rna loading buffer  (New England Biolabs)


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    Blue Loading Buffer Pack
    Description:
    Blue Loading Buffer Pack 8 0 ml
    Catalog Number:
    B7703S
    Price:
    29
    Category:
    Electrophoresis Tracking Dyes
    Size:
    8 0 ml
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    Structured Review

    New England Biolabs rna loading buffer
    Blue Loading Buffer Pack
    Blue Loading Buffer Pack 8 0 ml
    https://www.bioz.com/result/rna loading buffer/product/New England Biolabs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rna loading buffer - by Bioz Stars, 2021-07
    93/100 stars

    Images

    1) Product Images from "Assembly defects of the human tRNA splicing endonuclease contribute to impaired pre-tRNA processing in pontocerebellar hypoplasia"

    Article Title: Assembly defects of the human tRNA splicing endonuclease contribute to impaired pre-tRNA processing in pontocerebellar hypoplasia

    Journal: bioRxiv

    doi: 10.1101/2020.08.03.234229

    X-ray crystal structure of a TSEN15–34 heterodimer derived by limited proteolysis. a , Analysis of the limited tryptic digestion of the TSEN15–34 heterodimer by SDS-PAGE. b , Denaturing mass spectrum of the proteolytically stable fragments of the TSEN15–34 heterodimer from an aqueous ammonium acetate solution. The mass spectrum shows the presence of a TSEN34 fragment (orange circles), and two TSEN15 fragments (dark and light green circles) differing in mass only by a C-terminal arginine as revealed by LC-MS/MS. c , Bar diagrams of tryptic fragments of TSEN15 and TSEN34. Proteolyzed regions are indicated by dashed boxes. Positions of PCH mutations are shown. d , Purification of the re-cloned core of the TSEN15–34 heterodimer via SEC. The absorbance profile at 280 nm is shown. Fractions of the indicated retention range (grey area) were analyzed by SDS-PAGE. e , Asymmetric unit of the TSEN15–34 crystal. The biological unit (bracket) and the domain-swap area (dashed box) are indicated. α-helices and β-sheets are numbered for each subunit. f , Stick representation of amino acids of the domain-swap area with electron density (2F o -F c , 1.5σ). g , Molecular mass determination by size exclusion chromatography multi-angle light scattering (SEC-MALS) of the TSEN15–34 sample used for crystallization. The data reveal a dominant population of a dimer-of-a-heterodimer (13.2 ml, 56.6 kDa) and a minor populated heterodimer (14.9 ml, 30.0 kDa). Light scattering is shown as red dots. Mass determination by SEC-MALS was confirmed by two independent experiments. h , Superposition of the TSEN15–34 heterodimer and the pre-tRNA endonuclease from Archaeoglobus fulgidus at the interaction sites with the bulge-helix-bulge RNA (PDB ID 2GJW). Nucleotide positions of the RNA (black) and residues of the catalytic triads are shown. i , Representative thermal denaturation curves as shown in Fig. 3g of recombinant wt TSEN and mutant TSEN (T15 H116Y ) complexes derived from DSF. Sigmoidal Boltzmann fits are shown as red lines. Grey zones show standard deviations (SD) from technical triplicates. Denaturation temperature (T d ) is presented with error of fit. Unprocessed gels for a and d are shown in Source Data 6.
    Figure Legend Snippet: X-ray crystal structure of a TSEN15–34 heterodimer derived by limited proteolysis. a , Analysis of the limited tryptic digestion of the TSEN15–34 heterodimer by SDS-PAGE. b , Denaturing mass spectrum of the proteolytically stable fragments of the TSEN15–34 heterodimer from an aqueous ammonium acetate solution. The mass spectrum shows the presence of a TSEN34 fragment (orange circles), and two TSEN15 fragments (dark and light green circles) differing in mass only by a C-terminal arginine as revealed by LC-MS/MS. c , Bar diagrams of tryptic fragments of TSEN15 and TSEN34. Proteolyzed regions are indicated by dashed boxes. Positions of PCH mutations are shown. d , Purification of the re-cloned core of the TSEN15–34 heterodimer via SEC. The absorbance profile at 280 nm is shown. Fractions of the indicated retention range (grey area) were analyzed by SDS-PAGE. e , Asymmetric unit of the TSEN15–34 crystal. The biological unit (bracket) and the domain-swap area (dashed box) are indicated. α-helices and β-sheets are numbered for each subunit. f , Stick representation of amino acids of the domain-swap area with electron density (2F o -F c , 1.5σ). g , Molecular mass determination by size exclusion chromatography multi-angle light scattering (SEC-MALS) of the TSEN15–34 sample used for crystallization. The data reveal a dominant population of a dimer-of-a-heterodimer (13.2 ml, 56.6 kDa) and a minor populated heterodimer (14.9 ml, 30.0 kDa). Light scattering is shown as red dots. Mass determination by SEC-MALS was confirmed by two independent experiments. h , Superposition of the TSEN15–34 heterodimer and the pre-tRNA endonuclease from Archaeoglobus fulgidus at the interaction sites with the bulge-helix-bulge RNA (PDB ID 2GJW). Nucleotide positions of the RNA (black) and residues of the catalytic triads are shown. i , Representative thermal denaturation curves as shown in Fig. 3g of recombinant wt TSEN and mutant TSEN (T15 H116Y ) complexes derived from DSF. Sigmoidal Boltzmann fits are shown as red lines. Grey zones show standard deviations (SD) from technical triplicates. Denaturation temperature (T d ) is presented with error of fit. Unprocessed gels for a and d are shown in Source Data 6.

    Techniques Used: Derivative Assay, SDS Page, Liquid Chromatography with Mass Spectroscopy, Purification, Clone Assay, Size-exclusion Chromatography, Crystallization Assay, Recombinant, Mutagenesis

