blue loading buffer pack  (New England Biolabs)


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    Name:
    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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    New England Biolabs blue loading buffer pack
    Blue Loading Buffer Pack
    Blue Loading Buffer Pack 8 0 ml
    https://www.bioz.com/result/blue loading buffer pack/product/New England Biolabs
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    blue loading buffer pack - by Bioz Stars, 2021-06
    95/100 stars

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    Related Articles

    Western Blot:

    Article Title: Signal Recognition Particle RNA Contributes to Oxidative Stress Response in Deinococcus radiodurans by Modulating Catalase Localization
    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).

    Electrophoresis:

    Article Title: Signal Recognition Particle RNA Contributes to Oxidative Stress Response in Deinococcus radiodurans by Modulating Catalase Localization
    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).

    Article Title: Dissecting DNA Compaction by the Bacterial Condensin MukB
    Article Snippet: Add SDS to 0.2 % and Proteinase K to 0.2 mg/ml and continue incubation at 37 °C for 30 min ( see ). .. Load one set of reaction mixtures each on 0.8 % vertical agarose gels with or without chloroquine (do not add any loading dye) and electrophorese for 18 h at 1.8V/cm at room temperature using 1X TAE as the gel and electrophoresis buffer. .. Stain with SYBR Gold and scan as above.

    Article Title: Reconstituted System for the Examination of DNA Repair Synthesis in Homologous Recombination
    Article Snippet: - Dissolve the pellet with 10 μl of NEBuffer 4 supplemented with 0.1 mg/ml BSA. .. - Add 2.5 U of Ahd I or 10 U of Xmn I (20 U/μl) and incubate at 37°C for 10 min. - Terminate and deproteinize the reaction by adding 0.5 μl SDS and 1 μl PK and incubating it at 37°C for 10 min. - Add 5 μl of denaturing gel-loading buffer and incubate at 95°C for 5 min. - Resolve the DNA species by electrophoresis in the denaturing polyacrylamide gel at 55°C and 200 mA for 90 min using TBE buffer as the electrophoresis buffer. ..

    SDS Page:

    Article Title: Cohesin subunit SMC1 associates with mitotic microtubules at the spindle pole
    Article Snippet: The beads were then washed five times with 500 μl of lysis buffer. .. After the last wash, proteins were extracted by 50 μl of 1× SDS/PAGE Blue Loading buffer (New England Biolabs) and analyzed by SDS/PAGE. ..

    Concentration Assay:

    Article Title: Nasal neuron PET imaging quantifies neuron generation and degeneration
    Article Snippet: .. Samples were each diluted to a concentration of 1.5 μg/μl, mixed with one-third volume of 3× SDS loading buffer (B7703S; NEB), and heated to 95°C for 10 minutes. .. Ten micrograms of total protein was loaded in each lane and separated on Criterion Stain-Free 4%–20% gels (567-8095; Bio-Rad) at 150 V for 70 minutes.

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  • 95
    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: 95/100, 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 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rna loading buffer - by Bioz Stars, 2021-06
    95/100 stars
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    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

    Mapping of the SMC1 domain interacting with Rae1. Schematic drawing of the hinge-like structure known to be formed by the two cohesin subunits, SMC1 and SMC3; purple stars indicates the location of the two Ser residues that have been reported to be phosphorylated by ATM ( A Upper ). Domain structure of SMC1 and the constructs 1–5 covering the entire length of SMC1; numbers refer to amino acid residues ( A Lower ). Each of the five untagged SMC1 fragments (see above) and Flag-tagged Rae1 were synthesized in 35 S methionine containing reticulocyte lysate followed by pulldown using anti-Flag coated beads. Pulldown samples were resolved in a 4–20% SDS-polyacrylamide gradient gel and visualized by autoradiography. Note that Rae1 pulled down SMC1 fragment 5, containing residues 947-1233 ( A Lower ; B , lane 5). Numbers on left indicate molecular mass markers in kilodaltons. Further mapping of SMC1–5; three constructs (5A-C) representing residues 947-1100, 947-1025, and 947–967 were made; note that all three contain Ser 957 and Ser 966 , indicated in red ( C ). Fragments 5A-C were cosynthesized with Flag-Rae1 as in B and pulldowns were analyzed by SDS/PAGE by using an 18% acrylamide gel; arrows indicate pulled down fragments 5A, 5B, and 5C ( D ). In vivo expression of HA-tagged SMC1–5B and SMC1–5C and empty HA vector in HeLa cells, followed by anti-HA IP and immunoblotting with anti-Rae1 ( E ).

