nacl  (Thermo Fisher)


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    Structured Review

    Thermo Fisher nacl
    Purification of <t>Cas9</t> recombinant protein. (A) Purification of Cas9 by affinity chromatography using a Nickel-charged HiTrap Chelating HP. Gradient of Imidazole is indicated by the green line. (B) Coomassie-stained gel electrophoresis of peaks 1 and 2 after affinity chromatography (26 μl of each fraction) shows that peak 2 corresponds to Cas9. M: SeeBlue Plus2 Pre-Stained Protein Standard (Life Technologies). (C) Purification of Cas9 by cation-exchange chromatography using a MonoS 5/50 GL column. Gradient of <t>NaCl</t> is indicated by the green line. (D) Coomassie-stained gel electrophoresis of fractions correspondent to different peaks after cation-exchange chromatography (26 μl of each fraction, peaks 1–4: lanes 1–4). All peaks showed a band of the correct Cas9 molecular weight. Only the majoritarian peak (#2) was collected. Lane 5 corresponds to purified Cas9 after dialysis. M: SeeBlue Plus2 Pre-Stained Protein Standard (Life Technologies). (E) Purification of recombinant Cas9 produced in our laboratory showed endonuclease activity comparable to a commercial Cas9 (PNABio). 200 ng of PCR product of target gene was incubated with each individual sgRNA (lanes 1–12; 3.2 μM) in the presence of 3.8 μM Cas9 protein either purchased from PNABio (upper gel) or obtained in our laboratory (lower gel). As negative controls, 200 ng of PCR product alone (lane 13) or in combination with Cas9 protein (lane 14) were included. All samples were run on 2.2% agarose gels and visualized under UV light.
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    Images

    1) Product Images from "Optimization of sand fly embryo microinjection for gene editing by CRISPR/Cas9"

    Article Title: Optimization of sand fly embryo microinjection for gene editing by CRISPR/Cas9

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0006769

    Purification of Cas9 recombinant protein. (A) Purification of Cas9 by affinity chromatography using a Nickel-charged HiTrap Chelating HP. Gradient of Imidazole is indicated by the green line. (B) Coomassie-stained gel electrophoresis of peaks 1 and 2 after affinity chromatography (26 μl of each fraction) shows that peak 2 corresponds to Cas9. M: SeeBlue Plus2 Pre-Stained Protein Standard (Life Technologies). (C) Purification of Cas9 by cation-exchange chromatography using a MonoS 5/50 GL column. Gradient of NaCl is indicated by the green line. (D) Coomassie-stained gel electrophoresis of fractions correspondent to different peaks after cation-exchange chromatography (26 μl of each fraction, peaks 1–4: lanes 1–4). All peaks showed a band of the correct Cas9 molecular weight. Only the majoritarian peak (#2) was collected. Lane 5 corresponds to purified Cas9 after dialysis. M: SeeBlue Plus2 Pre-Stained Protein Standard (Life Technologies). (E) Purification of recombinant Cas9 produced in our laboratory showed endonuclease activity comparable to a commercial Cas9 (PNABio). 200 ng of PCR product of target gene was incubated with each individual sgRNA (lanes 1–12; 3.2 μM) in the presence of 3.8 μM Cas9 protein either purchased from PNABio (upper gel) or obtained in our laboratory (lower gel). As negative controls, 200 ng of PCR product alone (lane 13) or in combination with Cas9 protein (lane 14) were included. All samples were run on 2.2% agarose gels and visualized under UV light.
    Figure Legend Snippet: Purification of Cas9 recombinant protein. (A) Purification of Cas9 by affinity chromatography using a Nickel-charged HiTrap Chelating HP. Gradient of Imidazole is indicated by the green line. (B) Coomassie-stained gel electrophoresis of peaks 1 and 2 after affinity chromatography (26 μl of each fraction) shows that peak 2 corresponds to Cas9. M: SeeBlue Plus2 Pre-Stained Protein Standard (Life Technologies). (C) Purification of Cas9 by cation-exchange chromatography using a MonoS 5/50 GL column. Gradient of NaCl is indicated by the green line. (D) Coomassie-stained gel electrophoresis of fractions correspondent to different peaks after cation-exchange chromatography (26 μl of each fraction, peaks 1–4: lanes 1–4). All peaks showed a band of the correct Cas9 molecular weight. Only the majoritarian peak (#2) was collected. Lane 5 corresponds to purified Cas9 after dialysis. M: SeeBlue Plus2 Pre-Stained Protein Standard (Life Technologies). (E) Purification of recombinant Cas9 produced in our laboratory showed endonuclease activity comparable to a commercial Cas9 (PNABio). 200 ng of PCR product of target gene was incubated with each individual sgRNA (lanes 1–12; 3.2 μM) in the presence of 3.8 μM Cas9 protein either purchased from PNABio (upper gel) or obtained in our laboratory (lower gel). As negative controls, 200 ng of PCR product alone (lane 13) or in combination with Cas9 protein (lane 14) were included. All samples were run on 2.2% agarose gels and visualized under UV light.

