vitro protein synthesis kit  (New England Biolabs)


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  • 99
    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:
    Protein Expression and Purification Kits
    Score:
    85
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    Structured Review

    New England Biolabs vitro protein synthesis kit
    PURExpress In Vitro Protein Synthesis Kit
    PURExpress In Vitro Protein Synthesis Kit 100 rxns
    https://www.bioz.com/result/vitro protein synthesis kit/product/New England Biolabs
    Average 99 stars, based on 19 article reviews
    Price from $9.99 to $1999.99
    vitro protein synthesis kit - by Bioz Stars, 2019-10
    99/100 stars

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    Related Articles

    Clone Assay:

    Article Title: Sequence specificity is obtained from the majority of modular C2H2 zinc-finger arrays
    Article Snippet: ZFAs were cloned as SacI–BamHI fragments into pTH5325, a modified T7-driven GST expression vector (see Supplementary Document of the Supplementary Data ). .. Briefly, we used 150 ng of plasmid DNA in a 25 μl in vitro transcription/translation reaction using a PURExpress In Vitro Protein Synthesis Kit (New England BioLabs) supplemented with RNase inhibitor and 50 μM zinc acetate.

    Article Title: The Epstein-Barr virus B-ZIP protein Zta recognizes specific DNA sequences containing 5-methylcytosine and 5-hydroxymethylcytosine
    Article Snippet: Paragraph title: Cloning and expression of B-ZIP DNA binding domains ... Proteins were expressed using the PURExpress In vitro protein synthesis kit (NEB) according to the manufacturer’s protocol in a 25μL reaction volume containing 180 ng of plasmid .

    Centrifugation:

    Article Title: Redesign of extensive protein–DNA interfaces of meganucleases using iterative cycles of in vitro compartmentalization
    Article Snippet: Five hundred microliters of the upper phase was used to emulsify 30 µL of the in vitro protein synthesis mixture [25 μL of PURExpress (New England Biolabs), 20 units of RNase inhibitor, 1 mg/mL BSA, and a DNA library] by constant stirring at 1,400 r.p.m. for 3.5 min on ice ( and ). .. Eight nanograms of a DNA library were added in the aqueous phase in the first round of IVC and reduced to 1 ng or 0.5 ng in the second or third round, respectively.

    Luciferase:

    Article Title: Escherichia coli ItaT is a type II toxin that inhibits translation by acetylating isoleucyl-tRNAIle
    Article Snippet: Protein concentration was determined by Bradford method. .. The in vitro coupled transcription-translation reactions were carried out by PURExpress kit (NEB, USA) using DNA templates encoding the dihydrofolate reductase (DHFR) (NEB, USA) or the firefly luciferase (Promega, USA). .. Before template addition, translation reactions were incubated for 10 min at 37°C in the presence of 0.2 mM of cold acetyl coenzyme A (AcCoA) or radiolabeled [acetyl-1-14 C]-AcCoA (0.2 μCi, 60 mCi/mmol), Perkin Elmer, USA), with and without 2 μM ItaT.

    Synthesized:

    Article Title: Escherichia coli ItaT is a type II toxin that inhibits translation by acetylating isoleucyl-tRNAIle
    Article Snippet: The in vitro coupled transcription-translation reactions were carried out by PURExpress kit (NEB, USA) using DNA templates encoding the dihydrofolate reductase (DHFR) (NEB, USA) or the firefly luciferase (Promega, USA). .. Before template addition, translation reactions were incubated for 10 min at 37°C in the presence of 0.2 mM of cold acetyl coenzyme A (AcCoA) or radiolabeled [acetyl-1-14 C]-AcCoA (0.2 μCi, 60 mCi/mmol), Perkin Elmer, USA), with and without 2 μM ItaT.

    Article Title: The GET System Inserts the Tail-Anchored Protein, SYP72, into Endoplasmic Reticulum Membranes
    Article Snippet: Propidium iodide (P4864) was obtained from Sigma-Aldrich. .. 35 S-SYP72 substrates were synthesized using the T7-coupled PURExpress In Vitro Protein Synthesis Kit (New England Biolabs) in the presence of 35 S-Met and 25 μ m Get3. .. The Get3-SYP72 complex was purified on Streptactin sepharose and eluted with buffer containing 5 m m biotin.

    Article Title: Sequestration from protease adaptor confers differential stability to protease substrate
    Article Snippet: Purified proteins were used as 0.5 μM ClpS, 0.2 μM PhoP and 0.5 μM Mdh (Roche). .. In vitro synthesized ClpA, ClpP and MgtC-FLAG proteins were produced using the cell-free PURExpress in vitro protein synthesis system (NEB) at 37°C for 3 h. Then, in vitro synthesized proteins were diluted 10 fold in 1×TBS (Tris-buffered saline) buffer containing 10 % glycerol. .. 5 μl In vitro synthesized ClpA, ClpP and MgtC-FLAG were used for each reaction.

    Article Title: DNA binding strength increases the processivity and activity of a Y-Family DNA polymerase
    Article Snippet: The Sso7d gene was synthesized by IDT based on the published amino acid sequence , . .. The high copy plasmid DHFR (dihydrofolatereductase) supplied with PURExpress In Vitro Protein Synthesis Kit (New England Biolabs, Inc. (NEB)) was linearized by digestion with Nde I and BamH I restriction enzymes.

    Autoradiography:

    Article Title: Escherichia coli ItaT is a type II toxin that inhibits translation by acetylating isoleucyl-tRNAIle
    Article Snippet: The in vitro coupled transcription-translation reactions were carried out by PURExpress kit (NEB, USA) using DNA templates encoding the dihydrofolate reductase (DHFR) (NEB, USA) or the firefly luciferase (Promega, USA). .. The enzymatic activity of in vitro synthesized luciferase was measured using the Steady-Glo Luciferase Assay System (Promega, USA).

    Construct:

    Article Title: The Epstein-Barr virus B-ZIP protein Zta recognizes specific DNA sequences containing 5-methylcytosine and 5-hydroxymethylcytosine
    Article Snippet: The DBD of cFOS was obtained as a construct cloned into a pT5 expression plasmid . .. Proteins were expressed using the PURExpress In vitro protein synthesis kit (NEB) according to the manufacturer’s protocol in a 25μL reaction volume containing 180 ng of plasmid .

    Article Title: Calmodulin Binding to Dfi1p Promotes Invasiveness of Candida albicans
    Article Snippet: All primers contained linker sequences needed for the second round of PCR, which was performed following the manufacturer's recommendations and introduced a c-terminal STREP tag to the constructs (Qiagen EasyXpress Linear Template Kit). .. PCR reactions were gel purified using Qiaex II resin and 250 ng DNA used in each translation reaction (PURExpress In vitro Protein Synthesis Kit, New England Biolabs E6800S).

    Article Title: Redesign of extensive protein–DNA interfaces of meganucleases using iterative cycles of in vitro compartmentalization
    Article Snippet: PCR products were purified by extraction from agarose gels and assembled in the subsequent round of PCR with a sequence containing two copies of target sites for variant endonucleases to construct DNA libraries ( ). .. Five hundred microliters of the upper phase was used to emulsify 30 µL of the in vitro protein synthesis mixture [25 μL of PURExpress (New England Biolabs), 20 units of RNase inhibitor, 1 mg/mL BSA, and a DNA library] by constant stirring at 1,400 r.p.m. for 3.5 min on ice ( and ).

    SDS-Gel:

    Article Title: The ubiquitin ligase LIN41/TRIM71 targets p53 to antagonize cell death and differentiation pathways during stem cell differentiation
    Article Snippet: LIN41 protein for the in vitro interaction assays was generated using the PURExpress In Vitro Protein Synthesis Kit (New England Biolabs, Ipswich, MA, USA). .. An amount of 500 ng of the purified DNA in 10 mM Tris-HCl pH 8 was added to the in vitro expression reaction containing 10 μ l solution A, 7.5 μ l solution B, 20 units RNase inhibitor, ad 25 μ l with nuclease-free H2 O.

    Microarray:

    Article Title: Sequence specificity is obtained from the majority of modular C2H2 zinc-finger arrays
    Article Snippet: Paragraph title: Protein-binding microarray experiments ... Briefly, we used 150 ng of plasmid DNA in a 25 μl in vitro transcription/translation reaction using a PURExpress In Vitro Protein Synthesis Kit (New England BioLabs) supplemented with RNase inhibitor and 50 μM zinc acetate.