    Active involvement of the A–I base pair in coordinating pre-tRNA cleavage. a , SDS-PAGE of purified, recombinant inactive TSEN and inactive TSEN/CLP1 complexes (TSEN2 H377A and TSEN34 H255A double mutant). Protein size markers and protein identities are indicated. b , Two-colored pre-tRNA cleavage assay with TSEN-STREP and TSEN/CLP1-FLAG wt complexes and complexes carrying the TSEN2 H377A (T2 H377A ) or TSEN34 H255A (T34 H255A ) substitution. RNA cleavage products were separated on a denaturing Urea-PAGE and visualized by fluorescence of cyanine5 (Cy5) and Fluorescin (FITC). c , Thermodynamic competition parameters deduced from fluorescence anisotropy experiments. Inactive, tetrameric TSEN bound to fluorescently labeled pre-tRNA Phe GAA was titrated with unlabeled pre-tRNA. d , Electrophoretic mobility shift assay with fluorescently labeled pre-tRNA Phe GAA and inactive, tetrameric TSEN (TSEN inactive ). Free and bound fractions of pre-tRNA were analyzed via 4% TBE native PAGE with subsequent in-gel fluorescence measurement. e , Impact of A-I base pair mutations in pre-tRNA Tyr GTA 8-1 on endonucleolytic activity by tetrameric TSEN revealed by a pre-tRNA cleavage assay. CI 95 – 95% confidence interval, C 32 :G 52 – canonical A-I base pair, C 32 :C 52 and G 32 :G 52 – disrupted A-I base pair, G 32 :C 52 – inverted A-I base pair. Panels are representatives of three independent experiments. Unprocessed gels for a , b , d , and e are shown in Source Data 4.
    Figure Legend Snippet: Active involvement of the A–I base pair in coordinating pre-tRNA cleavage. a , SDS-PAGE of purified, recombinant inactive TSEN and inactive TSEN/CLP1 complexes (TSEN2 H377A and TSEN34 H255A double mutant). Protein size markers and protein identities are indicated. b , Two-colored pre-tRNA cleavage assay with TSEN-STREP and TSEN/CLP1-FLAG wt complexes and complexes carrying the TSEN2 H377A (T2 H377A ) or TSEN34 H255A (T34 H255A ) substitution. RNA cleavage products were separated on a denaturing Urea-PAGE and visualized by fluorescence of cyanine5 (Cy5) and Fluorescin (FITC). c , Thermodynamic competition parameters deduced from fluorescence anisotropy experiments. Inactive, tetrameric TSEN bound to fluorescently labeled pre-tRNA Phe GAA was titrated with unlabeled pre-tRNA. d , Electrophoretic mobility shift assay with fluorescently labeled pre-tRNA Phe GAA and inactive, tetrameric TSEN (TSEN inactive ). Free and bound fractions of pre-tRNA were analyzed via 4% TBE native PAGE with subsequent in-gel fluorescence measurement. e , Impact of A-I base pair mutations in pre-tRNA Tyr GTA 8-1 on endonucleolytic activity by tetrameric TSEN revealed by a pre-tRNA cleavage assay. CI 95 – 95% confidence interval, C 32 :G 52 – canonical A-I base pair, C 32 :C 52 and G 32 :G 52 – disrupted A-I base pair, G 32 :C 52 – inverted A-I base pair. Panels are representatives of three independent experiments. Unprocessed gels for a , b , d , and e are shown in Source Data 4.

    Techniques Used: SDS Page, Purification, Recombinant, Mutagenesis, Cleavage Assay, Polyacrylamide Gel Electrophoresis, Fluorescence, Labeling, Electrophoretic Mobility Shift Assay, Clear Native PAGE, Activity Assay

    Active involvement of the A–I base pair in coordinating pre-tRNA cleavage. a , Pre-tRNA cleavage assay comparing recombinant, inactive TSEN tetramer (TSEN2 H377A and TSEN34 H255A double mutant) to the TSEN/CLP1 complex. Cleavage products are visualized by denaturing Urea-PAGE with subsequent Toluidine blue staining. RNA size markers are indicated on the left of the gel. b , Pull-down assay with fluorescently labeled S.c. pre-tRNA Phe GAA and inactive, tetrameric TSEN captured on protein G agarose functionalized with an α-His-antibody. Protein size markers are indicated on the left of each immunoblot, protein and RNA identities on the right. Input and co-precipitated, labeled pre-tRNAs were visualized by in-gel fluorescence, TSEN subunits and the immunoglobulin G (IgG) heavy chain by immunoblotting. The IgG heavy chain served as loading control. c, Thermodynamic binding parameters of fluorescently labeled S.c. pre-tRNA Phe GAA and inactive, tetrameric TSEN revealed by fluorescence anisotropy. d , Thermodynamic binding parameters of fluorescently labeled tRNA Phe GAA and inactive, tetrameric TSEN revealed by fluorescence anisotropy. e , Schematic depiction of a pre-tRNA molecule showing ribonucleotides belonging to the mature domain (grey spheres), the intronic region (white spheres), the anticodon (black spheres), and the A-I base pair (yellow spheres). Proposed 5’ and 3’ splice sites (ss) are indicated. f , Impact of A-I base pair mutations in S.c. pre-tRNA Phe GAA on endonucleolytic activity of tetrameric TSEN revealed by a pre-tRNA cleavage assay. C 32 :G 54 – canonical A-I base pair, C 32 :C 54 – disrupted A-I base pair, G 32 :C 54 – inverted A-I base pair. All experiments are representatives of three independent assays. Unprocessed gels for a , b and f are shown in Source Data 3.
    Figure Legend Snippet: Active involvement of the A–I base pair in coordinating pre-tRNA cleavage. a , Pre-tRNA cleavage assay comparing recombinant, inactive TSEN tetramer (TSEN2 H377A and TSEN34 H255A double mutant) to the TSEN/CLP1 complex. Cleavage products are visualized by denaturing Urea-PAGE with subsequent Toluidine blue staining. RNA size markers are indicated on the left of the gel. b , Pull-down assay with fluorescently labeled S.c. pre-tRNA Phe GAA and inactive, tetrameric TSEN captured on protein G agarose functionalized with an α-His-antibody. Protein size markers are indicated on the left of each immunoblot, protein and RNA identities on the right. Input and co-precipitated, labeled pre-tRNAs were visualized by in-gel fluorescence, TSEN subunits and the immunoglobulin G (IgG) heavy chain by immunoblotting. The IgG heavy chain served as loading control. c, Thermodynamic binding parameters of fluorescently labeled S.c. pre-tRNA Phe GAA and inactive, tetrameric TSEN revealed by fluorescence anisotropy. d , Thermodynamic binding parameters of fluorescently labeled tRNA Phe GAA and inactive, tetrameric TSEN revealed by fluorescence anisotropy. e , Schematic depiction of a pre-tRNA molecule showing ribonucleotides belonging to the mature domain (grey spheres), the intronic region (white spheres), the anticodon (black spheres), and the A-I base pair (yellow spheres). Proposed 5’ and 3’ splice sites (ss) are indicated. f , Impact of A-I base pair mutations in S.c. pre-tRNA Phe GAA on endonucleolytic activity of tetrameric TSEN revealed by a pre-tRNA cleavage assay. C 32 :G 54 – canonical A-I base pair, C 32 :C 54 – disrupted A-I base pair, G 32 :C 54 – inverted A-I base pair. All experiments are representatives of three independent assays. Unprocessed gels for a , b and f are shown in Source Data 3.

