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tnt coupled reticulate lysate system  (Promega)

 
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    Promega tnt coupled reticulate lysate system
    Design and validation of rotavirus (RV) VP4 and nonstructural protein 3 (NSP3) plasmid constructs used to generate mutant viruses. (A) Schematic showing overall topology of SARS-CoV-2 spike protein in gray boxes: N-terminal domain (NTD), receptor binding domain (RBD), which contains the receptor binding motif (RBM), fusion peptide (FP), heptad repeats 1 and 2 (HR1 and HR2), transmembrane region (TM), and the intracellular domain (IC) (adapted from Lan et al. ). Dashed lines represent selected regions of the spike protein (colored boxes) that were inserted into the hypervariable region of the SA11 viral protein 4 (VP4) gene (top) and the C terminus of the RF NSP3 gene (bottom). SA11 VP4 was edited to incorporate either NTD, RBM.1, RBM.2, or HR2 spike peptide sequences. RF NSP3 was fused with either the RBD or RBM sequence with or without Thosea asigna virus 2A (T2A) (yellow box), represented by +/− sign. Both gene segments were flanked by the T7 promoter (T7P) and the antigenomic hepatitis delta virus (HDV) ribozyme (“HDV Rib,” green boxes) followed by T7 terminator (T7T). *Stop codons. Schematic not to scale. (B) Ribbon representation of VP4 (adapted from Settembre et al. ). Two orthogonal views are shown. The VP8* fragment is in magenta extending into the VP5* foot domains (in blue), the hypervariable region of VP4 is in gray and the region where SARS-CoV-2 peptides (omitted for clarity) were inserted is in green. VP5* β-barrel domains are in cyan and purple. (C and D) <t>Coupled</t> in vitro transcription and translation reactions of mutated SA11 VP4 (C) and RF NSP3 (D) segments were carried out using the <t>TnT</t> rabbit reticulocyte <t>lysate</t> <t>system</t> supplemented with [ 35 S]methionine. Samples were analyzed using SDS-PAGE and autoradiography. The molecular weight marker and the expected product sizes of each segment (in brackets) are indicated (kDa). Empty pCDNA 3.1 vector was used as a negative control. In panel D, black asterisks indicate T2A read-through product and red asterisks identify separated products. WT, wild type.
    Tnt Coupled Reticulate Lysate System, supplied by Promega, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/tnt+reticulate+lysates/tnt+coupled+reticulate+lysate+system/pmc09327695-292-13-12
    Average 90 stars, based on 1 article reviews
    tnt coupled reticulate lysate system - by Bioz Stars, 2026-09
    90/100 stars

    Images

    1) Product Images from "Using Species a Rotavirus Reverse Genetics to Engineer Chimeric Viruses Expressing SARS-CoV-2 Spike Epitopes"

    Article Title: Using Species a Rotavirus Reverse Genetics to Engineer Chimeric Viruses Expressing SARS-CoV-2 Spike Epitopes