    Techniques Used: Purification, Recombinant, Affinity Chromatography, Staining, Nucleic Acid Electrophoresis, Chromatography, Molecular Weight, Produced, Activity Assay, Polymerase Chain Reaction, Incubation

    2) Product Images from "Pharmacological restoration of autophagy reduces hypertension-related stroke occurrence"

    Article Title: Pharmacological restoration of autophagy reduces hypertension-related stroke occurrence

    Journal: Autophagy

    doi: 10.1080/15548627.2019.1687215

    NMN improved mitochondrial function and cell viability in vitro . ( A ) Evaluation of ultrastructural damage in mitochondria from A10 cells with Ndufc2 knockdown; representative micrographs of mitochondria (left) and graphical representation of the ultrastructural damage in either untreated or treated A10 cells (n = 3). Legend: Nu, nucleus; NM, nuclear membrane, PM, plasma membrane; rER, rough endoplasmic reticulum; Gx, grade of mitochondrial (Mt) damage; IMM, inner mitochondrial membrane; OMM, outer mitochondrial membrane. (B) Fluorescence microscope analysis of mitochondrial membrane potential (ΔΨm) levels through JC1 dye (n = 3); representative images (left) and corresponding quantification (right) are shown. (C) FACS analysis in A10 cells with Ndufc2 knockdown treated with NaCl without or with NMN (n = 3); CTR indicates non-silenced and untreated cells. Results are presented as mean values ± SEM; *p
    Figure Legend Snippet: NMN improved mitochondrial function and cell viability in vitro . ( A ) Evaluation of ultrastructural damage in mitochondria from A10 cells with Ndufc2 knockdown; representative micrographs of mitochondria (left) and graphical representation of the ultrastructural damage in either untreated or treated A10 cells (n = 3). Legend: Nu, nucleus; NM, nuclear membrane, PM, plasma membrane; rER, rough endoplasmic reticulum; Gx, grade of mitochondrial (Mt) damage; IMM, inner mitochondrial membrane; OMM, outer mitochondrial membrane. (B) Fluorescence microscope analysis of mitochondrial membrane potential (ΔΨm) levels through JC1 dye (n = 3); representative images (left) and corresponding quantification (right) are shown. (C) FACS analysis in A10 cells with Ndufc2 knockdown treated with NaCl without or with NMN (n = 3); CTR indicates non-silenced and untreated cells. Results are presented as mean values ± SEM; *p

    Techniques Used: In Vitro, Fluorescence, Microscopy, FACS

    3) Product Images from "Structural model of ubiquitin transfer onto an artificial RING finger as an E3 ligase"

    Article Title: Structural model of ubiquitin transfer onto an artificial RING finger as an E3 ligase

    Journal: Scientific Reports

    doi: 10.1038/srep06574

    CD spectra of the artificial WSTF PHD_EL5 RING finger and its five mutants. Spectra of 25 μM samples were collected in 20 mM Tris-HCl (pH 6.9), 50 mM NaCl, 1 mM dithiothreitol, and 50 μM ZnCl 2 at room temperature. (1) K4R, (2) K8R, (3) K9R, (4) K14R, and (5) K23R are denoted by solid lines, and the dotted line displays the wild-type.
    Figure Legend Snippet: CD spectra of the artificial WSTF PHD_EL5 RING finger and its five mutants. Spectra of 25 μM samples were collected in 20 mM Tris-HCl (pH 6.9), 50 mM NaCl, 1 mM dithiothreitol, and 50 μM ZnCl 2 at room temperature. (1) K4R, (2) K8R, (3) K9R, (4) K14R, and (5) K23R are denoted by solid lines, and the dotted line displays the wild-type.