    Techniques Used: Cleavage Assay, Recombinant, Mutagenesis, Polyacrylamide Gel Electrophoresis, Staining, Pull Down Assay, Labeling, Fluorescence, Binding Assay, Activity Assay

    Assembly and catalysis of recombinant human TSEN. a , Bar diagrams of TSEN subunits and CLP1 depicting positions of PCH mutations, predicted nuclease domains of TSEN2 and TSEN34, and the RNA kinase domain for CLP1. Total amino acids of each protein are indicated. b , SDS-PAGE of purified recombinant TSEN and TSEN/CLP1 complexes visualized by InstantBlue staining. Protein identities and size markers are shown. c , Native mass spectrum of tetrameric TSEN complex from an aqueous ammonium acetate solution. Charge states of the predominant TSEN2–15– 34–54 heterotetramer (blue circles), Heat Shock Protein (HSP) 70 (grey circles), the heterodimer TSEN15–34 (red circles) and TSEN15 (green circles) are indicated. d , Pre-tRNA cleavage assay using tetrameric TSEN complex with Saccharomyces cerevisia e ( S.c. ) pre-tRNA Phe GAA and mature tRNA Phe GAA . Input samples and cleavage products were separated via Urea-PAGE and visualized by Toluidine blue. RNA denominations are given on the right. e , Pre-tRNA cleavage assay with TSEN heterodimers and S.c. pre-tRNA Phe GAA . SDS-PAGE of the indicated heterodimers and the reconstituted TSEN tetramer is shown on the left (InstantBlue stain), Urea-PAGE of the cleavage products on the right (Toluidine blue stain). Gels are representative of three independent experiments. Unprocessed gels for b , c and e are shown in Source Data 1.
    Figure Legend Snippet: Assembly and catalysis of recombinant human TSEN. a , Bar diagrams of TSEN subunits and CLP1 depicting positions of PCH mutations, predicted nuclease domains of TSEN2 and TSEN34, and the RNA kinase domain for CLP1. Total amino acids of each protein are indicated. b , SDS-PAGE of purified recombinant TSEN and TSEN/CLP1 complexes visualized by InstantBlue staining. Protein identities and size markers are shown. c , Native mass spectrum of tetrameric TSEN complex from an aqueous ammonium acetate solution. Charge states of the predominant TSEN2–15– 34–54 heterotetramer (blue circles), Heat Shock Protein (HSP) 70 (grey circles), the heterodimer TSEN15–34 (red circles) and TSEN15 (green circles) are indicated. d , Pre-tRNA cleavage assay using tetrameric TSEN complex with Saccharomyces cerevisia e ( S.c. ) pre-tRNA Phe GAA and mature tRNA Phe GAA . Input samples and cleavage products were separated via Urea-PAGE and visualized by Toluidine blue. RNA denominations are given on the right. e , Pre-tRNA cleavage assay with TSEN heterodimers and S.c. pre-tRNA Phe GAA . SDS-PAGE of the indicated heterodimers and the reconstituted TSEN tetramer is shown on the left (InstantBlue stain), Urea-PAGE of the cleavage products on the right (Toluidine blue stain). Gels are representative of three independent experiments. Unprocessed gels for b , c and e are shown in Source Data 1.

    Techniques Used: Recombinant, SDS Page, Purification, Staining, Cleavage Assay, Polyacrylamide Gel Electrophoresis

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    Article Snippet: A BCA assay was performed on the soluble protein samples in order to determine the total protein concentration. .. Western blot analysis The soluble protein samples were heated for 5 min at 95° C in Blue Loading Buffer (New England Biolabs) with 40 mM of dithiothreitol and a total of 10 μg (β-actin), 20 μg (γH2AX), 25 μg (PS1), 35 μg (LRP), 40 μg (APP and H2AX), 50 μg (mTERT) and 60 μg (BACE1) of protein was then separated on AnykD™ Criterion™ TGX Stain-Free™ Protein Gels (Biorad). .. A prestained molecular weight marker (PageRuler Prestained Protein Ladder, Thermo Fisher) was loaded onto each gel.

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    Article Snippet: .. Western BlottingThree micrograms of proteins from the whole cell lysates or cytoplasmic/periplasmic fractions were mixed with 3 × SDS Blue Loading Buffer (New England Biolabs Inc.), denatured at 95°C for 10 min, and then loaded in 12% Mini-PROTEAN TGX Precast Protein Gels (Bio-Rad) for electrophoresis at 80–120 V for 90 min. .. The proteins were transferred onto a nitrocellulose membrane (Bio-Rad) at 25 V for 30 min using a Trans-Blot semidry transfer cell (Bio-Rad) in transfer buffer (14.41 g/L glycine, 3.03 g/L Tris base, 0.075 g/L SDS, and 20% methanol).

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    Staining:

    Article Title: LRP/LR specific antibody IgG1-iS18 impedes neurodegeneration in Alzheimer's disease mice
    Article Snippet: A BCA assay was performed on the soluble protein samples in order to determine the total protein concentration. .. Western blot analysis The soluble protein samples were heated for 5 min at 95° C in Blue Loading Buffer (New England Biolabs) with 40 mM of dithiothreitol and a total of 10 μg (β-actin), 20 μg (γH2AX), 25 μg (PS1), 35 μg (LRP), 40 μg (APP and H2AX), 50 μg (mTERT) and 60 μg (BACE1) of protein was then separated on AnykD™ Criterion™ TGX Stain-Free™ Protein Gels (Biorad). .. A prestained molecular weight marker (PageRuler Prestained Protein Ladder, Thermo Fisher) was loaded onto each gel.

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    Article Snippet: A BCA assay was performed on the soluble protein samples in order to determine the total protein concentration. .. The soluble protein samples were heated for 5 min at 95° C in Blue Loading Buffer (New England Biolabs) with 40 mM of dithiothreitol and a total of 10 μg (β-actin), 20 μg (γH2AX), 25 μg (PS1), 35 μg (LRP), 40 μg (APP and H2AX), 50 μg (mTERT) and 60 μg (BACE1) of protein was then separated on AnykD™ Criterion™ TGX Stain-Free™ Protein Gels (Biorad). .. A prestained molecular weight marker (PageRuler Prestained Protein Ladder, Thermo Fisher) was loaded onto each gel.

    Electrophoresis:

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    SDS Page:

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    Concentration Assay:

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    New England Biolabs dna loading buffer
    The bulk of HBc183 in capsids from human hepatoma cells is highly phosphorylated. (A) Enrichment of particulate HBc by Nycodenz gradient sedimentation. Cytoplasmic lysate from HBV producing HepG2.117 cells was sedimented through a Nycodenz gradient. Fractions were analyzed by SDS-PAGE and CB staining (top panel; asterisks mark a 21 kDa band possibly representing HBc183; the complete gel is shown in S12A Fig ); by NAGE followed by immunoblotting with the anti-HBc assembly domain mAb 312 as PO conjugate (middle panel); and by hybridization with a 32 P-labeled HBV probe. (B) Southern blot for capsid-borne HBV <t>DNA</t> in individual gradient fractions. M, DNA marker comprising HBV-specific fragments of the indicated sizes loaded in native ds form, or in heat-denatured ss form. (C) Phos-Tag SDS-PAGE immunoblot. Aliquots from the respective gradient fractions and recombinant HBc183 coexpressed with the indicated kinases, or not (ø), were separated by Phos-Tag SDS-PAGE and immuno-blotted with mAb 1D8. Short exposure showed one band with comparably strong retardation as SRPK1-phosphorylated HBc183. Longer exposure (left panel) revealed weak additional bands with mobilities similar to those of unmodified and PKC and PKA phosphorylated HBc183 (arrowheads). (D) Enveloped capsids contain phosphorylated HBc183. PEG-precipitated particles in supernatants from HepG2.117 cells, or from an HBc183 producing Huh7 line, H4-15 were analyzed by NAGE immunoblotting alongside E . coli HBc183 <t>CLPs.</t> The blot was sequentially probed with mAb T2212 (anti-phospho-CTD), mAb 312, and mAb 9H9 (anti-HBs). Note that T2212 detected only HBc from eukaryotic cells, including a low mobility species that comigrated with HBsAg and was absent from the H4-15 samples; it therefore represents enveloped capsids. Additional data employing mAb T2212 are presented in S12 Fig .
    Dna Loading Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs rna loading buffer
    X-ray crystal structure of a TSEN15–34 heterodimer derived by limited proteolysis. a , Analysis of the limited tryptic digestion of the TSEN15–34 heterodimer by SDS-PAGE. b , Denaturing mass spectrum of the proteolytically stable fragments of the TSEN15–34 heterodimer from an aqueous ammonium acetate solution. The mass spectrum shows the presence of a TSEN34 fragment (orange circles), and two TSEN15 fragments (dark and light green circles) differing in mass only by a C-terminal arginine as revealed by LC-MS/MS. c , Bar diagrams of tryptic fragments of TSEN15 and TSEN34. Proteolyzed regions are indicated by dashed boxes. Positions of PCH mutations are shown. d , Purification of the re-cloned core of the TSEN15–34 heterodimer via SEC. The absorbance profile at 280 nm is shown. Fractions of the indicated retention range (grey area) were analyzed by SDS-PAGE. e , Asymmetric unit of the TSEN15–34 crystal. The biological unit (bracket) and the domain-swap area (dashed box) are indicated. α-helices and β-sheets are numbered for each subunit. f , Stick representation of amino acids of the domain-swap area with electron density (2F o -F c , 1.5σ). g , Molecular mass determination by size exclusion chromatography multi-angle light scattering (SEC-MALS) of the TSEN15–34 sample used for crystallization. The data reveal a dominant population of a dimer-of-a-heterodimer (13.2 ml, 56.6 kDa) and a minor populated heterodimer (14.9 ml, 30.0 kDa). Light scattering is shown as red dots. Mass determination by SEC-MALS was confirmed by two independent experiments. h , Superposition of the TSEN15–34 heterodimer and the <t>pre-tRNA</t> endonuclease from Archaeoglobus fulgidus at the interaction sites with the bulge-helix-bulge <t>RNA</t> (PDB ID 2GJW). Nucleotide positions of the RNA (black) and residues of the catalytic triads are shown. i , Representative thermal denaturation curves as shown in Fig. 3g of recombinant wt TSEN and mutant TSEN (T15 H116Y ) complexes derived from DSF. Sigmoidal Boltzmann fits are shown as red lines. Grey zones show standard deviations (SD) from technical triplicates. Denaturation temperature (T d ) is presented with error of fit. Unprocessed gels for a and d are shown in Source Data 6.
    Rna Loading Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rna loading buffer/product/New England Biolabs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    New England Biolabs rna
    E rns is a nicking endoribonuclease. ( a ) Schematic representation of the RNase activity assay with the modified hybrids. A single-stranded <t>RNA</t> of positive polarity (ssRNA+) labeled in red together with either a single-stranded methylated RNA (metRNA) or a ssDNA of negative polarity, both labeled in green, were boiled and cooled down at room temperature for hybridization (RNA/metRNA or <t>RNA/DNA).</t> Strep-tag purified wild-type (C171), monomeric (R171) and RNase-inactive mutant (H30F) of E rns were incubated at the concentrations indicated with 625 nM single-stranded metRNA or DNA ( b ), double-stranded RNA/DNA− ( c ) or double-stranded RNA/metRNA-hybrids ( d ). Samples were separated by 14% SDS-PAGE and fluorescence was analysed with a Li-Cor Odyssey system. Due to the known, defined length of the directly labeled fragments (30 b for ssRNA, 30 bp for dsRNA), no size ladder was applied. Non-cropped gels as representative experiment out of three ( b ) or four ( c , d ) are shown.
    Rna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    The bulk of HBc183 in capsids from human hepatoma cells is highly phosphorylated. (A) Enrichment of particulate HBc by Nycodenz gradient sedimentation. Cytoplasmic lysate from HBV producing HepG2.117 cells was sedimented through a Nycodenz gradient. Fractions were analyzed by SDS-PAGE and CB staining (top panel; asterisks mark a 21 kDa band possibly representing HBc183; the complete gel is shown in S12A Fig ); by NAGE followed by immunoblotting with the anti-HBc assembly domain mAb 312 as PO conjugate (middle panel); and by hybridization with a 32 P-labeled HBV probe. (B) Southern blot for capsid-borne HBV DNA in individual gradient fractions. M, DNA marker comprising HBV-specific fragments of the indicated sizes loaded in native ds form, or in heat-denatured ss form. (C) Phos-Tag SDS-PAGE immunoblot. Aliquots from the respective gradient fractions and recombinant HBc183 coexpressed with the indicated kinases, or not (ø), were separated by Phos-Tag SDS-PAGE and immuno-blotted with mAb 1D8. Short exposure showed one band with comparably strong retardation as SRPK1-phosphorylated HBc183. Longer exposure (left panel) revealed weak additional bands with mobilities similar to those of unmodified and PKC and PKA phosphorylated HBc183 (arrowheads). (D) Enveloped capsids contain phosphorylated HBc183. PEG-precipitated particles in supernatants from HepG2.117 cells, or from an HBc183 producing Huh7 line, H4-15 were analyzed by NAGE immunoblotting alongside E . coli HBc183 CLPs. The blot was sequentially probed with mAb T2212 (anti-phospho-CTD), mAb 312, and mAb 9H9 (anti-HBs). Note that T2212 detected only HBc from eukaryotic cells, including a low mobility species that comigrated with HBsAg and was absent from the H4-15 samples; it therefore represents enveloped capsids. Additional data employing mAb T2212 are presented in S12 Fig .