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Cohesin subunit SMC1 associates with mitotic microtubules at the spindle pole

    doi: 10.1073/pnas.0807660105

    Figure Lengend Snippet: Mapping of the SMC1 domain interacting with Rae1. Schematic drawing of the hinge-like structure known to be formed by the two cohesin subunits, SMC1 and SMC3; purple stars indicates the location of the two Ser residues that have been reported to be phosphorylated by ATM ( A Upper ). Domain structure of SMC1 and the constructs 1–5 covering the entire length of SMC1; numbers refer to amino acid residues ( A Lower ). Each of the five untagged SMC1 fragments (see above) and Flag-tagged Rae1 were synthesized in 35 S methionine containing reticulocyte lysate followed by pulldown using anti-Flag coated beads. Pulldown samples were resolved in a 4–20% SDS-polyacrylamide gradient gel and visualized by autoradiography. Note that Rae1 pulled down SMC1 fragment 5, containing residues 947-1233 ( A Lower ; B , lane 5). Numbers on left indicate molecular mass markers in kilodaltons. Further mapping of SMC1–5; three constructs (5A-C) representing residues 947-1100, 947-1025, and 947–967 were made; note that all three contain Ser 957 and Ser 966 , indicated in red ( C ). Fragments 5A-C were cosynthesized with Flag-Rae1 as in B and pulldowns were analyzed by SDS/PAGE by using an 18% acrylamide gel; arrows indicate pulled down fragments 5A, 5B, and 5C ( D ). In vivo expression of HA-tagged SMC1–5B and SMC1–5C and empty HA vector in HeLa cells, followed by anti-HA IP and immunoblotting with anti-Rae1 ( E ).

    Article Snippet: After the last wash, proteins were extracted by 50 μl of 1× SDS/PAGE Blue Loading buffer (New England Biolabs) and analyzed by SDS/PAGE.

    Techniques: Construct, Synthesized, Autoradiography, SDS Page, Acrylamide Gel Assay, In Vivo, Expressing, Plasmid Preparation

    Human cohesin subunit SMC1 interacts with Rae1 during mitosis. IP from mitotic HeLa cell extracts with anti-Rae1, anti-SMC1, or nonspecific rabbit antibodies (IgG) were analyzed by SDS/PAGE, followed by immunoblotting with Rae1, SMC1, SMC3, Nup98 or tubulin (DM1A) antibodies. In lanes marked “2% input,” 5 μl of 250 μl of extract that was used per IP was analyzed directly ( A and B ). Anti-Rae1 IP was washed with 2.0 M guanidine·HCl before being analyzed by SDS/PAGE and immunoblotting with anti-SMC1 or -SMC3 antibodies ( C ).

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Cohesin subunit SMC1 associates with mitotic microtubules at the spindle pole

    doi: 10.1073/pnas.0807660105

    Figure Lengend Snippet: Human cohesin subunit SMC1 interacts with Rae1 during mitosis. IP from mitotic HeLa cell extracts with anti-Rae1, anti-SMC1, or nonspecific rabbit antibodies (IgG) were analyzed by SDS/PAGE, followed by immunoblotting with Rae1, SMC1, SMC3, Nup98 or tubulin (DM1A) antibodies. In lanes marked “2% input,” 5 μl of 250 μl of extract that was used per IP was analyzed directly ( A and B ). Anti-Rae1 IP was washed with 2.0 M guanidine·HCl before being analyzed by SDS/PAGE and immunoblotting with anti-SMC1 or -SMC3 antibodies ( C ).

    Article Snippet: After the last wash, proteins were extracted by 50 μl of 1× SDS/PAGE Blue Loading buffer (New England Biolabs) and analyzed by SDS/PAGE.

    Techniques: SDS Page