    Incubation:

    Article Title: Sequence specificity is obtained from the majority of modular C2H2 zinc-finger arrays
    Article Snippet: Briefly, we used 150 ng of plasmid DNA in a 25 μl in vitro transcription/translation reaction using a PURExpress In Vitro Protein Synthesis Kit (New England BioLabs) supplemented with RNase inhibitor and 50 μM zinc acetate. .. Briefly, we used 150 ng of plasmid DNA in a 25 μl in vitro transcription/translation reaction using a PURExpress In Vitro Protein Synthesis Kit (New England BioLabs) supplemented with RNase inhibitor and 50 μM zinc acetate.

    Article Title: Context influences on TALE–DNA binding revealed by quantitative profiling
    Article Snippet: Proteins were expressed using the PURExpress In Vitro Transcription and Translation Kit (New England Biolabs). .. Proteins were expressed using the PURExpress In Vitro Transcription and Translation Kit (New England Biolabs).

    Article Title: Ribosome Rescue Inhibitors Kill Actively Growing and Nonreplicating Persister Mycobacterium tuberculosis Cells
    Article Snippet: Assays were set up using the PURExpress in vitro protein synthesis kit (NEB, Ipswich, MA) according to the manufacturer’s protocols. .. The reactions were performed with no DNA template, a nonstop DHFR template, the full length DHFR gene, full length DHFR gene with 0 bases after the stop codon, or full length DHFR gene with 33 bases after the stop codon., KKL-2098 (final concentration of 1 μM) was added to a mixture of assay components, and the samples were incubated at room temperature for 1 h. Samples were placed on ice, irradiated with 312 nm UV light for 10 min, and used to set up click conjugation assays in the presence of KKL-2107 (final concentration of 0.5 mM).

    Article Title: The RNA Helicase DeaD Stimulates ExsA Translation To Promote Expression of the Pseudomonas aeruginosa Type III Secretion System
    Article Snippet: A PURExpress in vitro protein synthesis system (New England BioLabs, Ipswich, MA) was used to measure DeaD activity as follows. .. Reaction mixtures (17 μl) contained 1 μl (10.2 μCi) of [35 S]methionine (Perkin-Elmer, Waltham, MA), 5 μg of DNA template, 0.5 μl (20 U) of RNAseOUT (Life Technologies, Grand Island, NY), and purified DeaDHis or DeaDHis E168A as indicated.

    Article Title: Regulation of Hfq by the RNA CrcZ in Pseudomonas aeruginosa Carbon Catabolite Repression
    Article Snippet: In vitro translation was performed with the PURExpress in vitro protein synthesis kit (New England BioLabs). .. For competition CrcZ (5 and 10 pmol) or RsmZ (10 pmol) RNA were added.

    Article Title: Post-Translational Sortase-Mediated Attachment of High-Strength Force Spectroscopy Handles
    Article Snippet: All protein solutions were concentrated using Amicon centrifugal filter units (10k MWCO, Merck KGaA, Darmstadt, Germany), followed by buffer exchange to Ca-TBS buffer (25 mM Tris, pH 7.2, 75 mM NaCl, and 1 mM CaCl2 ) using polyacrylamide spin desalting columns. .. For cell-free expression, 25 μM reactions of PURExpress In Vitro Protein Synthesis Kit (New England Biolabs, Ipswich, Massachusetts) were incubated for 2 h at 37 °C, containing 300 ng plasmid DNA coding for the POIs. .. In case of MGGG-His-Doc, the N-terminal methionine cleavage in E. coli was sufficient for the preparation of GGG-His-Doc, so that no additional protease digestion was necessary.

    Article Title: Redesign of extensive protein–DNA interfaces of meganucleases using iterative cycles of in vitro compartmentalization
    Article Snippet: The oil–surfactant mixture [2% (vol/vol) ABIL EM 90 (gift from Evonik Industries AG Personal Care) and 0.05% Triton X-100 in light mineral oil] was thoroughly mixed with the saturation buffer [100 mM potassium glutamate (pH 7.5), 10 mM magnesium acetate (pH 7.5), 1 mM DTT, and 5 mg/mL BSA], incubated at 37 °C for 20 min, and centrifuged at 16,000 × g for 15 min at 4 °C. .. Five hundred microliters of the upper phase was used to emulsify 30 µL of the in vitro protein synthesis mixture [25 μL of PURExpress (New England Biolabs), 20 units of RNase inhibitor, 1 mg/mL BSA, and a DNA library] by constant stirring at 1,400 r.p.m. for 3.5 min on ice ( and ).

    Amplification:

    Article Title: The stress-related, rhizobial small RNA RcsR1 destabilizes the autoinducer synthase encoding mRNA sinI in Sinorhizobium meliloti
    Article Snippet: A sinI'-egfp in vitro transcript was generated using primers sinIivfwd and egfpivrev and plasmid pLK64 for PCR amplification of the template and subsequent in vitro transcription with the MEGAshortscript T7 kit (Ambion) (see Tables S4 and S5). .. Five μg of sinI'-egfp transcript were used for in vitro translation carried out by the PURExpress In Vitro Protein Synthesis kit (New England Biolabs) following the manufacturer's instructions.

    Article Title: Calmodulin Binding to Dfi1p Promotes Invasiveness of Candida albicans
    Article Snippet: Two-round PCR protocols (Qiagen) were used to generate DNA fragments for in vitro translation reactions. pGEX-GST-ctDFI1 constructs were amplified using primers PZ326 and either PZ352 (GST-STREP control) or PZ330 (GST-ctDFI1-STREP and all mutants). .. PCR reactions were gel purified using Qiaex II resin and 250 ng DNA used in each translation reaction (PURExpress In vitro Protein Synthesis Kit, New England Biolabs E6800S).

    Activity Assay:

    Article Title: Escherichia coli ItaT is a type II toxin that inhibits translation by acetylating isoleucyl-tRNAIle
    Article Snippet: The in vitro coupled transcription-translation reactions were carried out by PURExpress kit (NEB, USA) using DNA templates encoding the dihydrofolate reductase (DHFR) (NEB, USA) or the firefly luciferase (Promega, USA). .. Before template addition, translation reactions were incubated for 10 min at 37°C in the presence of 0.2 mM of cold acetyl coenzyme A (AcCoA) or radiolabeled [acetyl-1-14 C]-AcCoA (0.2 μCi, 60 mCi/mmol), Perkin Elmer, USA), with and without 2 μM ItaT.

    Article Title: The RNA Helicase DeaD Stimulates ExsA Translation To Promote Expression of the Pseudomonas aeruginosa Type III Secretion System
    Article Snippet: The protein concentrations were determined using a DC protein assay (Bio-Rad Laboratories, Hercules, CA). .. A PURExpress in vitro protein synthesis system (New England BioLabs, Ipswich, MA) was used to measure DeaD activity as follows. .. DNA templates for ExsA, Vfr, and LcrF were PCR amplified from pET23b-exsA1, pET23b-Vfr, and pET23b-LcrF using the primers 127176830 and 124849942.

    Article Title: The ubiquitin ligase LIN41/TRIM71 targets p53 to antagonize cell death and differentiation pathways during stem cell differentiation
    Article Snippet: LIN41 protein for the in vitro interaction assays was generated using the PURExpress In Vitro Protein Synthesis Kit (New England Biolabs, Ipswich, MA, USA). .. For creating a Lin41 template containing the T7 promoter, ribosome-binding site and T7 terminator required for a one-step transcription and translation, the DHFR gene from the positive control plasmid of the kit was replaced with Flag-tagged full-length Lin41 cDNA.

    Article Title: DNA binding strength increases the processivity and activity of a Y-Family DNA polymerase
    Article Snippet: Previous work has shown that this tag does not significantly alter the polymerization activity of Y-family pols . .. The high copy plasmid DHFR (dihydrofolatereductase) supplied with PURExpress In Vitro Protein Synthesis Kit (New England Biolabs, Inc. (NEB)) was linearized by digestion with Nde I and BamH I restriction enzymes.

    Expressing:

    Article Title: Sequence specificity is obtained from the majority of modular C2H2 zinc-finger arrays
    Article Snippet: ZFAs were cloned as SacI–BamHI fragments into pTH5325, a modified T7-driven GST expression vector (see Supplementary Document of the Supplementary Data ). .. Briefly, we used 150 ng of plasmid DNA in a 25 μl in vitro transcription/translation reaction using a PURExpress In Vitro Protein Synthesis Kit (New England BioLabs) supplemented with RNase inhibitor and 50 μM zinc acetate.

    Article Title: The Epstein-Barr virus B-ZIP protein Zta recognizes specific DNA sequences containing 5-methylcytosine and 5-hydroxymethylcytosine
    Article Snippet: Paragraph title: Cloning and expression of B-ZIP DNA binding domains ... Proteins were expressed using the PURExpress In vitro protein synthesis kit (NEB) according to the manufacturer’s protocol in a 25μL reaction volume containing 180 ng of plasmid .