    Journal: PLoS Pathogens

    Article Title: Hepatitis B virus core protein phosphorylation: Identification of the SRPK1 target sites and impact of their occupancy on RNA binding and capsid structure

    doi: 10.1371/journal.ppat.1007488

    Figure Lengend Snippet: The bulk of HBc183 in capsids from human hepatoma cells is highly phosphorylated. (A) Enrichment of particulate HBc by Nycodenz gradient sedimentation. Cytoplasmic lysate from HBV producing HepG2.117 cells was sedimented through a Nycodenz gradient. Fractions were analyzed by SDS-PAGE and CB staining (top panel; asterisks mark a 21 kDa band possibly representing HBc183; the complete gel is shown in S12A Fig ); by NAGE followed by immunoblotting with the anti-HBc assembly domain mAb 312 as PO conjugate (middle panel); and by hybridization with a 32 P-labeled HBV probe. (B) Southern blot for capsid-borne HBV DNA in individual gradient fractions. M, DNA marker comprising HBV-specific fragments of the indicated sizes loaded in native ds form, or in heat-denatured ss form. (C) Phos-Tag SDS-PAGE immunoblot. Aliquots from the respective gradient fractions and recombinant HBc183 coexpressed with the indicated kinases, or not (ø), were separated by Phos-Tag SDS-PAGE and immuno-blotted with mAb 1D8. Short exposure showed one band with comparably strong retardation as SRPK1-phosphorylated HBc183. Longer exposure (left panel) revealed weak additional bands with mobilities similar to those of unmodified and PKC and PKA phosphorylated HBc183 (arrowheads). (D) Enveloped capsids contain phosphorylated HBc183. PEG-precipitated particles in supernatants from HepG2.117 cells, or from an HBc183 producing Huh7 line, H4-15 were analyzed by NAGE immunoblotting alongside E . coli HBc183 CLPs. The blot was sequentially probed with mAb T2212 (anti-phospho-CTD), mAb 312, and mAb 9H9 (anti-HBs). Note that T2212 detected only HBc from eukaryotic cells, including a low mobility species that comigrated with HBsAg and was absent from the H4-15 samples; it therefore represents enveloped capsids. Additional data employing mAb T2212 are presented in S12 Fig .

    Article Snippet: HBc CLP sensitivity against SDS About 5 μg of sucrose gradient enriched HBc CLPs were incubated for 30 min with increasing amounts of a 6x DNA loading buffer containing 0.48% SDS (NEB Purple) to yield final SDS concentrations from 0.024% to 0.24%; samples incubated in a non-denaturing 6x loading buffer served as control.

    Techniques: Sedimentation, SDS Page, Staining, Hybridization, Labeling, Southern Blot, Marker, Recombinant

    Little impact on SDS sensitivity of CLPs by high phosphorylation, low RNA content and F97L mutation. About 5 μg HBc protein from the indicated CLP preparations were directly loaded (Ø), or after 30 min incubation in non-denaturing 6x DNA loading buffer (Ø*) or in SDS-containing DNA loading buffer (NEB Purple) at the indicated final SDS concentrations, then analyzed by NAGE. A 1 kb DNA ladder (M) plus 1 μg of untreated HBc183 CLPs served as markers. EB fluorescence signals (top) were recorded using a laser scanner (excitation 532 nm/O580 nm filter). Proteins were subsequently stained by CB. In all samples the intact CLP bands became fuzzier at 0.024% SDS compared to the untreated controls, and a distinct upward mobility shift occurred at 0.048% SDS. For the non-phosphorylated CLPs (-SRPK1) this was accompanied by visible release of RNA.

    Journal: PLoS Pathogens

    Article Title: Hepatitis B virus core protein phosphorylation: Identification of the SRPK1 target sites and impact of their occupancy on RNA binding and capsid structure

    doi: 10.1371/journal.ppat.1007488

    Figure Lengend Snippet: Little impact on SDS sensitivity of CLPs by high phosphorylation, low RNA content and F97L mutation. About 5 μg HBc protein from the indicated CLP preparations were directly loaded (Ø), or after 30 min incubation in non-denaturing 6x DNA loading buffer (Ø*) or in SDS-containing DNA loading buffer (NEB Purple) at the indicated final SDS concentrations, then analyzed by NAGE. A 1 kb DNA ladder (M) plus 1 μg of untreated HBc183 CLPs served as markers. EB fluorescence signals (top) were recorded using a laser scanner (excitation 532 nm/O580 nm filter). Proteins were subsequently stained by CB. In all samples the intact CLP bands became fuzzier at 0.024% SDS compared to the untreated controls, and a distinct upward mobility shift occurred at 0.048% SDS. For the non-phosphorylated CLPs (-SRPK1) this was accompanied by visible release of RNA.

    Article Snippet: HBc CLP sensitivity against SDS About 5 μg of sucrose gradient enriched HBc CLPs were incubated for 30 min with increasing amounts of a 6x DNA loading buffer containing 0.48% SDS (NEB Purple) to yield final SDS concentrations from 0.024% to 0.24%; samples incubated in a non-denaturing 6x loading buffer served as control.

    Techniques: Mutagenesis, Incubation, Fluorescence, Staining, Mobility Shift

    Implications of the high HBc phosphorylation—Low CLP RNA content correlation for specific pgRNA encapsidation in HBV infection. In E . coli nonphosphorylated HBc183 CTDs have maximal positive charge and thus maximal electrostatic RNA binding capacity, generating RNA-filled CLPs ( Fig 1B ). Seven-fold phosphorylation by SRPK1 neutralizes most positive charges, leading to virtually empty capsids. The similarly strong Phos-tag retardation of most HBc183 from human cells indicates similarly high phosphorylation, whether by SRPK1 or a combination of kinases. Strongly reduced RNA binding favors formation of empty capsids and then empty virions, perhaps the dominant pathway in vivo [ 52 ]. The backside of avoiding irrelevant RNA packaging is a loss in specific pgRNA interaction capability. As blocking just one of the seven SRPK1 phosphorylation sites restored substantial RNA packaging in bacteria we propose that the pgRNA/P protein complex, besides other host factors, also carries a protein phosphatase (PPase) activity. This PPase would dephosphorylate only nearby HBc CTDs, and thus locally unleash their RNA binding potential in proximity to pgRNA. This would go on with further HBc dimers until the shell is completed. Progressive dephosphorylation of the now internal CTDs by the PPase activity could maintain electrostatic homeostasis, especially during the near doubling of negative charges associated with dsDNA formation. Targeted nucleocapsid destabilization upon infection of a new cell could occur by CTD re-phosphorylation and completion of plus-strand DNA.

    Journal: PLoS Pathogens

    Article Title: Hepatitis B virus core protein phosphorylation: Identification of the SRPK1 target sites and impact of their occupancy on RNA binding and capsid structure

    doi: 10.1371/journal.ppat.1007488

    Figure Lengend Snippet: Implications of the high HBc phosphorylation—Low CLP RNA content correlation for specific pgRNA encapsidation in HBV infection. In E . coli nonphosphorylated HBc183 CTDs have maximal positive charge and thus maximal electrostatic RNA binding capacity, generating RNA-filled CLPs ( Fig 1B ). Seven-fold phosphorylation by SRPK1 neutralizes most positive charges, leading to virtually empty capsids. The similarly strong Phos-tag retardation of most HBc183 from human cells indicates similarly high phosphorylation, whether by SRPK1 or a combination of kinases. Strongly reduced RNA binding favors formation of empty capsids and then empty virions, perhaps the dominant pathway in vivo [ 52 ]. The backside of avoiding irrelevant RNA packaging is a loss in specific pgRNA interaction capability. As blocking just one of the seven SRPK1 phosphorylation sites restored substantial RNA packaging in bacteria we propose that the pgRNA/P protein complex, besides other host factors, also carries a protein phosphatase (PPase) activity. This PPase would dephosphorylate only nearby HBc CTDs, and thus locally unleash their RNA binding potential in proximity to pgRNA. This would go on with further HBc dimers until the shell is completed. Progressive dephosphorylation of the now internal CTDs by the PPase activity could maintain electrostatic homeostasis, especially during the near doubling of negative charges associated with dsDNA formation. Targeted nucleocapsid destabilization upon infection of a new cell could occur by CTD re-phosphorylation and completion of plus-strand DNA.