    Techniques Used:

    4) Product Images from "The RNA-binding proteins HYL1 and SE promote accurate in vitro processing of pri-miRNA by DCL1"

    Article Title: The RNA-binding proteins HYL1 and SE promote accurate in vitro processing of pri-miRNA by DCL1

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

    doi: 10.1073/pnas.0803356105

    Recombinant DCL1 cleavage of dsRNA. ( A ) Domains of DCL1, HYL1, and SE. The 6× His, HA, and FLAG epitope tags are indicated. ( B ) Purified recombinant His-HA-DCL1-FLAG, His-HYL1, and His-SE proteins were fractionated on 4–12% SDS/PAGE and stained with Coomassie blue G-250. ( C–F ) DCL1 cleavage of dsRNA substrates. Recombinant DCL1 protein was incubated with 200 ng of a 94-bp dsRNA substrate with a 2-nt 3′ overhang (see Methods ). The reaction products were phenol/chloroform purified and analyzed on 15% 8 M urea PAGE. ( C ) Time course of cleavage using 200 ng of DCL1. ( D ) dsRNA cleavage as a function of DCL1 protein concentration for 60 min. ( E ) The effect of NaCl concentration on dsRNA cleavage for 60 min with 200 ng of DCL1. ( F ) The effect of ATP and Mg 2+ on DCL1 cleavage of dsRNA. EDTA (2 mM) was added to the reaction lacking the Mg 2+ (last lane). The arrowheads indicates the dsRNA substrate, and the arrows indicate the 21-nt RNA products.
    Figure Legend Snippet: Recombinant DCL1 cleavage of dsRNA. ( A ) Domains of DCL1, HYL1, and SE. The 6× His, HA, and FLAG epitope tags are indicated. ( B ) Purified recombinant His-HA-DCL1-FLAG, His-HYL1, and His-SE proteins were fractionated on 4–12% SDS/PAGE and stained with Coomassie blue G-250. ( C–F ) DCL1 cleavage of dsRNA substrates. Recombinant DCL1 protein was incubated with 200 ng of a 94-bp dsRNA substrate with a 2-nt 3′ overhang (see Methods ). The reaction products were phenol/chloroform purified and analyzed on 15% 8 M urea PAGE. ( C ) Time course of cleavage using 200 ng of DCL1. ( D ) dsRNA cleavage as a function of DCL1 protein concentration for 60 min. ( E ) The effect of NaCl concentration on dsRNA cleavage for 60 min with 200 ng of DCL1. ( F ) The effect of ATP and Mg 2+ on DCL1 cleavage of dsRNA. EDTA (2 mM) was added to the reaction lacking the Mg 2+ (last lane). The arrowheads indicates the dsRNA substrate, and the arrows indicate the 21-nt RNA products.

    Techniques Used: Recombinant, FLAG-tag, Purification, SDS Page, Staining, Incubation, Polyacrylamide Gel Electrophoresis, Protein Concentration, Concentration Assay

    5) Product Images from "Single-molecule DREEM imaging reveals DNA wrapping around human mitochondrial single-stranded DNA binding protein"

    Article Title: Single-molecule DREEM imaging reveals DNA wrapping around human mitochondrial single-stranded DNA binding protein

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky875

    DREEM imaging shows wrapping paths of a 90-nt ssDNA oligomer around Escherichia coli SSB. Samples containing 80 nM E. coli SSB and 159 nM 90-nt ssDNA were incubated for 10 min at 37°C in Incubation Buffer containing 20 mM HEPES (pH 7.5) and 100 mM NaCl and imaged in AFM Imaging Buffer containing 20 mM HEPES (pH 7.5), 100 mM NaCl, 10 mM Mg(OAc) 2 as described in ‘Materials and Methods’ section. AFM topography (left panels) and DREEM phase (right panels) images of E. coli SSB ( N = 60) show ( A ) one strand (35%) or ( B ) two strands (65%) of ssDNA crossing the face of the E. coli tetramer. DREEM-based cartoon models of E. coli SSB–DNA complexes depict E. coli SSB in green and the 90-nt ssDNA in yellow. ( C ) Cross-sectional analysis of the edge-to-edge distance for two strands (top panel) or one strand (bottom panel) of ssDNA. DNA edges are identified at half maxima of the DNA DREEM signal (blue circles). X-Y Scale bar = 10 nm.
    Figure Legend Snippet: DREEM imaging shows wrapping paths of a 90-nt ssDNA oligomer around Escherichia coli SSB. Samples containing 80 nM E. coli SSB and 159 nM 90-nt ssDNA were incubated for 10 min at 37°C in Incubation Buffer containing 20 mM HEPES (pH 7.5) and 100 mM NaCl and imaged in AFM Imaging Buffer containing 20 mM HEPES (pH 7.5), 100 mM NaCl, 10 mM Mg(OAc) 2 as described in ‘Materials and Methods’ section. AFM topography (left panels) and DREEM phase (right panels) images of E. coli SSB ( N = 60) show ( A ) one strand (35%) or ( B ) two strands (65%) of ssDNA crossing the face of the E. coli tetramer. DREEM-based cartoon models of E. coli SSB–DNA complexes depict E. coli SSB in green and the 90-nt ssDNA in yellow. ( C ) Cross-sectional analysis of the edge-to-edge distance for two strands (top panel) or one strand (bottom panel) of ssDNA. DNA edges are identified at half maxima of the DNA DREEM signal (blue circles). X-Y Scale bar = 10 nm.