    Article Title: The ubiquitin ligase LIN41/TRIM71 targets p53 to antagonize cell death and differentiation pathways during stem cell differentiation
    Article Snippet: LIN41 protein for the in vitro interaction assays was generated using the PURExpress In Vitro Protein Synthesis Kit (New England Biolabs, Ipswich, MA, USA). .. To remove residual RNaseA activity from plasmid DNA preparation, ethanol precipitation was carried out.

    Article Title: Post-Translational Sortase-Mediated Attachment of High-Strength Force Spectroscopy Handles
    Article Snippet: All protein solutions were concentrated using Amicon centrifugal filter units (10k MWCO, Merck KGaA, Darmstadt, Germany), followed by buffer exchange to Ca-TBS buffer (25 mM Tris, pH 7.2, 75 mM NaCl, and 1 mM CaCl2 ) using polyacrylamide spin desalting columns. .. For cell-free expression, 25 μM reactions of PURExpress In Vitro Protein Synthesis Kit (New England Biolabs, Ipswich, Massachusetts) were incubated for 2 h at 37 °C, containing 300 ng plasmid DNA coding for the POIs. .. In case of MGGG-His-Doc, the N-terminal methionine cleavage in E. coli was sufficient for the preparation of GGG-His-Doc, so that no additional protease digestion was necessary.

    Modification:

    Article Title: Sequence specificity is obtained from the majority of modular C2H2 zinc-finger arrays
    Article Snippet: ZFAs were cloned as SacI–BamHI fragments into pTH5325, a modified T7-driven GST expression vector (see Supplementary Document of the Supplementary Data ). .. Briefly, we used 150 ng of plasmid DNA in a 25 μl in vitro transcription/translation reaction using a PURExpress In Vitro Protein Synthesis Kit (New England BioLabs) supplemented with RNase inhibitor and 50 μM zinc acetate.

    Article Title: The Epstein-Barr virus B-ZIP protein Zta recognizes specific DNA sequences containing 5-methylcytosine and 5-hydroxymethylcytosine
    Article Snippet: Zta was obtained as a GST construct cloned into a modified pDEST15 MAGIC vector (N-terminal GST) vector . .. Proteins were expressed using the PURExpress In vitro protein synthesis kit (NEB) according to the manufacturer’s protocol in a 25μL reaction volume containing 180 ng of plasmid .

    Western Blot:

    Article Title: The stress-related, rhizobial small RNA RcsR1 destabilizes the autoinducer synthase encoding mRNA sinI in Sinorhizobium meliloti
    Article Snippet: Five μg of sinI'-egfp transcript were used for in vitro translation carried out by the PURExpress In Vitro Protein Synthesis kit (New England Biolabs) following the manufacturer's instructions. .. If applied, equal amounts of RcsR1 were incubated with sinI'-egfp 20 min at 30 °C prior to in vitro translation.

    Article Title: Sequestration from protease adaptor confers differential stability to protease substrate
    Article Snippet: In vitro synthesized ClpA, ClpP and MgtC-FLAG proteins were produced using the cell-free PURExpress in vitro protein synthesis system (NEB) at 37°C for 3 h. Then, in vitro synthesized proteins were diluted 10 fold in 1×TBS (Tris-buffered saline) buffer containing 10 % glycerol. .. In vitro synthesized ClpA, ClpP and MgtC-FLAG proteins were produced using the cell-free PURExpress in vitro protein synthesis system (NEB) at 37°C for 3 h. Then, in vitro synthesized proteins were diluted 10 fold in 1×TBS (Tris-buffered saline) buffer containing 10 % glycerol.

    Article Title: Calmodulin Binding to Dfi1p Promotes Invasiveness of Candida albicans
    Article Snippet: PCR reactions were gel purified using Qiaex II resin and 250 ng DNA used in each translation reaction (PURExpress In vitro Protein Synthesis Kit, New England Biolabs E6800S). .. PCR reactions were gel purified using Qiaex II resin and 250 ng DNA used in each translation reaction (PURExpress In vitro Protein Synthesis Kit, New England Biolabs E6800S).

    Article Title: Regulation of Hfq by the RNA CrcZ in Pseudomonas aeruginosa Carbon Catabolite Repression
    Article Snippet: In vitro translation was performed with the PURExpress in vitro protein synthesis kit (New England BioLabs). .. For competition CrcZ (5 and 10 pmol) or RsmZ (10 pmol) RNA were added.

    Conjugation Assay:

    Article Title: Ribosome Rescue Inhibitors Kill Actively Growing and Nonreplicating Persister Mycobacterium tuberculosis Cells
    Article Snippet: Paragraph title: In Vitro Photolabeling and Click Conjugation ... Assays were set up using the PURExpress in vitro protein synthesis kit (NEB, Ipswich, MA) according to the manufacturer’s protocols.

    Buffer Exchange:

    Article Title: Post-Translational Sortase-Mediated Attachment of High-Strength Force Spectroscopy Handles
    Article Snippet: All protein solutions were concentrated using Amicon centrifugal filter units (10k MWCO, Merck KGaA, Darmstadt, Germany), followed by buffer exchange to Ca-TBS buffer (25 mM Tris, pH 7.2, 75 mM NaCl, and 1 mM CaCl2 ) using polyacrylamide spin desalting columns. .. For cell-free expression, 25 μM reactions of PURExpress In Vitro Protein Synthesis Kit (New England Biolabs, Ipswich, Massachusetts) were incubated for 2 h at 37 °C, containing 300 ng plasmid DNA coding for the POIs.

    Generated:

    Article Title: The stress-related, rhizobial small RNA RcsR1 destabilizes the autoinducer synthase encoding mRNA sinI in Sinorhizobium meliloti
    Article Snippet: A sinI'-egfp in vitro transcript was generated using primers sinIivfwd and egfpivrev and plasmid pLK64 for PCR amplification of the template and subsequent in vitro transcription with the MEGAshortscript T7 kit (Ambion) (see Tables S4 and S5). .. Five μg of sinI'-egfp transcript were used for in vitro translation carried out by the PURExpress In Vitro Protein Synthesis kit (New England Biolabs) following the manufacturer's instructions.

    Article Title: The ubiquitin ligase LIN41/TRIM71 targets p53 to antagonize cell death and differentiation pathways during stem cell differentiation
    Article Snippet: Beads were recovered by centrifugation at 2500 × g for 2 min at 4 °C, washed three times with STE buffer and resuspended in 30 μ l 2 × SDS sample buffer for elution by boiling. .. LIN41 protein for the in vitro interaction assays was generated using the PURExpress In Vitro Protein Synthesis Kit (New England Biolabs, Ipswich, MA, USA). .. For creating a Lin41 template containing the T7 promoter, ribosome-binding site and T7 terminator required for a one-step transcription and translation, the DHFR gene from the positive control plasmid of the kit was replaced with Flag-tagged full-length Lin41 cDNA.

    Article Title: Redesign of extensive protein–DNA interfaces of meganucleases using iterative cycles of in vitro compartmentalization
    Article Snippet: Five hundred microliters of the upper phase was used to emulsify 30 µL of the in vitro protein synthesis mixture [25 μL of PURExpress (New England Biolabs), 20 units of RNase inhibitor, 1 mg/mL BSA, and a DNA library] by constant stirring at 1,400 r.p.m. for 3.5 min on ice ( and ). .. The emulsion was incubated at 30 °C for 4 h in the first round of IVC and then heated at 75 °C for 15 min. After an addition of 170 µL of 10 mM Tris⋅HCl (pH 8.0), emulsified droplets were collected by centrifugation at 16,000 × g for 15 min at 4 °C and broken by an addition of phenol/chloroform/isoamyl alcohol.

    Protein Concentration:

    Article Title: The ubiquitin ligase LIN41/TRIM71 targets p53 to antagonize cell death and differentiation pathways during stem cell differentiation
    Article Snippet: LIN41 protein for the in vitro interaction assays was generated using the PURExpress In Vitro Protein Synthesis Kit (New England Biolabs, Ipswich, MA, USA). .. An amount of 500 ng of the purified DNA in 10 mM Tris-HCl pH 8 was added to the in vitro expression reaction containing 10 μ l solution A, 7.5 μ l solution B, 20 units RNase inhibitor, ad 25 μ l with nuclease-free H2 O.

    Sequencing:

    Article Title: The stress-related, rhizobial small RNA RcsR1 destabilizes the autoinducer synthase encoding mRNA sinI in Sinorhizobium meliloti
    Article Snippet: This transcript contains the 5' UTR of sinI including the Shine-Dalgarno sequence and the first 9 codons translationally fused to full-length egfp . .. Five μg of sinI'-egfp transcript were used for in vitro translation carried out by the PURExpress In Vitro Protein Synthesis kit (New England Biolabs) following the manufacturer's instructions.