    Journal: Journal of Virology

    doi: 10.1128/jvi.00488-22

    Design and validation of rotavirus (RV) VP4 and nonstructural protein 3 (NSP3) plasmid constructs used to generate mutant viruses. (A) Schematic showing overall topology of SARS-CoV-2 spike protein in gray boxes: N-terminal domain (NTD), receptor binding domain (RBD), which contains the receptor binding motif (RBM), fusion peptide (FP), heptad repeats 1 and 2 (HR1 and HR2), transmembrane region (TM), and the intracellular domain (IC) (adapted from Lan et al. ). Dashed lines represent selected regions of the spike protein (colored boxes) that were inserted into the hypervariable region of the SA11 viral protein 4 (VP4) gene (top) and the C terminus of the RF NSP3 gene (bottom). SA11 VP4 was edited to incorporate either NTD, RBM.1, RBM.2, or HR2 spike peptide sequences. RF NSP3 was fused with either the RBD or RBM sequence with or without Thosea asigna virus 2A (T2A) (yellow box), represented by +/− sign. Both gene segments were flanked by the T7 promoter (T7P) and the antigenomic hepatitis delta virus (HDV) ribozyme (“HDV Rib,” green boxes) followed by T7 terminator (T7T). *Stop codons. Schematic not to scale. (B) Ribbon representation of VP4 (adapted from Settembre et al. ). Two orthogonal views are shown. The VP8* fragment is in magenta extending into the VP5* foot domains (in blue), the hypervariable region of VP4 is in gray and the region where SARS-CoV-2 peptides (omitted for clarity) were inserted is in green. VP5* β-barrel domains are in cyan and purple. (C and D) Coupled in vitro transcription and translation reactions of mutated SA11 VP4 (C) and RF NSP3 (D) segments were carried out using the TnT rabbit reticulocyte lysate system supplemented with [ 35 S]methionine. Samples were analyzed using SDS-PAGE and autoradiography. The molecular weight marker and the expected product sizes of each segment (in brackets) are indicated (kDa). Empty pCDNA 3.1 vector was used as a negative control. In panel D, black asterisks indicate T2A read-through product and red asterisks identify separated products. WT, wild type.
    Figure Legend Snippet: Design and validation of rotavirus (RV) VP4 and nonstructural protein 3 (NSP3) plasmid constructs used to generate mutant viruses. (A) Schematic showing overall topology of SARS-CoV-2 spike protein in gray boxes: N-terminal domain (NTD), receptor binding domain (RBD), which contains the receptor binding motif (RBM), fusion peptide (FP), heptad repeats 1 and 2 (HR1 and HR2), transmembrane region (TM), and the intracellular domain (IC) (adapted from Lan et al. ). Dashed lines represent selected regions of the spike protein (colored boxes) that were inserted into the hypervariable region of the SA11 viral protein 4 (VP4) gene (top) and the C terminus of the RF NSP3 gene (bottom). SA11 VP4 was edited to incorporate either NTD, RBM.1, RBM.2, or HR2 spike peptide sequences. RF NSP3 was fused with either the RBD or RBM sequence with or without Thosea asigna virus 2A (T2A) (yellow box), represented by +/− sign. Both gene segments were flanked by the T7 promoter (T7P) and the antigenomic hepatitis delta virus (HDV) ribozyme (“HDV Rib,” green boxes) followed by T7 terminator (T7T). *Stop codons. Schematic not to scale. (B) Ribbon representation of VP4 (adapted from Settembre et al. ). Two orthogonal views are shown. The VP8* fragment is in magenta extending into the VP5* foot domains (in blue), the hypervariable region of VP4 is in gray and the region where SARS-CoV-2 peptides (omitted for clarity) were inserted is in green. VP5* β-barrel domains are in cyan and purple. (C and D) Coupled in vitro transcription and translation reactions of mutated SA11 VP4 (C) and RF NSP3 (D) segments were carried out using the TnT rabbit reticulocyte lysate system supplemented with [ 35 S]methionine. Samples were analyzed using SDS-PAGE and autoradiography. The molecular weight marker and the expected product sizes of each segment (in brackets) are indicated (kDa). Empty pCDNA 3.1 vector was used as a negative control. In panel D, black asterisks indicate T2A read-through product and red asterisks identify separated products. WT, wild type.

    Techniques Used: Biomarker Discovery, Plasmid Preparation, Construct, Mutagenesis, Binding Assay, Sequencing, Virus, In Vitro, SDS Page, Autoradiography, Molecular Weight, Marker, Negative Control

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    Article Title: ATM Protein Physically and Functionally Interacts with Proliferating Cell Nuclear Antigen to Regulate DNA Synthesis
    Article Snippet: Proteinswere labeledwith 35S-labeledmethionine using TNT reticulate lysates, following the standard protocol provided by Promega (Madison, WI).

    Article Title: Regulation of KLF4 turnover reveals an unexpected tissue specific role of pVHL in tumorigenesis
    Article Snippet: Wild type KLF4 and deletion mutants were labelled with 35 S-methionine using TNT reticulate lysates, following the standard protocol provided by Promega.

    Labeling:

    Article Title: Multivalent Binding of p53 to the STAGA Complex Mediates Coactivator Recruitment after UV Damage
    Article Snippet: .. Proteins were labeled with [ 35 S]methionine using TNT reticulate lysates, following the standard protocol provided by Promega. ..

    Article Title: Multivalent Binding of p53 to the STAGA Complex Mediates Coactivator Recruitment after UV Damage
    Article Snippet: .. Proteins were labeled with [35S]methionine using TNT reticulate lysates, following the standard protocol provided by Promega. ..

    Article Title: The STAGA Subunit ADA2b Is an Important Regulator of Human GCN5 Catalysis
    Article Snippet: .. Proteins were labeled with [ 35 S]methionine by using TNT reticulate lysates according to the standard protocol provided by Promega. ..

    Article Title: GCN5 acetylates and regulates the stability of the oncoprotein E2A-PBX1 in acute lymphoblastic leukemia.
    Article Snippet: .. In Figure 1e, GCN5 was labeled with [35S]-methionine using TNT reticulate lysates (Promega, Madison, WI, USA), according to the manufacturer’s standard protocol. ..