    Techniques Used: Imaging, Incubation

    AFM images of Escherichia coli SSB bound to single-stranded M13mp18 DNA. AFM topographic images were collected for E. coli SSB (8.4 nM) interacting with circular single-stranded M13mp18 DNA (7249 nt) at a stoichiometric ratio of 10 E. coli SSB tetramers/M13mp18 DNA. Samples were incubated in Incubation Buffer containing 20 mM HEPES (pH 7.5) and 100 mM NaCl at 37°C for 10 min and imaged in AFM Imaging Buffer containing 20 mM HEPES (pH 7.5), 100 mM NaCl, 10 mM Mg(OAc) 2 as described in ‘Materials and Methods’ section. X-Y scale bar = 200 nm.
    Figure Legend Snippet: AFM images of Escherichia coli SSB bound to single-stranded M13mp18 DNA. AFM topographic images were collected for E. coli SSB (8.4 nM) interacting with circular single-stranded M13mp18 DNA (7249 nt) at a stoichiometric ratio of 10 E. coli SSB tetramers/M13mp18 DNA. Samples were incubated in Incubation Buffer containing 20 mM HEPES (pH 7.5) and 100 mM NaCl at 37°C for 10 min and imaged in AFM Imaging Buffer containing 20 mM HEPES (pH 7.5), 100 mM NaCl, 10 mM Mg(OAc) 2 as described in ‘Materials and Methods’ section. X-Y scale bar = 200 nm.

    Techniques Used: Incubation, Imaging

    Binding of mtSSB tetramers to ssDNA gap shortens DNA lengths. ( A ) Cartoon representation of the process used to generate the circular and linear DNA substrate containing a 37-nt ssDNA gap, as described in ‘Materials and Methods’ section. Two inserts show AFM images of circular and linearized gapped DNA. X-Y scale bars = 100 nm. Circular plasmid DNA (4060 bp) contains a head-to-tail duplication, and digestion with Sca1 generates linear DNA fragments (2030 bp). ( B ) AFM images of mtSSB bound to linear gapped DNA. X-Y Scale bars = 50 nm. ( C ) The position distribution of mtSSB ( N = 113) binding on the linear-gapped DNA was fit with a Gaussian function (red line, R 2 = 0.92), which is centered 22.6% (±4.6%) from the closer DNA end. ( D ) AFM volumes of mtSSB bound to the linear DNA substrate containing a 37-nt ssDNA gap ( N = 112). The data were fit to a Gaussian function (red line, R 2 = 0.91) centered at 113.8 (±86.9) nm 3 . ( E ) Lengths of linear DNA without treatment, after gapping procedures, and upon binding mtSSB. The data were fit to Gaussian functions ( R 2 > 0.91) with peaks centered at 671.0 (±26.4) nm for non-gapped DNA alone (blue), 663.8 (±19.4) nm for gapped DNA alone (gray), and 650.8 (±15.7) nm for gapped DNA bound with mtSSB (red). All the samples were incubated in Incubation Buffer containing 20 mM HEPES (pH 7.5) and 100 mM NaCl and imaged in AFM Imaging Buffer containing 20 mM HEPES (pH 7.5), 100 mM NaCl, 10 mM Mg(OAc) 2 as described in ‘Materials and Methods’ section.
    Figure Legend Snippet: Binding of mtSSB tetramers to ssDNA gap shortens DNA lengths. ( A ) Cartoon representation of the process used to generate the circular and linear DNA substrate containing a 37-nt ssDNA gap, as described in ‘Materials and Methods’ section. Two inserts show AFM images of circular and linearized gapped DNA. X-Y scale bars = 100 nm. Circular plasmid DNA (4060 bp) contains a head-to-tail duplication, and digestion with Sca1 generates linear DNA fragments (2030 bp). ( B ) AFM images of mtSSB bound to linear gapped DNA. X-Y Scale bars = 50 nm. ( C ) The position distribution of mtSSB ( N = 113) binding on the linear-gapped DNA was fit with a Gaussian function (red line, R 2 = 0.92), which is centered 22.6% (±4.6%) from the closer DNA end. ( D ) AFM volumes of mtSSB bound to the linear DNA substrate containing a 37-nt ssDNA gap ( N = 112). The data were fit to a Gaussian function (red line, R 2 = 0.91) centered at 113.8 (±86.9) nm 3 . ( E ) Lengths of linear DNA without treatment, after gapping procedures, and upon binding mtSSB. The data were fit to Gaussian functions ( R 2 > 0.91) with peaks centered at 671.0 (±26.4) nm for non-gapped DNA alone (blue), 663.8 (±19.4) nm for gapped DNA alone (gray), and 650.8 (±15.7) nm for gapped DNA bound with mtSSB (red). All the samples were incubated in Incubation Buffer containing 20 mM HEPES (pH 7.5) and 100 mM NaCl and imaged in AFM Imaging Buffer containing 20 mM HEPES (pH 7.5), 100 mM NaCl, 10 mM Mg(OAc) 2 as described in ‘Materials and Methods’ section.