    Article Title: DNA binding strength increases the processivity and activity of a Y-Family DNA polymerase
    Article Snippet: The Sso7d gene was synthesized by IDT based on the published amino acid sequence , . .. The high copy plasmid DHFR (dihydrofolatereductase) supplied with PURExpress In Vitro Protein Synthesis Kit (New England Biolabs, Inc. (NEB)) was linearized by digestion with Nde I and BamH I restriction enzymes.

    Article Title: Redesign of extensive protein–DNA interfaces of meganucleases using iterative cycles of in vitro compartmentalization
    Article Snippet: PCR products were purified by extraction from agarose gels and assembled in the subsequent round of PCR with a sequence containing two copies of target sites for variant endonucleases to construct DNA libraries ( ). .. Five hundred microliters of the upper phase was used to emulsify 30 µL of the in vitro protein synthesis mixture [25 μL of PURExpress (New England Biolabs), 20 units of RNase inhibitor, 1 mg/mL BSA, and a DNA library] by constant stirring at 1,400 r.p.m. for 3.5 min on ice ( and ).

    Sonication:

    Article Title: Post-Translational Sortase-Mediated Attachment of High-Strength Force Spectroscopy Handles
    Article Snippet: After pelleting, the cells were lysed by sonication and then centrifuged at 4 °C, 39 000 rcf for 60 min. .. For cell-free expression, 25 μM reactions of PURExpress In Vitro Protein Synthesis Kit (New England Biolabs, Ipswich, Massachusetts) were incubated for 2 h at 37 °C, containing 300 ng plasmid DNA coding for the POIs.

    Recombinant:

    Article Title: The GET System Inserts the Tail-Anchored Protein, SYP72, into Endoplasmic Reticulum Membranes
    Article Snippet: 35 S-SYP72 substrates were synthesized using the T7-coupled PURExpress In Vitro Protein Synthesis Kit (New England Biolabs) in the presence of 35 S-Met and 25 μ m Get3. .. The Get3-SYP72 complex was purified on Streptactin sepharose and eluted with buffer containing 5 m m biotin.

    Fluorescence:

    Article Title: Ribosome Rescue Inhibitors Kill Actively Growing and Nonreplicating Persister Mycobacterium tuberculosis Cells
    Article Snippet: Assays were set up using the PURExpress in vitro protein synthesis kit (NEB, Ipswich, MA) according to the manufacturer’s protocols. .. Assays were set up using the PURExpress in vitro protein synthesis kit (NEB, Ipswich, MA) according to the manufacturer’s protocols.

    Electron Microscopy:

    Article Title: Redesign of extensive protein–DNA interfaces of meganucleases using iterative cycles of in vitro compartmentalization
    Article Snippet: The oil–surfactant mixture [2% (vol/vol) ABIL EM 90 (gift from Evonik Industries AG Personal Care) and 0.05% Triton X-100 in light mineral oil] was thoroughly mixed with the saturation buffer [100 mM potassium glutamate (pH 7.5), 10 mM magnesium acetate (pH 7.5), 1 mM DTT, and 5 mg/mL BSA], incubated at 37 °C for 20 min, and centrifuged at 16,000 × g for 15 min at 4 °C. .. Five hundred microliters of the upper phase was used to emulsify 30 µL of the in vitro protein synthesis mixture [25 μL of PURExpress (New England Biolabs), 20 units of RNase inhibitor, 1 mg/mL BSA, and a DNA library] by constant stirring at 1,400 r.p.m. for 3.5 min on ice ( and ).

    Mutagenesis:

    Article Title: YaeJ is a novel ribosome-associated protein in Escherichia coli that can hydrolyze peptidyl-tRNA on stalled ribosomes
    Article Snippet: PURExpress (New England Biolabs) based on PUREsystem technology and FluroTect GreenLys in vitro translation labeling system (Promega) were used for in vitro translation experiments. .. For example, to prepare wild-type mRNA of the crp gene, stop primers were used for the first PCR, followed by universal primer and stop (R) primer for the second PCR ( ).

    Article Title: Translational Repression of the RpoS Antiadapter IraD by CsrA Is Mediated via Translational Coupling to a Short Upstream Open Reading Frame
    Article Snippet: In vitro coupled transcription-translation assays using PURExpress (New England Biolabs) followed a published procedure ( ). .. Plasmid pT7-P1iraD '- 'lacZ contains a T7 promoter driving transcription of the translational fusion from the natural iraD P1 transcription start site ( ).

    Article Title: Redesign of extensive protein–DNA interfaces of meganucleases using iterative cycles of in vitro compartmentalization
    Article Snippet: Two to three rounds of IVC were conducted following each round of site-directed saturation mutagenesis to enrich active variant genes. .. Five hundred microliters of the upper phase was used to emulsify 30 µL of the in vitro protein synthesis mixture [25 μL of PURExpress (New England Biolabs), 20 units of RNase inhibitor, 1 mg/mL BSA, and a DNA library] by constant stirring at 1,400 r.p.m. for 3.5 min on ice ( and ).

    Labeling:

    Article Title: YaeJ is a novel ribosome-associated protein in Escherichia coli that can hydrolyze peptidyl-tRNA on stalled ribosomes
    Article Snippet: The absence of the YaeJ protein and tmRNA was confirmed by western blotting or PCR. .. PURExpress (New England Biolabs) based on PUREsystem technology and FluroTect GreenLys in vitro translation labeling system (Promega) were used for in vitro translation experiments. .. Template DNA fragments were prepared using a two-step PCR reaction.

    Purification:

    Article Title: The GET System Inserts the Tail-Anchored Protein, SYP72, into Endoplasmic Reticulum Membranes
    Article Snippet: 35 S-SYP72 substrates were synthesized using the T7-coupled PURExpress In Vitro Protein Synthesis Kit (New England Biolabs) in the presence of 35 S-Met and 25 μ m Get3. .. The Get3-SYP72 complex was purified on Streptactin sepharose and eluted with buffer containing 5 m m biotin.

    Article Title: Sequestration from protease adaptor confers differential stability to protease substrate
    Article Snippet: Purified proteins were used as 0.5 μM ClpS, 0.2 μM PhoP and 0.5 μM Mdh (Roche). .. In vitro synthesized ClpA, ClpP and MgtC-FLAG proteins were produced using the cell-free PURExpress in vitro protein synthesis system (NEB) at 37°C for 3 h. Then, in vitro synthesized proteins were diluted 10 fold in 1×TBS (Tris-buffered saline) buffer containing 10 % glycerol.

    Article Title: The RNA Helicase DeaD Stimulates ExsA Translation To Promote Expression of the Pseudomonas aeruginosa Type III Secretion System
    Article Snippet: A PURExpress in vitro protein synthesis system (New England BioLabs, Ipswich, MA) was used to measure DeaD activity as follows. .. DNA templates for ExsA, Vfr, and LcrF were PCR amplified from pET23b-exsA1, pET23b-Vfr, and pET23b-LcrF using the primers 127176830 and 124849942.

    Article Title: Calmodulin Binding to Dfi1p Promotes Invasiveness of Candida albicans
    Article Snippet: All primers contained linker sequences needed for the second round of PCR, which was performed following the manufacturer's recommendations and introduced a c-terminal STREP tag to the constructs (Qiagen EasyXpress Linear Template Kit). .. PCR reactions were gel purified using Qiaex II resin and 250 ng DNA used in each translation reaction (PURExpress In vitro Protein Synthesis Kit, New England Biolabs E6800S). .. Translation reactions were performed with 20 U of murine RNAse inhibitor (NEB M0314S) according to the manufacturer's instructions.

    Article Title: Regulation of Hfq by the RNA CrcZ in Pseudomonas aeruginosa Carbon Catabolite Repression
    Article Snippet: In vitro translation was performed with the PURExpress in vitro protein synthesis kit (New England BioLabs). .. 5 pmol in vitro transcribed amiE Flag mRNA was used in a 12.5 µl reaction.

    Article Title: The ubiquitin ligase LIN41/TRIM71 targets p53 to antagonize cell death and differentiation pathways during stem cell differentiation
    Article Snippet: LIN41 protein for the in vitro interaction assays was generated using the PURExpress In Vitro Protein Synthesis Kit (New England Biolabs, Ipswich, MA, USA). .. To remove residual RNaseA activity from plasmid DNA preparation, ethanol precipitation was carried out.

    Article Title: Post-Translational Sortase-Mediated Attachment of High-Strength Force Spectroscopy Handles
    Article Snippet: Paragraph title: Protein Expression and Purification ... For cell-free expression, 25 μM reactions of PURExpress In Vitro Protein Synthesis Kit (New England Biolabs, Ipswich, Massachusetts) were incubated for 2 h at 37 °C, containing 300 ng plasmid DNA coding for the POIs.

