ion chromatogram for SBP in shb17 Δ and wild type S. cerevisiae . Inset: Mass spectrum displaying the accurate mass for the parent ion (M) and natural 13 C abundance ion (M+1) observed for SBP in negative ionization mode via LC/Exactive Orbitrap MS. (D) Table of [M-H] ions with altered abundance between shb17 Δ and wild type.

    Article Snippet: Initial enzymatic screens for enzymatic activity were performed using in vitro synthesized, untagged Shb17 (New England Biolabs Inc. PURExpress® In Vitro Protein Synthesis Kit).

    Techniques: Quantitation Assay, Mass Spectrometry

    Chromozyme cleavage assay (Roche) for a truncated version of tissue plasminogen activator protein (vtPA) synthesized in the PURExpress reactions with and without PURExpress Disulfide Bond Enhancer (PDBE). This tissue plasminogen activator contains 9 disulfide

    Journal: Current protocols in molecular biology / edited by Frederick M. Ausubel ... [et al.]

    Article Title: Protein Synthesis Using A Reconstituted Cell-Free System

    doi: 10.1002/0471142727.mb1631s108

    Figure Lengend Snippet: Chromozyme cleavage assay (Roche) for a truncated version of tissue plasminogen activator protein (vtPA) synthesized in the PURExpress reactions with and without PURExpress Disulfide Bond Enhancer (PDBE). This tissue plasminogen activator contains 9 disulfide

    Article Snippet: A PURExpress In vitro Protein Synthesis kit (New England Biolabs, ) containing: Solution A (yellow tube) Solution B (red tube) DHFR control template PURExpress Disulfide Bond Enhancer (New England Biolabs, ) containing: Disulfide Bond Enhancer Solution 1 Disulfide Bond Enhancer Solution 2 microcentrifuge tubes or microtiter plate Nuclease-free H2 O (Integrated DNA technologies) Template DNA (See Support Protocol 1 and 2) or mRNA (See Support Protocol 3) Murine RNase inhibitor (40 U/µl, New England Biolabs) or RNasin Ribonuclease inhibitor (20–40 U/µl, Promega) Microcentrifuge Air incubator set at 37°C 3x SDS-PAGE loading buffer (New England Biolabs, CPMB Chapter X) SDS-PAGE gel (4–20% Tris-glycine, Life Technologies, CPMB Chapter X)

    Techniques: Cleavage Assay, Synthesized

    SDS-PAGE analysis of the reverse purification of the DHFR control protein synthesized in the PURExpress reaction. M: molecular weight standards (kDa); Lane 1: Control PURExpress reaction with no input template, Lane 2: PURExpress reaction with the DHFR

    Journal: Current protocols in molecular biology / edited by Frederick M. Ausubel ... [et al.]

    Article Title: Protein Synthesis Using A Reconstituted Cell-Free System

    doi: 10.1002/0471142727.mb1631s108

    Figure Lengend Snippet: SDS-PAGE analysis of the reverse purification of the DHFR control protein synthesized in the PURExpress reaction. M: molecular weight standards (kDa); Lane 1: Control PURExpress reaction with no input template, Lane 2: PURExpress reaction with the DHFR

    Article Snippet: A PURExpress In vitro Protein Synthesis kit (New England Biolabs, ) containing: Solution A (yellow tube) Solution B (red tube) DHFR control template PURExpress Disulfide Bond Enhancer (New England Biolabs, ) containing: Disulfide Bond Enhancer Solution 1 Disulfide Bond Enhancer Solution 2 microcentrifuge tubes or microtiter plate Nuclease-free H2 O (Integrated DNA technologies) Template DNA (See Support Protocol 1 and 2) or mRNA (See Support Protocol 3) Murine RNase inhibitor (40 U/µl, New England Biolabs) or RNasin Ribonuclease inhibitor (20–40 U/µl, Promega) Microcentrifuge Air incubator set at 37°C 3x SDS-PAGE loading buffer (New England Biolabs, CPMB Chapter X) SDS-PAGE gel (4–20% Tris-glycine, Life Technologies, CPMB Chapter X)

    Techniques: SDS Page, Purification, Synthesized, Molecular Weight

    Scanned image of a SDS-PAGE gel of proteins synthesized in the PURExpress reactions and labeled with FluoroTect™ Green Lys . Lane 1: DHFR; lane 2: GFP; lane 3: Renilla luciferase; lane 4: Firefly luciferase; lane 5: E. coli β-galactosidase.

    Journal: Current protocols in molecular biology / edited by Frederick M. Ausubel ... [et al.]

    Article Title: Protein Synthesis Using A Reconstituted Cell-Free System

    doi: 10.1002/0471142727.mb1631s108

    Figure Lengend Snippet: Scanned image of a SDS-PAGE gel of proteins synthesized in the PURExpress reactions and labeled with FluoroTect™ Green Lys . Lane 1: DHFR; lane 2: GFP; lane 3: Renilla luciferase; lane 4: Firefly luciferase; lane 5: E. coli β-galactosidase.

    Article Snippet: A PURExpress In vitro Protein Synthesis kit (New England Biolabs, ) containing: Solution A (yellow tube) Solution B (red tube) DHFR control template PURExpress Disulfide Bond Enhancer (New England Biolabs, ) containing: Disulfide Bond Enhancer Solution 1 Disulfide Bond Enhancer Solution 2 microcentrifuge tubes or microtiter plate Nuclease-free H2 O (Integrated DNA technologies) Template DNA (See Support Protocol 1 and 2) or mRNA (See Support Protocol 3) Murine RNase inhibitor (40 U/µl, New England Biolabs) or RNasin Ribonuclease inhibitor (20–40 U/µl, Promega) Microcentrifuge Air incubator set at 37°C 3x SDS-PAGE loading buffer (New England Biolabs, CPMB Chapter X) SDS-PAGE gel (4–20% Tris-glycine, Life Technologies, CPMB Chapter X)

    Techniques: SDS Page, Synthesized, Labeling, Luciferase

    Testing agsA thermometer function in a cell-free protein synthesis system using purified pre-transcribed mRNA. (a) Fluorescence trajectories over time for translation of SFGFP from agsA constructs and control mRNA in the PURExpress protein synthesis system at 30 °C and 42 °C. Shading represents standard deviation over three replicates. (b) SFGFP production rates, calculated from the trajectories in (a), during the linear synthesis regime for agsA constructs and control at 30 °C (45–50 minutes) and 42 °C (30–35 minutes), with error bars representing standard deviation.

    Journal: Biochemistry

    Article Title: Characterizing the structure-function relationship of a naturally-occurring RNA thermometer

    doi: 10.1021/acs.biochem.7b01170

    Figure Lengend Snippet: Testing agsA thermometer function in a cell-free protein synthesis system using purified pre-transcribed mRNA. (a) Fluorescence trajectories over time for translation of SFGFP from agsA constructs and control mRNA in the PURExpress protein synthesis system at 30 °C and 42 °C. Shading represents standard deviation over three replicates. (b) SFGFP production rates, calculated from the trajectories in (a), during the linear synthesis regime for agsA constructs and control at 30 °C (45–50 minutes) and 42 °C (30–35 minutes), with error bars representing standard deviation.

    Article Snippet: The PURExpress probing experiments without ribosomes were carried out in a similar fashion using the PURExpress ΔRibosome Kit (New England Biolabs), with each 10 μL reaction containing 4 μL kit Solution A, 1.2 μL Factor Mix, and 3.24 picomoles of mRNA.

    Techniques: Purification, Fluorescence, Construct, Standard Deviation