    Techniques Used: Binding Assay, Plasmid Preparation, Incubation, Imaging

    MtSSB binding to ssDNA is not cooperative. AFM topographic images were collected for ( A ) circular single-stranded M13mp18 DNA (7249 nt), and ( B – E ) mtSSB bound to single-stranded M13mp18 DNA at stoichiometric ratios (R = mtSSB tetramer/M13mp18 DNA) of (B) R = 2, (C) R = 5, (D) R = 10 or (E) R = 20. X-Y scale bar = 50 nm. Samples were incubated in Incubation Buffer containing 20 mM HEPES (pH 7.5) and 100 mM NaCl at 37°C for 10 min and imaged in AFM Imaging Buffer containing 20 mM HEPES (pH 7.5), 100 mM NaCl, 10 mM Mg(OAc) 2 as described in ‘Materials and Methods’ section.
    Figure Legend Snippet: MtSSB binding to ssDNA is not cooperative. AFM topographic images were collected for ( A ) circular single-stranded M13mp18 DNA (7249 nt), and ( B – E ) mtSSB bound to single-stranded M13mp18 DNA at stoichiometric ratios (R = mtSSB tetramer/M13mp18 DNA) of (B) R = 2, (C) R = 5, (D) R = 10 or (E) R = 20. X-Y scale bar = 50 nm. Samples were incubated in Incubation Buffer containing 20 mM HEPES (pH 7.5) and 100 mM NaCl at 37°C for 10 min and imaged in AFM Imaging Buffer containing 20 mM HEPES (pH 7.5), 100 mM NaCl, 10 mM Mg(OAc) 2 as described in ‘Materials and Methods’ section.

    Techniques Used: Binding Assay, Incubation, Imaging

    DNA binding affinity of mtSSB and Escherichia coli SSB. Changes in fluorescence anisotropy of a fluorescein-conjugated 90-nt oligonucleotide substrate were measured in response to the step-wise addition of ( A ) mtSSB or ( B ) E. coli SSB proteins, as described in ‘Materials and Methods’ section. Binding buffer contained 30 mM HEPES-KOH (pH 7.5), 1 mM 2-mercaptoethanol, 5 mM MgCl 2 , 0.01% NP-40, 50 mM NaCl, 20 nM oligonucleotide and the indicated amounts of proteins. Protein concentrations are expressed as tetramers. Error bars are standard deviations of triplicate determinations. ( C ) DNA binding cooperativity was estimated by replotting binding data for mtSSB (blue circles) and E. coli SSB (red squares) as a Hill plot.
    Figure Legend Snippet: DNA binding affinity of mtSSB and Escherichia coli SSB. Changes in fluorescence anisotropy of a fluorescein-conjugated 90-nt oligonucleotide substrate were measured in response to the step-wise addition of ( A ) mtSSB or ( B ) E. coli SSB proteins, as described in ‘Materials and Methods’ section. Binding buffer contained 30 mM HEPES-KOH (pH 7.5), 1 mM 2-mercaptoethanol, 5 mM MgCl 2 , 0.01% NP-40, 50 mM NaCl, 20 nM oligonucleotide and the indicated amounts of proteins. Protein concentrations are expressed as tetramers. Error bars are standard deviations of triplicate determinations. ( C ) DNA binding cooperativity was estimated by replotting binding data for mtSSB (blue circles) and E. coli SSB (red squares) as a Hill plot.