    Article Title: Redesign of extensive protein–DNA interfaces of meganucleases using iterative cycles of in vitro compartmentalization
    Article Snippet: A successfully assembled DNA fragment was again purified by gel extraction ( ). .. Five hundred microliters of the upper phase was used to emulsify 30 µL of the in vitro protein synthesis mixture [25 μL of PURExpress (New England Biolabs), 20 units of RNase inhibitor, 1 mg/mL BSA, and a DNA library] by constant stirring at 1,400 r.p.m. for 3.5 min on ice ( and ).

    Polymerase Chain Reaction:

    Article Title: YaeJ is a novel ribosome-associated protein in Escherichia coli that can hydrolyze peptidyl-tRNA on stalled ribosomes
    Article Snippet: PURExpress (New England Biolabs) based on PUREsystem technology and FluroTect GreenLys in vitro translation labeling system (Promega) were used for in vitro translation experiments. .. Template DNA fragments were prepared using a two-step PCR reaction.

    Article Title: The stress-related, rhizobial small RNA RcsR1 destabilizes the autoinducer synthase encoding mRNA sinI in Sinorhizobium meliloti
    Article Snippet: A sinI'-egfp in vitro transcript was generated using primers sinIivfwd and egfpivrev and plasmid pLK64 for PCR amplification of the template and subsequent in vitro transcription with the MEGAshortscript T7 kit (Ambion) (see Tables S4 and S5). .. Five μg of sinI'-egfp transcript were used for in vitro translation carried out by the PURExpress In Vitro Protein Synthesis kit (New England Biolabs) following the manufacturer's instructions.

    Article Title: Escherichia coli ItaT is a type II toxin that inhibits translation by acetylating isoleucyl-tRNAIle
    Article Snippet: The in vitro coupled transcription-translation reactions were carried out by PURExpress kit (NEB, USA) using DNA templates encoding the dihydrofolate reductase (DHFR) (NEB, USA) or the firefly luciferase (Promega, USA). .. The products of DHFR in vitro translation reactions were analyzed by SDS-PAGE or by 12% denaturing acid-urea PAGE followed by autoradiography ( ).

    Article Title: Calmodulin Binding to Dfi1p Promotes Invasiveness of Candida albicans
    Article Snippet: All primers contained linker sequences needed for the second round of PCR, which was performed following the manufacturer's recommendations and introduced a c-terminal STREP tag to the constructs (Qiagen EasyXpress Linear Template Kit). .. PCR reactions were gel purified using Qiaex II resin and 250 ng DNA used in each translation reaction (PURExpress In vitro Protein Synthesis Kit, New England Biolabs E6800S). .. Translation reactions were performed with 20 U of murine RNAse inhibitor (NEB M0314S) according to the manufacturer's instructions.

    Article Title: DNA binding strength increases the processivity and activity of a Y-Family DNA polymerase
    Article Snippet: The overlapping fragments of Dbh and Sso7d were PCR-amplified by oligonucleotides Fdbh-linker, Rdbh-dhfr, Fsso-dhfr and Rsso-linker, respectively (Table ). .. The high copy plasmid DHFR (dihydrofolatereductase) supplied with PURExpress In Vitro Protein Synthesis Kit (New England Biolabs, Inc. (NEB)) was linearized by digestion with Nde I and BamH I restriction enzymes.

    Article Title: Redesign of extensive protein–DNA interfaces of meganucleases using iterative cycles of in vitro compartmentalization
    Article Snippet: PCR products were purified by extraction from agarose gels and assembled in the subsequent round of PCR with a sequence containing two copies of target sites for variant endonucleases to construct DNA libraries ( ). .. Five hundred microliters of the upper phase was used to emulsify 30 µL of the in vitro protein synthesis mixture [25 μL of PURExpress (New England Biolabs), 20 units of RNase inhibitor, 1 mg/mL BSA, and a DNA library] by constant stirring at 1,400 r.p.m. for 3.5 min on ice ( and ).

    Polyacrylamide Gel Electrophoresis:

    Article Title: Escherichia coli ItaT is a type II toxin that inhibits translation by acetylating isoleucyl-tRNAIle
    Article Snippet: The in vitro coupled transcription-translation reactions were carried out by PURExpress kit (NEB, USA) using DNA templates encoding the dihydrofolate reductase (DHFR) (NEB, USA) or the firefly luciferase (Promega, USA). .. The enzymatic activity of in vitro synthesized luciferase was measured using the Steady-Glo Luciferase Assay System (Promega, USA).

    Staining:

    Article Title: Ribosome Rescue Inhibitors Kill Actively Growing and Nonreplicating Persister Mycobacterium tuberculosis Cells
    Article Snippet: Assays were set up using the PURExpress in vitro protein synthesis kit (NEB, Ipswich, MA) according to the manufacturer’s protocols. .. Assays were set up using the PURExpress in vitro protein synthesis kit (NEB, Ipswich, MA) according to the manufacturer’s protocols.

    Article Title: Sequestration from protease adaptor confers differential stability to protease substrate
    Article Snippet: In vitro synthesized ClpA, ClpP and MgtC-FLAG proteins were produced using the cell-free PURExpress in vitro protein synthesis system (NEB) at 37°C for 3 h. Then, in vitro synthesized proteins were diluted 10 fold in 1×TBS (Tris-buffered saline) buffer containing 10 % glycerol. .. In vitro synthesized ClpA, ClpP and MgtC-FLAG proteins were produced using the cell-free PURExpress in vitro protein synthesis system (NEB) at 37°C for 3 h. Then, in vitro synthesized proteins were diluted 10 fold in 1×TBS (Tris-buffered saline) buffer containing 10 % glycerol.

    Article Title: The ubiquitin ligase LIN41/TRIM71 targets p53 to antagonize cell death and differentiation pathways during stem cell differentiation
    Article Snippet: LIN41 protein for the in vitro interaction assays was generated using the PURExpress In Vitro Protein Synthesis Kit (New England Biolabs, Ipswich, MA, USA). .. An amount of 500 ng of the purified DNA in 10 mM Tris-HCl pH 8 was added to the in vitro expression reaction containing 10 μ l solution A, 7.5 μ l solution B, 20 units RNase inhibitor, ad 25 μ l with nuclease-free H2 O.

    SDS Page:

    Article Title: Escherichia coli ItaT is a type II toxin that inhibits translation by acetylating isoleucyl-tRNAIle
    Article Snippet: The in vitro coupled transcription-translation reactions were carried out by PURExpress kit (NEB, USA) using DNA templates encoding the dihydrofolate reductase (DHFR) (NEB, USA) or the firefly luciferase (Promega, USA). .. The enzymatic activity of in vitro synthesized luciferase was measured using the Steady-Glo Luciferase Assay System (Promega, USA).

    Article Title: Sequestration from protease adaptor confers differential stability to protease substrate
    Article Snippet: In vitro synthesized ClpA, ClpP and MgtC-FLAG proteins were produced using the cell-free PURExpress in vitro protein synthesis system (NEB) at 37°C for 3 h. Then, in vitro synthesized proteins were diluted 10 fold in 1×TBS (Tris-buffered saline) buffer containing 10 % glycerol. .. In vitro synthesized ClpA, ClpP and MgtC-FLAG proteins were produced using the cell-free PURExpress in vitro protein synthesis system (NEB) at 37°C for 3 h. Then, in vitro synthesized proteins were diluted 10 fold in 1×TBS (Tris-buffered saline) buffer containing 10 % glycerol.

    Article Title: The RNA Helicase DeaD Stimulates ExsA Translation To Promote Expression of the Pseudomonas aeruginosa Type III Secretion System
    Article Snippet: A PURExpress in vitro protein synthesis system (New England BioLabs, Ipswich, MA) was used to measure DeaD activity as follows. .. A PURExpress in vitro protein synthesis system (New England BioLabs, Ipswich, MA) was used to measure DeaD activity as follows.

    Article Title: Protein Synthesis Using A Reconstituted Cell-Free System
    Article Snippet: When the protocols specify other commercial reagents, we indicate commercial and noncommercial alternatives. .. A 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 plate 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) or RNasin Ribonuclease inhibitor (20–40 U/µl, Promega) Microcentrifuge Air incubator set at 37°C 3x SDS-PAGE loading buffer (New England Biolabs) SDS-PAGE gel (4–20 % Tris-glycine, Life Technologies) .. Do not thaw Solutions A and B at room or higher temperatures as this would result in a loss in activity.