    Article Snippet: HBc CLP sensitivity against SDS About 5 μg of sucrose gradient enriched HBc CLPs were incubated for 30 min with increasing amounts of a 6x DNA loading buffer containing 0.48% SDS (NEB Purple) to yield final SDS concentrations from 0.024% to 0.24%; samples incubated in a non-denaturing 6x loading buffer served as control.

    Techniques: Infection, RNA Binding Assay, In Vivo, Blocking Assay, Activity Assay, De-Phosphorylation Assay

    X-ray crystal structure of a TSEN15–34 heterodimer derived by limited proteolysis. a , Analysis of the limited tryptic digestion of the TSEN15–34 heterodimer by SDS-PAGE. b , Denaturing mass spectrum of the proteolytically stable fragments of the TSEN15–34 heterodimer from an aqueous ammonium acetate solution. The mass spectrum shows the presence of a TSEN34 fragment (orange circles), and two TSEN15 fragments (dark and light green circles) differing in mass only by a C-terminal arginine as revealed by LC-MS/MS. c , Bar diagrams of tryptic fragments of TSEN15 and TSEN34. Proteolyzed regions are indicated by dashed boxes. Positions of PCH mutations are shown. d , Purification of the re-cloned core of the TSEN15–34 heterodimer via SEC. The absorbance profile at 280 nm is shown. Fractions of the indicated retention range (grey area) were analyzed by SDS-PAGE. e , Asymmetric unit of the TSEN15–34 crystal. The biological unit (bracket) and the domain-swap area (dashed box) are indicated. α-helices and β-sheets are numbered for each subunit. f , Stick representation of amino acids of the domain-swap area with electron density (2F o -F c , 1.5σ). g , Molecular mass determination by size exclusion chromatography multi-angle light scattering (SEC-MALS) of the TSEN15–34 sample used for crystallization. The data reveal a dominant population of a dimer-of-a-heterodimer (13.2 ml, 56.6 kDa) and a minor populated heterodimer (14.9 ml, 30.0 kDa). Light scattering is shown as red dots. Mass determination by SEC-MALS was confirmed by two independent experiments. h , Superposition of the TSEN15–34 heterodimer and the pre-tRNA endonuclease from Archaeoglobus fulgidus at the interaction sites with the bulge-helix-bulge RNA (PDB ID 2GJW). Nucleotide positions of the RNA (black) and residues of the catalytic triads are shown. i , Representative thermal denaturation curves as shown in Fig. 3g of recombinant wt TSEN and mutant TSEN (T15 H116Y ) complexes derived from DSF. Sigmoidal Boltzmann fits are shown as red lines. Grey zones show standard deviations (SD) from technical triplicates. Denaturation temperature (T d ) is presented with error of fit. Unprocessed gels for a and d are shown in Source Data 6.

    Journal: bioRxiv

    Article Title: Assembly defects of the human tRNA splicing endonuclease contribute to impaired pre-tRNA processing in pontocerebellar hypoplasia

    doi: 10.1101/2020.08.03.234229

    Figure Lengend Snippet: X-ray crystal structure of a TSEN15–34 heterodimer derived by limited proteolysis. a , Analysis of the limited tryptic digestion of the TSEN15–34 heterodimer by SDS-PAGE. b , Denaturing mass spectrum of the proteolytically stable fragments of the TSEN15–34 heterodimer from an aqueous ammonium acetate solution. The mass spectrum shows the presence of a TSEN34 fragment (orange circles), and two TSEN15 fragments (dark and light green circles) differing in mass only by a C-terminal arginine as revealed by LC-MS/MS. c , Bar diagrams of tryptic fragments of TSEN15 and TSEN34. Proteolyzed regions are indicated by dashed boxes. Positions of PCH mutations are shown. d , Purification of the re-cloned core of the TSEN15–34 heterodimer via SEC. The absorbance profile at 280 nm is shown. Fractions of the indicated retention range (grey area) were analyzed by SDS-PAGE. e , Asymmetric unit of the TSEN15–34 crystal. The biological unit (bracket) and the domain-swap area (dashed box) are indicated. α-helices and β-sheets are numbered for each subunit. f , Stick representation of amino acids of the domain-swap area with electron density (2F o -F c , 1.5σ). g , Molecular mass determination by size exclusion chromatography multi-angle light scattering (SEC-MALS) of the TSEN15–34 sample used for crystallization. The data reveal a dominant population of a dimer-of-a-heterodimer (13.2 ml, 56.6 kDa) and a minor populated heterodimer (14.9 ml, 30.0 kDa). Light scattering is shown as red dots. Mass determination by SEC-MALS was confirmed by two independent experiments. h , Superposition of the TSEN15–34 heterodimer and the pre-tRNA endonuclease from Archaeoglobus fulgidus at the interaction sites with the bulge-helix-bulge RNA (PDB ID 2GJW). Nucleotide positions of the RNA (black) and residues of the catalytic triads are shown. i , Representative thermal denaturation curves as shown in Fig. 3g of recombinant wt TSEN and mutant TSEN (T15 H116Y ) complexes derived from DSF. Sigmoidal Boltzmann fits are shown as red lines. Grey zones show standard deviations (SD) from technical triplicates. Denaturation temperature (T d ) is presented with error of fit. Unprocessed gels for a and d are shown in Source Data 6.

    Article Snippet: Prior to use, pre-tRNA stock was diluted 1 in 2 into RNA loading buffer (New England Biolabs) and separated on a 10% acrylamide urea-TBE denaturing gel, with the band corresponding to pre-tRNA excised.

    Techniques: Derivative Assay, SDS Page, Liquid Chromatography with Mass Spectroscopy, Purification, Clone Assay, Size-exclusion Chromatography, Crystallization Assay, Recombinant, Mutagenesis

    Active involvement of the A–I base pair in coordinating pre-tRNA cleavage. a , SDS-PAGE of purified, recombinant inactive TSEN and inactive TSEN/CLP1 complexes (TSEN2 H377A and TSEN34 H255A double mutant). Protein size markers and protein identities are indicated. b , Two-colored pre-tRNA cleavage assay with TSEN-STREP and TSEN/CLP1-FLAG wt complexes and complexes carrying the TSEN2 H377A (T2 H377A ) or TSEN34 H255A (T34 H255A ) substitution. RNA cleavage products were separated on a denaturing Urea-PAGE and visualized by fluorescence of cyanine5 (Cy5) and Fluorescin (FITC). c , Thermodynamic competition parameters deduced from fluorescence anisotropy experiments. Inactive, tetrameric TSEN bound to fluorescently labeled pre-tRNA Phe GAA was titrated with unlabeled pre-tRNA. d , Electrophoretic mobility shift assay with fluorescently labeled pre-tRNA Phe GAA and inactive, tetrameric TSEN (TSEN inactive ). Free and bound fractions of pre-tRNA were analyzed via 4% TBE native PAGE with subsequent in-gel fluorescence measurement. e , Impact of A-I base pair mutations in pre-tRNA Tyr GTA 8-1 on endonucleolytic activity by tetrameric TSEN revealed by a pre-tRNA cleavage assay. CI 95 – 95% confidence interval, C 32 :G 52 – canonical A-I base pair, C 32 :C 52 and G 32 :G 52 – disrupted A-I base pair, G 32 :C 52 – inverted A-I base pair. Panels are representatives of three independent experiments. Unprocessed gels for a , b , d , and e are shown in Source Data 4.