    Techniques Used: Binding Assay, Fluorescence

    AFM and DREEM imaging of mtSSB on a circular DNA substrate containing a 407-nt ssDNA gap. AFM topography images of circular plasmid DNA containing a 407-nt ssDNA gap in the ( A ) absence and ( B ) presence of mtSSB (white arrows). X-Y scale bar = 100 nm. ( C ) AFM topography (left panels), DREEM phase (middle panels) and DREEM amplitude (right panels) images of mtSSB interacting with circular DNA containing a 407-nt ssDNA gap. The volume of mtSSB on DNA with a 407-nt ssDNA gap was measured as 246 nm 3 (top left panels), 466 nm 3 (bottom left panels) and 368 nm 3 (right panel). X-Y scale bar = 20 nm. Samples were prepared for AFM and DREEM in Incubation Buffer containing 20 mM HEPES (pH 7.5) and 100 mM NaCl at 37°C for 10 min and imaged in AFM Imaging Buffer containing 20 mM HEPES (pH 7.5), 100 mM NaCl, 10 mM Mg(OAc) 2 as described in ‘Materials and Methods’ section.
    Figure Legend Snippet: AFM and DREEM imaging of mtSSB on a circular DNA substrate containing a 407-nt ssDNA gap. AFM topography images of circular plasmid DNA containing a 407-nt ssDNA gap in the ( A ) absence and ( B ) presence of mtSSB (white arrows). X-Y scale bar = 100 nm. ( C ) AFM topography (left panels), DREEM phase (middle panels) and DREEM amplitude (right panels) images of mtSSB interacting with circular DNA containing a 407-nt ssDNA gap. The volume of mtSSB on DNA with a 407-nt ssDNA gap was measured as 246 nm 3 (top left panels), 466 nm 3 (bottom left panels) and 368 nm 3 (right panel). X-Y scale bar = 20 nm. Samples were prepared for AFM and DREEM in Incubation Buffer containing 20 mM HEPES (pH 7.5) and 100 mM NaCl at 37°C for 10 min and imaged in AFM Imaging Buffer containing 20 mM HEPES (pH 7.5), 100 mM NaCl, 10 mM Mg(OAc) 2 as described in ‘Materials and Methods’ section.

    Techniques Used: Imaging, Plasmid Preparation, Incubation

    DREEM imaging shows wrapping paths of a 90-nt ssDNA oligomer around mtSSB. Samples containing 80 nM mtSSB and 159 nM 90-nt ssDNA were incubated for 10 min at 37°C in Incubation Buffer containing 20 mM HEPES (pH 7.5) and 100 mM NaCl and imaged in AFM Imaging Buffer containing 20 mM HEPES (pH 7.5), 100 mM NaCl, 10 mM Mg(OAc) 2 as described in ‘Materials and Methods’ section. AFM topography (left panels) and DREEM phase (right panels) images of mtSSB ( N = 59) show ( A ) one strand (85%) or ( B ) two strands (15%) of ssDNA crossing the face of the mtSSB tetramer. DREEM-based cartoon models of mtSSB–DNA complexes depict mtSSB in red and the 90-nt ssDNA in yellow. ( C ) Cross-sectional analysis of the edge-to-edge distance for one strand (top panel) or two strands (bottom panel) of ssDNA. DNA edges are identified at half maxima of the DNA DREEM signal (blue circles). X-Y scale bar = 25 nm.
    Figure Legend Snippet: DREEM imaging shows wrapping paths of a 90-nt ssDNA oligomer around mtSSB. Samples containing 80 nM mtSSB and 159 nM 90-nt ssDNA were incubated for 10 min at 37°C in Incubation Buffer containing 20 mM HEPES (pH 7.5) and 100 mM NaCl and imaged in AFM Imaging Buffer containing 20 mM HEPES (pH 7.5), 100 mM NaCl, 10 mM Mg(OAc) 2 as described in ‘Materials and Methods’ section. AFM topography (left panels) and DREEM phase (right panels) images of mtSSB ( N = 59) show ( A ) one strand (85%) or ( B ) two strands (15%) of ssDNA crossing the face of the mtSSB tetramer. DREEM-based cartoon models of mtSSB–DNA complexes depict mtSSB in red and the 90-nt ssDNA in yellow. ( C ) Cross-sectional analysis of the edge-to-edge distance for one strand (top panel) or two strands (bottom panel) of ssDNA. DNA edges are identified at half maxima of the DNA DREEM signal (blue circles). X-Y scale bar = 25 nm.