    Article Title: Protein Synthesis Using A Reconstituted Cell-Free System
    Article Snippet: This basic protocol describes the use of additional components, supplied as "PURExpress Disulfide Bond Enhancer", to help to correctly fold proteins that are known or expected to form disulfide bonds.. .. 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) .. Thaw Solutions A and B and Disulfide Bond Enhancer Solution 1 and 2 on ice.

    Plasmid Preparation:

    Article Title: Sequence specificity is obtained from the majority of modular C2H2 zinc-finger arrays
    Article Snippet: ZFAs were cloned as SacI–BamHI fragments into pTH5325, a modified T7-driven GST expression vector (see Supplementary Document of the Supplementary Data ). .. Briefly, we used 150 ng of plasmid DNA in a 25 μl in vitro transcription/translation reaction using a PURExpress In Vitro Protein Synthesis Kit (New England BioLabs) supplemented with RNase inhibitor and 50 μM zinc acetate. .. After a 2-h incubation at 37°C, 12.5 μl of the mix was added to 137.5 μl of protein-binding solution for a final mix of PBS/2% skim milk/0.2 mg per ml BSA/50 μM zinc acetate/0.1% Tween-20.

    Article Title: The stress-related, rhizobial small RNA RcsR1 destabilizes the autoinducer synthase encoding mRNA sinI in Sinorhizobium meliloti
    Article Snippet: A sinI'-egfp in vitro transcript was generated using primers sinIivfwd and egfpivrev and plasmid pLK64 for PCR amplification of the template and subsequent in vitro transcription with the MEGAshortscript T7 kit (Ambion) (see Tables S4 and S5). .. Five μg of sinI'-egfp transcript were used for in vitro translation carried out by the PURExpress In Vitro Protein Synthesis kit (New England Biolabs) following the manufacturer's instructions.

    Article Title: The Epstein-Barr virus B-ZIP protein Zta recognizes specific DNA sequences containing 5-methylcytosine and 5-hydroxymethylcytosine
    Article Snippet: The DBD of cFOS was obtained as a construct cloned into a pT5 expression plasmid . .. Proteins were expressed using the PURExpress In vitro protein synthesis kit (NEB) according to the manufacturer’s protocol in a 25μL reaction volume containing 180 ng of plasmid . .. The amino acid sequences of the B-ZIP domains with the alpha helical DNA binding region in bold are shown below: Zta: STVQTAAAVVFACPGANQGQQLADIGVPQPAPVAAPARRTRKPQQPESLEECDS ELEIKRYKNRVASRKCRAKFKQLLQ HYREVAAAKSSENDRLRLLLKQMCPSLDVDSIIPRTPDVLHEDLLNF CREB1: VVMASSPALPTQPAEE AARKREVRLMKNREAARECRRKKKEYVKCLEN RVAVLENQNKTLIEELKALKDLYCHKSD CEBPB: PPAAPAKAKAKKTVDKL SDEYKMRRERNNIAVRKSRDKAKMRNLET QHKVLELTAENERLQKKVEQLSRELSTLRNLFKQLPEPLLASAGHC cJun: MPGETPPLSPIDMESQERI KAERKRMRNRIAASKCRKRKLERIARL EEKVKTLKAQNSELASTANMLREQVAQLKQKVMNHVNSGCQLMLTQQLQ cFos: AQSIGRRGKVEQLSPEEEE KRRIRRERNKMAAAKCRNRRRELTD TLQAETDQLEDEKSALQTEIANLLKEKEKLEFILAAHRPACKIPDDLGFPE

    Article Title: The ubiquitin ligase LIN41/TRIM71 targets p53 to antagonize cell death and differentiation pathways during stem cell differentiation
    Article Snippet: LIN41 protein for the in vitro interaction assays was generated using the PURExpress In Vitro Protein Synthesis Kit (New England Biolabs, Ipswich, MA, USA). .. For creating a Lin41 template containing the T7 promoter, ribosome-binding site and T7 terminator required for a one-step transcription and translation, the DHFR gene from the positive control plasmid of the kit was replaced with Flag-tagged full-length Lin41 cDNA.

    Article Title: DNA binding strength increases the processivity and activity of a Y-Family DNA polymerase
    Article Snippet: The overlapping fragments of Dbh and Sso7d were PCR-amplified by oligonucleotides Fdbh-linker, Rdbh-dhfr, Fsso-dhfr and Rsso-linker, respectively (Table ). .. The high copy plasmid DHFR (dihydrofolatereductase) supplied with PURExpress In Vitro Protein Synthesis Kit (New England Biolabs, Inc. (NEB)) was linearized by digestion with Nde I and BamH I restriction enzymes. .. The recombinant plasmids DHFR-dbh and DHFR-Sdbh were consequently constructed according to the Gibson assembly protocol (NEB) and verified by DNA sequencing.

    Article Title: Translational Repression of the RpoS Antiadapter IraD by CsrA Is Mediated via Translational Coupling to a Short Upstream Open Reading Frame
    Article Snippet: In vitro coupled transcription-translation assays using PURExpress (New England Biolabs) followed a published procedure ( ). .. Plasmid pT7-P1iraD '- 'lacZ contains a T7 promoter driving transcription of the translational fusion from the natural iraD P1 transcription start site ( ).

    Article Title: Post-Translational Sortase-Mediated Attachment of High-Strength Force Spectroscopy Handles
    Article Snippet: All protein solutions were concentrated using Amicon centrifugal filter units (10k MWCO, Merck KGaA, Darmstadt, Germany), followed by buffer exchange to Ca-TBS buffer (25 mM Tris, pH 7.2, 75 mM NaCl, and 1 mM CaCl2 ) using polyacrylamide spin desalting columns. .. For cell-free expression, 25 μM reactions of PURExpress In Vitro Protein Synthesis Kit (New England Biolabs, Ipswich, Massachusetts) were incubated for 2 h at 37 °C, containing 300 ng plasmid DNA coding for the POIs. .. In case of MGGG-His-Doc, the N-terminal methionine cleavage in E. coli was sufficient for the preparation of GGG-His-Doc, so that no additional protease digestion was necessary.

    Binding Assay:

    Article Title: The Epstein-Barr virus B-ZIP protein Zta recognizes specific DNA sequences containing 5-methylcytosine and 5-hydroxymethylcytosine
    Article Snippet: Paragraph title: Cloning and expression of B-ZIP DNA binding domains ... Proteins were expressed using the PURExpress In vitro protein synthesis kit (NEB) according to the manufacturer’s protocol in a 25μL reaction volume containing 180 ng of plasmid .

    Article Title: Calmodulin Binding to Dfi1p Promotes Invasiveness of Candida albicans
    Article Snippet: Paragraph title: In vitro calmodulin binding assay ... PCR reactions were gel purified using Qiaex II resin and 250 ng DNA used in each translation reaction (PURExpress In vitro Protein Synthesis Kit, New England Biolabs E6800S).

    Agarose Gel Electrophoresis:

    Article Title: Ribosome Rescue Inhibitors Kill Actively Growing and Nonreplicating Persister Mycobacterium tuberculosis Cells
    Article Snippet: Assays were set up using the PURExpress in vitro protein synthesis kit (NEB, Ipswich, MA) according to the manufacturer’s protocols. .. After incubating for 30 min, an equal volume of 2× formamide loading buffer was added, and the tubes were incubated at 65 °C for 10 min.

    In Vitro:

    Article Title: YaeJ is a novel ribosome-associated protein in Escherichia coli that can hydrolyze peptidyl-tRNA on stalled ribosomes
    Article Snippet: The absence of the YaeJ protein and tmRNA was confirmed by western blotting or PCR. .. PURExpress (New England Biolabs) based on PUREsystem technology and FluroTect GreenLys in vitro translation labeling system (Promega) were used for in vitro translation experiments. .. Template DNA fragments were prepared using a two-step PCR reaction.

    Article Title: Sequence specificity is obtained from the majority of modular C2H2 zinc-finger arrays
    Article Snippet: ZFAs were cloned as SacI–BamHI fragments into pTH5325, a modified T7-driven GST expression vector (see Supplementary Document of the Supplementary Data ). .. Briefly, we used 150 ng of plasmid DNA in a 25 μl in vitro transcription/translation reaction using a PURExpress In Vitro Protein Synthesis Kit (New England BioLabs) supplemented with RNase inhibitor and 50 μM zinc acetate. .. After a 2-h incubation at 37°C, 12.5 μl of the mix was added to 137.5 μl of protein-binding solution for a final mix of PBS/2% skim milk/0.2 mg per ml BSA/50 μM zinc acetate/0.1% Tween-20.

    Article Title: Context influences on TALE–DNA binding revealed by quantitative profiling
    Article Snippet: Probe set descriptions, including the array design versions on which they are included, are provided in . .. Proteins were expressed using the PURExpress In Vitro Transcription and Translation Kit (New England Biolabs). .. Protein concentrations were determined by anti-GST western blots with a dilution series of recombinant GST (Sigma).