    Journal: bioRxiv

    Article Title: Assembly defects of the human tRNA splicing endonuclease contribute to impaired pre-tRNA processing in pontocerebellar hypoplasia

    doi: 10.1101/2020.08.03.234229

    Figure Lengend Snippet: Active involvement of the A–I base pair in coordinating pre-tRNA cleavage. a , SDS-PAGE of purified, recombinant inactive TSEN and inactive TSEN/CLP1 complexes (TSEN2 H377A and TSEN34 H255A double mutant). Protein size markers and protein identities are indicated. b , Two-colored pre-tRNA cleavage assay with TSEN-STREP and TSEN/CLP1-FLAG wt complexes and complexes carrying the TSEN2 H377A (T2 H377A ) or TSEN34 H255A (T34 H255A ) substitution. RNA cleavage products were separated on a denaturing Urea-PAGE and visualized by fluorescence of cyanine5 (Cy5) and Fluorescin (FITC). c , Thermodynamic competition parameters deduced from fluorescence anisotropy experiments. Inactive, tetrameric TSEN bound to fluorescently labeled pre-tRNA Phe GAA was titrated with unlabeled pre-tRNA. d , Electrophoretic mobility shift assay with fluorescently labeled pre-tRNA Phe GAA and inactive, tetrameric TSEN (TSEN inactive ). Free and bound fractions of pre-tRNA were analyzed via 4% TBE native PAGE with subsequent in-gel fluorescence measurement. e , Impact of A-I base pair mutations in pre-tRNA Tyr GTA 8-1 on endonucleolytic activity by tetrameric TSEN revealed by a pre-tRNA cleavage assay. CI 95 – 95% confidence interval, C 32 :G 52 – canonical A-I base pair, C 32 :C 52 and G 32 :G 52 – disrupted A-I base pair, G 32 :C 52 – inverted A-I base pair. Panels are representatives of three independent experiments. Unprocessed gels for a , b , d , and e are shown in Source Data 4.

    Article Snippet: Prior to use, pre-tRNA stock was diluted 1 in 2 into RNA loading buffer (New England Biolabs) and separated on a 10% acrylamide urea-TBE denaturing gel, with the band corresponding to pre-tRNA excised.

    Techniques: SDS Page, Purification, Recombinant, Mutagenesis, Cleavage Assay, Polyacrylamide Gel Electrophoresis, Fluorescence, Labeling, Electrophoretic Mobility Shift Assay, Clear Native PAGE, Activity Assay

    Active involvement of the A–I base pair in coordinating pre-tRNA cleavage. a , Pre-tRNA cleavage assay comparing recombinant, inactive TSEN tetramer (TSEN2 H377A and TSEN34 H255A double mutant) to the TSEN/CLP1 complex. Cleavage products are visualized by denaturing Urea-PAGE with subsequent Toluidine blue staining. RNA size markers are indicated on the left of the gel. b , Pull-down assay with fluorescently labeled S.c. pre-tRNA Phe GAA and inactive, tetrameric TSEN captured on protein G agarose functionalized with an α-His-antibody. Protein size markers are indicated on the left of each immunoblot, protein and RNA identities on the right. Input and co-precipitated, labeled pre-tRNAs were visualized by in-gel fluorescence, TSEN subunits and the immunoglobulin G (IgG) heavy chain by immunoblotting. The IgG heavy chain served as loading control. c, Thermodynamic binding parameters of fluorescently labeled S.c. pre-tRNA Phe GAA and inactive, tetrameric TSEN revealed by fluorescence anisotropy. d , Thermodynamic binding parameters of fluorescently labeled tRNA Phe GAA and inactive, tetrameric TSEN revealed by fluorescence anisotropy. e , Schematic depiction of a pre-tRNA molecule showing ribonucleotides belonging to the mature domain (grey spheres), the intronic region (white spheres), the anticodon (black spheres), and the A-I base pair (yellow spheres). Proposed 5’ and 3’ splice sites (ss) are indicated. f , Impact of A-I base pair mutations in S.c. pre-tRNA Phe GAA on endonucleolytic activity of tetrameric TSEN revealed by a pre-tRNA cleavage assay. C 32 :G 54 – canonical A-I base pair, C 32 :C 54 – disrupted A-I base pair, G 32 :C 54 – inverted A-I base pair. All experiments are representatives of three independent assays. Unprocessed gels for a , b and f are shown in Source Data 3.

    Journal: bioRxiv

    Article Title: Assembly defects of the human tRNA splicing endonuclease contribute to impaired pre-tRNA processing in pontocerebellar hypoplasia

    doi: 10.1101/2020.08.03.234229

    Figure Lengend Snippet: Active involvement of the A–I base pair in coordinating pre-tRNA cleavage. a , Pre-tRNA cleavage assay comparing recombinant, inactive TSEN tetramer (TSEN2 H377A and TSEN34 H255A double mutant) to the TSEN/CLP1 complex. Cleavage products are visualized by denaturing Urea-PAGE with subsequent Toluidine blue staining. RNA size markers are indicated on the left of the gel. b , Pull-down assay with fluorescently labeled S.c. pre-tRNA Phe GAA and inactive, tetrameric TSEN captured on protein G agarose functionalized with an α-His-antibody. Protein size markers are indicated on the left of each immunoblot, protein and RNA identities on the right. Input and co-precipitated, labeled pre-tRNAs were visualized by in-gel fluorescence, TSEN subunits and the immunoglobulin G (IgG) heavy chain by immunoblotting. The IgG heavy chain served as loading control. c, Thermodynamic binding parameters of fluorescently labeled S.c. pre-tRNA Phe GAA and inactive, tetrameric TSEN revealed by fluorescence anisotropy. d , Thermodynamic binding parameters of fluorescently labeled tRNA Phe GAA and inactive, tetrameric TSEN revealed by fluorescence anisotropy. e , Schematic depiction of a pre-tRNA molecule showing ribonucleotides belonging to the mature domain (grey spheres), the intronic region (white spheres), the anticodon (black spheres), and the A-I base pair (yellow spheres). Proposed 5’ and 3’ splice sites (ss) are indicated. f , Impact of A-I base pair mutations in S.c. pre-tRNA Phe GAA on endonucleolytic activity of tetrameric TSEN revealed by a pre-tRNA cleavage assay. C 32 :G 54 – canonical A-I base pair, C 32 :C 54 – disrupted A-I base pair, G 32 :C 54 – inverted A-I base pair. All experiments are representatives of three independent assays. Unprocessed gels for a , b and f are shown in Source Data 3.