    Techniques Used: Imaging, Incubation

    DREEM imaging of mtSSB on a linear DNA fragment containing a 37-nt ssDNA gap. ( A ) AFM topography (left panels), DREEM phase (middle panels) and DREEM amplitude (right panels) images of mtSSB interacting with the linear DNA substrate containing a 37-nt ssDNA gap. X-Y scale bar = 20 nm. The respective volumes of mtSSB–ssDNA complexes were measured as 147 nm 3 (top row), 192 nm 3 (second row) and 145 nm 3 (third row). DREEM-based cartoon models of mtSSB–DNA complexes depict mtSSB in red, the 37-nt ssDNA gap in green and flanking dsDNA arms in blue. In DREEM phase images, proteins and DNA show negative signals relative to the mica surface, but the proteins produce greater contrast (darker regions) compared to the DNA. ( B ) The distribution of distances between mtSSB and the closer DNA end in DREEM images. The data were fit to a Gaussian function (red line, R 2 = 0.77) centered at 160.4 ± 11.6 nm ( N = 42). The ssDNA gap is located 470 bp (23%) from one end of the DNA fragment (2030 bp). All the samples were incubated in Incubation Buffer containing 20 mM HEPES (pH 7.5) and 100 mM NaCl at 37°C for 10 min and imaged in AFM Imaging Buffer containing 20 mM HEPES (pH 7.5), 100 mM NaCl, 10 mM Mg(OAc) 2 , as described in ‘Materials and Methods’ section.
    Figure Legend Snippet: DREEM imaging of mtSSB on a linear DNA fragment containing a 37-nt ssDNA gap. ( A ) AFM topography (left panels), DREEM phase (middle panels) and DREEM amplitude (right panels) images of mtSSB interacting with the linear DNA substrate containing a 37-nt ssDNA gap. X-Y scale bar = 20 nm. The respective volumes of mtSSB–ssDNA complexes were measured as 147 nm 3 (top row), 192 nm 3 (second row) and 145 nm 3 (third row). DREEM-based cartoon models of mtSSB–DNA complexes depict mtSSB in red, the 37-nt ssDNA gap in green and flanking dsDNA arms in blue. In DREEM phase images, proteins and DNA show negative signals relative to the mica surface, but the proteins produce greater contrast (darker regions) compared to the DNA. ( B ) The distribution of distances between mtSSB and the closer DNA end in DREEM images. The data were fit to a Gaussian function (red line, R 2 = 0.77) centered at 160.4 ± 11.6 nm ( N = 42). The ssDNA gap is located 470 bp (23%) from one end of the DNA fragment (2030 bp). All the samples were incubated in Incubation Buffer containing 20 mM HEPES (pH 7.5) and 100 mM NaCl at 37°C for 10 min and imaged in AFM Imaging Buffer containing 20 mM HEPES (pH 7.5), 100 mM NaCl, 10 mM Mg(OAc) 2 , as described in ‘Materials and Methods’ section.

    Techniques Used: Imaging, Incubation

    6) Product Images from "Using pyrene-labeled HIV-1 TAR to measure RNA-small molecule binding"

    Article Title: Using pyrene-labeled HIV-1 TAR to measure RNA-small molecule binding