    Article Title: The stress-related, rhizobial small RNA RcsR1 destabilizes the autoinducer synthase encoding mRNA sinI in Sinorhizobium meliloti
    Article Snippet: This transcript contains the 5' UTR of sinI including the Shine-Dalgarno sequence and the first 9 codons translationally fused to full-length egfp . .. Five μg of sinI'-egfp transcript were used for in vitro translation carried out by the PURExpress In Vitro Protein Synthesis kit (New England Biolabs) following the manufacturer's instructions. .. If applied, equal amounts of RcsR1 were incubated with sinI'-egfp 20 min at 30 °C prior to in vitro translation.

    Article Title: Ribosome Rescue Inhibitors Kill Actively Growing and Nonreplicating Persister Mycobacterium tuberculosis Cells
    Article Snippet: Samples were analyzed by SDS PAGE. .. Assays were set up using the PURExpress in vitro protein synthesis kit (NEB, Ipswich, MA) according to the manufacturer’s protocols. .. The reactions were performed with no DNA template, a nonstop DHFR template, the full length DHFR gene, full length DHFR gene with 0 bases after the stop codon, or full length DHFR gene with 33 bases after the stop codon., KKL-2098 (final concentration of 1 μM) was added to a mixture of assay components, and the samples were incubated at room temperature for 1 h. Samples were placed on ice, irradiated with 312 nm UV light for 10 min, and used to set up click conjugation assays in the presence of KKL-2107 (final concentration of 0.5 mM).

    Article Title: Escherichia coli ItaT is a type II toxin that inhibits translation by acetylating isoleucyl-tRNAIle
    Article Snippet: Protein concentration was determined by Bradford method. .. The in vitro coupled transcription-translation reactions were carried out by PURExpress kit (NEB, USA) using DNA templates encoding the dihydrofolate reductase (DHFR) (NEB, USA) or the firefly luciferase (Promega, USA). .. Before template addition, translation reactions were incubated for 10 min at 37°C in the presence of 0.2 mM of cold acetyl coenzyme A (AcCoA) or radiolabeled [acetyl-1-14 C]-AcCoA (0.2 μCi, 60 mCi/mmol), Perkin Elmer, USA), with and without 2 μM ItaT.

    Article Title: The GET System Inserts the Tail-Anchored Protein, SYP72, into Endoplasmic Reticulum Membranes
    Article Snippet: Propidium iodide (P4864) was obtained from Sigma-Aldrich. .. 35 S-SYP72 substrates were synthesized using the T7-coupled PURExpress In Vitro Protein Synthesis Kit (New England Biolabs) in the presence of 35 S-Met and 25 μ m Get3. .. The Get3-SYP72 complex was purified on Streptactin sepharose and eluted with buffer containing 5 m m biotin.

    Article Title: Sequestration from protease adaptor confers differential stability to protease substrate
    Article Snippet: Purified proteins were used as 0.5 μM ClpS, 0.2 μM PhoP and 0.5 μM Mdh (Roche). .. In vitro synthesized ClpA, ClpP and MgtC-FLAG proteins were produced using the cell-free PURExpress in vitro protein synthesis system (NEB) at 37°C for 3 h. Then, in vitro synthesized proteins were diluted 10 fold in 1×TBS (Tris-buffered saline) buffer containing 10 % glycerol. .. 5 μl In vitro synthesized ClpA, ClpP and MgtC-FLAG were used for each reaction.

    Article Title: The Epstein-Barr virus B-ZIP protein Zta recognizes specific DNA sequences containing 5-methylcytosine and 5-hydroxymethylcytosine
    Article Snippet: The DBD of cFOS was obtained as a construct cloned into a pT5 expression plasmid . .. Proteins were expressed using the PURExpress In vitro protein synthesis kit (NEB) according to the manufacturer’s protocol in a 25μL reaction volume containing 180 ng of plasmid . .. The amino acid sequences of the B-ZIP domains with the alpha helical DNA binding region in bold are shown below: Zta: STVQTAAAVVFACPGANQGQQLADIGVPQPAPVAAPARRTRKPQQPESLEECDS ELEIKRYKNRVASRKCRAKFKQLLQ HYREVAAAKSSENDRLRLLLKQMCPSLDVDSIIPRTPDVLHEDLLNF CREB1: VVMASSPALPTQPAEE AARKREVRLMKNREAARECRRKKKEYVKCLEN RVAVLENQNKTLIEELKALKDLYCHKSD CEBPB: PPAAPAKAKAKKTVDKL SDEYKMRRERNNIAVRKSRDKAKMRNLET QHKVLELTAENERLQKKVEQLSRELSTLRNLFKQLPEPLLASAGHC cJun: MPGETPPLSPIDMESQERI KAERKRMRNRIAASKCRKRKLERIARL EEKVKTLKAQNSELASTANMLREQVAQLKQKVMNHVNSGCQLMLTQQLQ cFos: AQSIGRRGKVEQLSPEEEE KRRIRRERNKMAAAKCRNRRRELTD TLQAETDQLEDEKSALQTEIANLLKEKEKLEFILAAHRPACKIPDDLGFPE

    Article Title: The RNA Helicase DeaD Stimulates ExsA Translation To Promote Expression of the Pseudomonas aeruginosa Type III Secretion System
    Article Snippet: The protein concentrations were determined using a DC protein assay (Bio-Rad Laboratories, Hercules, CA). .. A PURExpress in vitro protein synthesis system (New England BioLabs, Ipswich, MA) was used to measure DeaD activity as follows. .. DNA templates for ExsA, Vfr, and LcrF were PCR amplified from pET23b-exsA1, pET23b-Vfr, and pET23b-LcrF using the primers 127176830 and 124849942.

    Article Title: Calmodulin Binding to Dfi1p Promotes Invasiveness of Candida albicans
    Article Snippet: All primers contained linker sequences needed for the second round of PCR, which was performed following the manufacturer's recommendations and introduced a c-terminal STREP tag to the constructs (Qiagen EasyXpress Linear Template Kit). .. PCR reactions were gel purified using Qiaex II resin and 250 ng DNA used in each translation reaction (PURExpress In vitro Protein Synthesis Kit, New England Biolabs E6800S). .. Translation reactions were performed with 20 U of murine RNAse inhibitor (NEB M0314S) according to the manufacturer's instructions.

    Article Title: Regulation of Hfq by the RNA CrcZ in Pseudomonas aeruginosa Carbon Catabolite Repression
    Article Snippet: The radioactively labeled bands were visualized with a PhosphorImager (Molecular Dynamics) and quantified with ImageQuant software 5.2. .. In vitro translation was performed with the PURExpress in vitro protein synthesis kit (New England BioLabs). .. 5 pmol in vitro transcribed amiE Flag mRNA was used in a 12.5 µl reaction.

    Article Title: The ubiquitin ligase LIN41/TRIM71 targets p53 to antagonize cell death and differentiation pathways during stem cell differentiation
    Article Snippet: Beads were recovered by centrifugation at 2500 × g for 2 min at 4 °C, washed three times with STE buffer and resuspended in 30 μ l 2 × SDS sample buffer for elution by boiling. .. LIN41 protein for the in vitro interaction assays was generated using the PURExpress In Vitro Protein Synthesis Kit (New England Biolabs, Ipswich, MA, USA). .. For creating a Lin41 template containing the T7 promoter, ribosome-binding site and T7 terminator required for a one-step transcription and translation, the DHFR gene from the positive control plasmid of the kit was replaced with Flag-tagged full-length Lin41 cDNA.

    Article Title: DNA binding strength increases the processivity and activity of a Y-Family DNA polymerase
    Article Snippet: The overlapping fragments of Dbh and Sso7d were PCR-amplified by oligonucleotides Fdbh-linker, Rdbh-dhfr, Fsso-dhfr and Rsso-linker, respectively (Table ). .. The high copy plasmid DHFR (dihydrofolatereductase) supplied with PURExpress In Vitro Protein Synthesis Kit (New England Biolabs, Inc. (NEB)) was linearized by digestion with Nde I and BamH I restriction enzymes. .. The recombinant plasmids DHFR-dbh and DHFR-Sdbh were consequently constructed according to the Gibson assembly protocol (NEB) and verified by DNA sequencing.

    Article Title: Translational Repression of the RpoS Antiadapter IraD by CsrA Is Mediated via Translational Coupling to a Short Upstream Open Reading Frame
    Article Snippet: Toeprint patterns were visualized with a phosphorimager. .. In vitro coupled transcription-translation assays using PURExpress (New England Biolabs) followed a published procedure ( ). .. Plasmid pT7-P1iraD '- 'lacZ contains a T7 promoter driving transcription of the translational fusion from the natural iraD P1 transcription start site ( ).