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    Image Search Results


    Design and validation of rotavirus (RV) VP4 and nonstructural protein 3 (NSP3) plasmid constructs used to generate mutant viruses. (A) Schematic showing overall topology of SARS-CoV-2 spike protein in gray boxes: N-terminal domain (NTD), receptor binding domain (RBD), which contains the receptor binding motif (RBM), fusion peptide (FP), heptad repeats 1 and 2 (HR1 and HR2), transmembrane region (TM), and the intracellular domain (IC) (adapted from Lan et al. ). Dashed lines represent selected regions of the spike protein (colored boxes) that were inserted into the hypervariable region of the SA11 viral protein 4 (VP4) gene (top) and the C terminus of the RF NSP3 gene (bottom). SA11 VP4 was edited to incorporate either NTD, RBM.1, RBM.2, or HR2 spike peptide sequences. RF NSP3 was fused with either the RBD or RBM sequence with or without Thosea asigna virus 2A (T2A) (yellow box), represented by +/− sign. Both gene segments were flanked by the T7 promoter (T7P) and the antigenomic hepatitis delta virus (HDV) ribozyme (“HDV Rib,” green boxes) followed by T7 terminator (T7T). *Stop codons. Schematic not to scale. (B) Ribbon representation of VP4 (adapted from Settembre et al. ). Two orthogonal views are shown. The VP8* fragment is in magenta extending into the VP5* foot domains (in blue), the hypervariable region of VP4 is in gray and the region where SARS-CoV-2 peptides (omitted for clarity) were inserted is in green. VP5* β-barrel domains are in cyan and purple. (C and D) Coupled in vitro transcription and translation reactions of mutated SA11 VP4 (C) and RF NSP3 (D) segments were carried out using the TnT rabbit reticulocyte lysate system supplemented with [ 35 S]methionine. Samples were analyzed using SDS-PAGE and autoradiography. The molecular weight marker and the expected product sizes of each segment (in brackets) are indicated (kDa). Empty pCDNA 3.1 vector was used as a negative control. In panel D, black asterisks indicate T2A read-through product and red asterisks identify separated products. WT, wild type.

    Journal: Journal of Virology

    Article Title: Using Species a Rotavirus Reverse Genetics to Engineer Chimeric Viruses Expressing SARS-CoV-2 Spike Epitopes

    doi: 10.1128/jvi.00488-22

    Figure Lengend Snippet: Design and validation of rotavirus (RV) VP4 and nonstructural protein 3 (NSP3) plasmid constructs used to generate mutant viruses. (A) Schematic showing overall topology of SARS-CoV-2 spike protein in gray boxes: N-terminal domain (NTD), receptor binding domain (RBD), which contains the receptor binding motif (RBM), fusion peptide (FP), heptad repeats 1 and 2 (HR1 and HR2), transmembrane region (TM), and the intracellular domain (IC) (adapted from Lan et al. ). Dashed lines represent selected regions of the spike protein (colored boxes) that were inserted into the hypervariable region of the SA11 viral protein 4 (VP4) gene (top) and the C terminus of the RF NSP3 gene (bottom). SA11 VP4 was edited to incorporate either NTD, RBM.1, RBM.2, or HR2 spike peptide sequences. RF NSP3 was fused with either the RBD or RBM sequence with or without Thosea asigna virus 2A (T2A) (yellow box), represented by +/− sign. Both gene segments were flanked by the T7 promoter (T7P) and the antigenomic hepatitis delta virus (HDV) ribozyme (“HDV Rib,” green boxes) followed by T7 terminator (T7T). *Stop codons. Schematic not to scale. (B) Ribbon representation of VP4 (adapted from Settembre et al. ). Two orthogonal views are shown. The VP8* fragment is in magenta extending into the VP5* foot domains (in blue), the hypervariable region of VP4 is in gray and the region where SARS-CoV-2 peptides (omitted for clarity) were inserted is in green. VP5* β-barrel domains are in cyan and purple. (C and D) Coupled in vitro transcription and translation reactions of mutated SA11 VP4 (C) and RF NSP3 (D) segments were carried out using the TnT rabbit reticulocyte lysate system supplemented with [ 35 S]methionine. Samples were analyzed using SDS-PAGE and autoradiography. The molecular weight marker and the expected product sizes of each segment (in brackets) are indicated (kDa). Empty pCDNA 3.1 vector was used as a negative control. In panel D, black asterisks indicate T2A read-through product and red asterisks identify separated products. WT, wild type.

    Article Snippet: Coupled in vitro transcription and translation reactions were carried out using the Promega TnT Coupled Reticulate Lysate System labeled with radioactive [ 35 S]methionine (PerkinElmer Inc.) according to the manufacturer’s protocol.

    Techniques: Biomarker Discovery, Plasmid Preparation, Construct, Mutagenesis, Binding Assay, Sequencing, Virus, In Vitro, SDS Page, Autoradiography, Molecular Weight, Marker, Negative Control