    Article Snippet: Prior to use, pre-tRNA stock was diluted 1 in 2 into RNA loading buffer (New England Biolabs) and separated on a 10% acrylamide urea-TBE denaturing gel, with the band corresponding to pre-tRNA excised.

    Techniques: Cleavage Assay, Recombinant, Mutagenesis, Polyacrylamide Gel Electrophoresis, Staining, Pull Down Assay, Labeling, Fluorescence, Binding Assay, Activity Assay

    Assembly and catalysis of recombinant human TSEN. a , Bar diagrams of TSEN subunits and CLP1 depicting positions of PCH mutations, predicted nuclease domains of TSEN2 and TSEN34, and the RNA kinase domain for CLP1. Total amino acids of each protein are indicated. b , SDS-PAGE of purified recombinant TSEN and TSEN/CLP1 complexes visualized by InstantBlue staining. Protein identities and size markers are shown. c , Native mass spectrum of tetrameric TSEN complex from an aqueous ammonium acetate solution. Charge states of the predominant TSEN2–15– 34–54 heterotetramer (blue circles), Heat Shock Protein (HSP) 70 (grey circles), the heterodimer TSEN15–34 (red circles) and TSEN15 (green circles) are indicated. d , Pre-tRNA cleavage assay using tetrameric TSEN complex with Saccharomyces cerevisia e ( S.c. ) pre-tRNA Phe GAA and mature tRNA Phe GAA . Input samples and cleavage products were separated via Urea-PAGE and visualized by Toluidine blue. RNA denominations are given on the right. e , Pre-tRNA cleavage assay with TSEN heterodimers and S.c. pre-tRNA Phe GAA . SDS-PAGE of the indicated heterodimers and the reconstituted TSEN tetramer is shown on the left (InstantBlue stain), Urea-PAGE of the cleavage products on the right (Toluidine blue stain). Gels are representative of three independent experiments. Unprocessed gels for b , c and e are shown in Source Data 1.

    Journal: bioRxiv

    Article Title: Assembly defects of the human tRNA splicing endonuclease contribute to impaired pre-tRNA processing in pontocerebellar hypoplasia

    doi: 10.1101/2020.08.03.234229

    Figure Lengend Snippet: Assembly and catalysis of recombinant human TSEN. a , Bar diagrams of TSEN subunits and CLP1 depicting positions of PCH mutations, predicted nuclease domains of TSEN2 and TSEN34, and the RNA kinase domain for CLP1. Total amino acids of each protein are indicated. b , SDS-PAGE of purified recombinant TSEN and TSEN/CLP1 complexes visualized by InstantBlue staining. Protein identities and size markers are shown. c , Native mass spectrum of tetrameric TSEN complex from an aqueous ammonium acetate solution. Charge states of the predominant TSEN2–15– 34–54 heterotetramer (blue circles), Heat Shock Protein (HSP) 70 (grey circles), the heterodimer TSEN15–34 (red circles) and TSEN15 (green circles) are indicated. d , Pre-tRNA cleavage assay using tetrameric TSEN complex with Saccharomyces cerevisia e ( S.c. ) pre-tRNA Phe GAA and mature tRNA Phe GAA . Input samples and cleavage products were separated via Urea-PAGE and visualized by Toluidine blue. RNA denominations are given on the right. e , Pre-tRNA cleavage assay with TSEN heterodimers and S.c. pre-tRNA Phe GAA . SDS-PAGE of the indicated heterodimers and the reconstituted TSEN tetramer is shown on the left (InstantBlue stain), Urea-PAGE of the cleavage products on the right (Toluidine blue stain). Gels are representative of three independent experiments. Unprocessed gels for b , c and e are shown in Source Data 1.

    Article Snippet: Prior to use, pre-tRNA stock was diluted 1 in 2 into RNA loading buffer (New England Biolabs) and separated on a 10% acrylamide urea-TBE denaturing gel, with the band corresponding to pre-tRNA excised.

    Techniques: Recombinant, SDS Page, Purification, Staining, Cleavage Assay, Polyacrylamide Gel Electrophoresis

    E rns is a nicking endoribonuclease. ( a ) Schematic representation of the RNase activity assay with the modified hybrids. A single-stranded RNA of positive polarity (ssRNA+) labeled in red together with either a single-stranded methylated RNA (metRNA) or a ssDNA of negative polarity, both labeled in green, were boiled and cooled down at room temperature for hybridization (RNA/metRNA or RNA/DNA). Strep-tag purified wild-type (C171), monomeric (R171) and RNase-inactive mutant (H30F) of E rns were incubated at the concentrations indicated with 625 nM single-stranded metRNA or DNA ( b ), double-stranded RNA/DNA− ( c ) or double-stranded RNA/metRNA-hybrids ( d ). Samples were separated by 14% SDS-PAGE and fluorescence was analysed with a Li-Cor Odyssey system. Due to the known, defined length of the directly labeled fragments (30 b for ssRNA, 30 bp for dsRNA), no size ladder was applied. Non-cropped gels as representative experiment out of three ( b ) or four ( c , d ) are shown.

    Journal: Scientific Reports

    Article Title: Homodimerisation-independent cleavage of dsRNA by a pestiviral nicking endoribonuclease

    doi: 10.1038/s41598-018-26557-4

    Figure Lengend Snippet: E rns is a nicking endoribonuclease. ( a ) Schematic representation of the RNase activity assay with the modified hybrids. A single-stranded RNA of positive polarity (ssRNA+) labeled in red together with either a single-stranded methylated RNA (metRNA) or a ssDNA of negative polarity, both labeled in green, were boiled and cooled down at room temperature for hybridization (RNA/metRNA or RNA/DNA). Strep-tag purified wild-type (C171), monomeric (R171) and RNase-inactive mutant (H30F) of E rns were incubated at the concentrations indicated with 625 nM single-stranded metRNA or DNA ( b ), double-stranded RNA/DNA− ( c ) or double-stranded RNA/metRNA-hybrids ( d ). Samples were separated by 14% SDS-PAGE and fluorescence was analysed with a Li-Cor Odyssey system. Due to the known, defined length of the directly labeled fragments (30 b for ssRNA, 30 bp for dsRNA), no size ladder was applied. Non-cropped gels as representative experiment out of three ( b ) or four ( c , d ) are shown.

    Article Snippet: GoTaq DNA Polymerase, BenchTop 100 bp DNA ladder and RNasin were from Promega, whereas the RNA loading dye was obtained from New England Biolabs (Ipswich, MA).

    Techniques: Activity Assay, Modification, Labeling, Methylation, Hybridization, Strep-tag, Purification, Mutagenesis, Incubation, SDS Page, Fluorescence