    Article Title: Post-Translational Sortase-Mediated Attachment of High-Strength Force Spectroscopy Handles
    Article Snippet: All protein solutions were concentrated using Amicon centrifugal filter units (10k MWCO, Merck KGaA, Darmstadt, Germany), followed by buffer exchange to Ca-TBS buffer (25 mM Tris, pH 7.2, 75 mM NaCl, and 1 mM CaCl2 ) using polyacrylamide spin desalting columns. .. For cell-free expression, 25 μM reactions of PURExpress In Vitro Protein Synthesis Kit (New England Biolabs, Ipswich, Massachusetts) were incubated for 2 h at 37 °C, containing 300 ng plasmid DNA coding for the POIs. .. In case of MGGG-His-Doc, the N-terminal methionine cleavage in E. coli was sufficient for the preparation of GGG-His-Doc, so that no additional protease digestion was necessary.

    Article Title: Protein Synthesis Using A Reconstituted Cell-Free System
    Article Snippet: When the protocols specify other commercial reagents, we indicate commercial and noncommercial alternatives. .. A 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 plate 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) or RNasin Ribonuclease inhibitor (20–40 U/µl, Promega) Microcentrifuge Air incubator set at 37°C 3x SDS-PAGE loading buffer (New England Biolabs) SDS-PAGE gel (4–20 % Tris-glycine, Life Technologies) .. Do not thaw Solutions A and B at room or higher temperatures as this would result in a loss in activity.

    Article Title: Protein Synthesis Using A Reconstituted Cell-Free System
    Article Snippet: This basic protocol describes the use of additional components, supplied as "PURExpress Disulfide Bond Enhancer", to help to correctly fold proteins that are known or expected to form disulfide bonds.. .. 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) .. Thaw Solutions A and B and Disulfide Bond Enhancer Solution 1 and 2 on ice.

    Article Title: Protein Synthesis Using A Reconstituted Cell-Free System
    Article Snippet: These two treatments leave the synthesized protein in the flow through fraction. shows a Coomassie-blue stained SDS-PAGE of fractions from a typical reverse purification of the DHFR control protein synthesized in the PURExpress reaction. .. 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) .. Set up a synthesis reaction with your template encoding a protein of interest (see Basic Protocol 1 steps 1 and 2.

    Article Title: Redesign of extensive protein–DNA interfaces of meganucleases using iterative cycles of in vitro compartmentalization
    Article Snippet: The oil–surfactant mixture [2% (vol/vol) ABIL EM 90 (gift from Evonik Industries AG Personal Care) and 0.05% Triton X-100 in light mineral oil] was thoroughly mixed with the saturation buffer [100 mM potassium glutamate (pH 7.5), 10 mM magnesium acetate (pH 7.5), 1 mM DTT, and 5 mg/mL BSA], incubated at 37 °C for 20 min, and centrifuged at 16,000 × g for 15 min at 4 °C. .. Five hundred microliters of the upper phase was used to emulsify 30 µL of the in vitro protein synthesis mixture [25 μL of PURExpress (New England Biolabs), 20 units of RNase inhibitor, 1 mg/mL BSA, and a DNA library] by constant stirring at 1,400 r.p.m. for 3.5 min on ice ( and ). .. Eight nanograms of a DNA library were added in the aqueous phase in the first round of IVC and reduced to 1 ng or 0.5 ng in the second or third round, respectively.

    Protein Binding:

    Article Title: Sequence specificity is obtained from the majority of modular C2H2 zinc-finger arrays
    Article Snippet: Paragraph title: Protein-binding microarray experiments ... Briefly, we used 150 ng of plasmid DNA in a 25 μl in vitro transcription/translation reaction using a PURExpress In Vitro Protein Synthesis Kit (New England BioLabs) supplemented with RNase inhibitor and 50 μM zinc acetate.

    Produced:

    Article Title: Sequestration from protease adaptor confers differential stability to protease substrate
    Article Snippet: Purified proteins were used as 0.5 μM ClpS, 0.2 μM PhoP and 0.5 μM Mdh (Roche). .. In vitro synthesized ClpA, ClpP and MgtC-FLAG proteins were produced using the cell-free PURExpress in vitro protein synthesis system (NEB) at 37°C for 3 h. Then, in vitro synthesized proteins were diluted 10 fold in 1×TBS (Tris-buffered saline) buffer containing 10 % glycerol. .. 5 μl In vitro synthesized ClpA, ClpP and MgtC-FLAG were used for each reaction.

    Concentration Assay:

    Article Title: Context influences on TALE–DNA binding revealed by quantitative profiling
    Article Snippet: Proteins were expressed using the PURExpress In Vitro Transcription and Translation Kit (New England Biolabs). .. Proteins were expressed using the PURExpress In Vitro Transcription and Translation Kit (New England Biolabs).

    Ethanol Precipitation:

    Article Title: The ubiquitin ligase LIN41/TRIM71 targets p53 to antagonize cell death and differentiation pathways during stem cell differentiation
    Article Snippet: LIN41 protein for the in vitro interaction assays was generated using the PURExpress In Vitro Protein Synthesis Kit (New England Biolabs, Ipswich, MA, USA). .. For creating a Lin41 template containing the T7 promoter, ribosome-binding site and T7 terminator required for a one-step transcription and translation, the DHFR gene from the positive control plasmid of the kit was replaced with Flag-tagged full-length Lin41 cDNA.

    Strep-tag:

    Article Title: Calmodulin Binding to Dfi1p Promotes Invasiveness of Candida albicans
    Article Snippet: All primers contained linker sequences needed for the second round of PCR, which was performed following the manufacturer's recommendations and introduced a c-terminal STREP tag to the constructs (Qiagen EasyXpress Linear Template Kit). .. PCR reactions were gel purified using Qiaex II resin and 250 ng DNA used in each translation reaction (PURExpress In vitro Protein Synthesis Kit, New England Biolabs E6800S).

    Gel Extraction:

    Article Title: Redesign of extensive protein–DNA interfaces of meganucleases using iterative cycles of in vitro compartmentalization
    Article Snippet: A successfully assembled DNA fragment was again purified by gel extraction ( ). .. Five hundred microliters of the upper phase was used to emulsify 30 µL of the in vitro protein synthesis mixture [25 μL of PURExpress (New England Biolabs), 20 units of RNase inhibitor, 1 mg/mL BSA, and a DNA library] by constant stirring at 1,400 r.p.m. for 3.5 min on ice ( and ).

    Variant Assay:

    Article Title: Redesign of extensive protein–DNA interfaces of meganucleases using iterative cycles of in vitro compartmentalization
    Article Snippet: Two to three rounds of IVC were conducted following each round of site-directed saturation mutagenesis to enrich active variant genes. .. Five hundred microliters of the upper phase was used to emulsify 30 µL of the in vitro protein synthesis mixture [25 μL of PURExpress (New England Biolabs), 20 units of RNase inhibitor, 1 mg/mL BSA, and a DNA library] by constant stirring at 1,400 r.p.m. for 3.5 min on ice ( and ).

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    New England Biolabs purexpress in vitro protein synthesis kit
    Chromozyme cleavage assay (Roche) for a truncated version of tissue plasminogen activator protein (vtPA) synthesized in the <t>PURExpress</t> reactions with and without PURExpress Disulfide Bond Enhancer (PDBE). This tissue plasminogen activator contains 9 disulfide
    Purexpress In Vitro Protein Synthesis Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 48 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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:

    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 Nuclease free microcentrifuge tubes or microtiter plate 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) or RNasin Ribonuclease inhibitor (20–40 U/µl, Promega) Microcentrifuge Air incubator set at 37°C 3x SDS-PAGE loading buffer (New England Biolabs) SDS-PAGE gel (4–20 % Tris-glycine, Life Technologies)

    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:

    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 Nuclease free microcentrifuge tubes or microtiter plate 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) or RNasin Ribonuclease inhibitor (20–40 U/µl, Promega) Microcentrifuge Air incubator set at 37°C 3x SDS-PAGE loading buffer (New England Biolabs) SDS-PAGE gel (4–20 % Tris-glycine, Life Technologies)

    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:

    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 Nuclease free microcentrifuge tubes or microtiter plate 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) or RNasin Ribonuclease inhibitor (20–40 U/µl, Promega) Microcentrifuge Air incubator set at 37°C 3x SDS-PAGE loading buffer (New England Biolabs) SDS-PAGE gel (4–20 % Tris-glycine, Life Technologies)

    Techniques: SDS Page, Synthesized, Labeling, Luciferase