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkg755

    ( A ) The fluorescence spectrum of TAR-2′pyr(25) in the absence and presence of purified neomycin B, in 50 mM Tris pH 7.4, 1 mM MgCl 2 and 100 mM NaCl. The dotted line indicates the fluorescence spectrum of the buffer alone. This spectrum, with an excitation wavelength of 340 nm and slit widths of 12 nm for excitation and 10 nm for emission, has a λ max of 390 nm. ( B ) The fluorescence spectrum of a 5′-U-(2′-amino- butyryl-pyrene-uridine)-U 3mer in the presence and absence of purified neomycin B, in 50 mM Tris pH 7.4, 1 mM MgCl 2 and 100 mM NaCl. The slight decrease in intensity reflects dilution. ( C ), after correcting for dilution and for the residual fluorescence of neomycin B at higher concentrations (shown in filled squares). The open squares demonstrate that the fluorescence of a U-(2′-amino-butyryl-pyrene-uridine)-U 3mer does not increase over this range of neomycin concentrations. Error bars indicate the standard deviation of at least three independent measurements at each aminoglycoside concentration.
    Figure Legend Snippet: ( A ) The fluorescence spectrum of TAR-2′pyr(25) in the absence and presence of purified neomycin B, in 50 mM Tris pH 7.4, 1 mM MgCl 2 and 100 mM NaCl. The dotted line indicates the fluorescence spectrum of the buffer alone. This spectrum, with an excitation wavelength of 340 nm and slit widths of 12 nm for excitation and 10 nm for emission, has a λ max of 390 nm. ( B ) The fluorescence spectrum of a 5′-U-(2′-amino- butyryl-pyrene-uridine)-U 3mer in the presence and absence of purified neomycin B, in 50 mM Tris pH 7.4, 1 mM MgCl 2 and 100 mM NaCl. The slight decrease in intensity reflects dilution. ( C ), after correcting for dilution and for the residual fluorescence of neomycin B at higher concentrations (shown in filled squares). The open squares demonstrate that the fluorescence of a U-(2′-amino-butyryl-pyrene-uridine)-U 3mer does not increase over this range of neomycin concentrations. Error bars indicate the standard deviation of at least three independent measurements at each aminoglycoside concentration.

    Techniques Used: Fluorescence, Purification, Standard Deviation, Concentration Assay

    7) Product Images from "NMR-Assisted Prediction of RNA Secondary Structure: Identification of a Probable Pseudoknot in the Coding Region of an R2 Retrotransposon"

    Article Title: NMR-Assisted Prediction of RNA Secondary Structure: Identification of a Probable Pseudoknot in the Coding Region of an R2 Retrotransposon

    Journal: Journal of the American Chemical Society

    doi: 10.1021/ja8026696

    Comparison of predicted structures and NMR evidence at 15 °C for a 74-nt fragment of the B. mori R2 retrotransposon coding region. (a) Possible secondary structures; colored dots indicate base pair type (blue = AU, red = GC, green = GU). (b) NMR spectral data. Upper frames depict the imino proton region from a 200 msec 2D-NOESY spectrum of an unlabeled sample; lower frames are taken from a 2D-NHSQC spectrum of a G- and U-labeled sample. Typical chemical shift ranges are indicated by brackets above and to the right of the spectra. Dashed lines and asterisk indicate an imino proton connection that is not apparent from this plot alone, but is confirmed by a 1D NOE experiment (see Figure S3 of Supporting Information ). The connections in the upper left frame depict a helical walk that rules out structure 3. Similarly, the upper right frame depicts a helix that does not match any in structure 2 or 3, but does coincide with the helices in structure 1 indicated by purple boxes (including a connection across a flush coaxial stack). Additional helical walks connecting two and five consecutive GC pairs are also observed but not depicted here (see Figure 2 ). Data were acquired on a 600 MHz spectrometer at 15 °C in NMR buffer of 150 mM NaCl, 2 mM MgCl 2 , 10 mM NaH 2 PO 4 , 0.05 mM EDTA, pH 6.0. Similar spectra were acquired at 25 °C with no differences in the observed patterns (see Figure S4 of Supporting Information ).
    Figure Legend Snippet: Comparison of predicted structures and NMR evidence at 15 °C for a 74-nt fragment of the B. mori R2 retrotransposon coding region. (a) Possible secondary structures; colored dots indicate base pair type (blue = AU, red = GC, green = GU). (b) NMR spectral data. Upper frames depict the imino proton region from a 200 msec 2D-NOESY spectrum of an unlabeled sample; lower frames are taken from a 2D-NHSQC spectrum of a G- and U-labeled sample. Typical chemical shift ranges are indicated by brackets above and to the right of the spectra. Dashed lines and asterisk indicate an imino proton connection that is not apparent from this plot alone, but is confirmed by a 1D NOE experiment (see Figure S3 of Supporting Information ). The connections in the upper left frame depict a helical walk that rules out structure 3. Similarly, the upper right frame depicts a helix that does not match any in structure 2 or 3, but does coincide with the helices in structure 1 indicated by purple boxes (including a connection across a flush coaxial stack). Additional helical walks connecting two and five consecutive GC pairs are also observed but not depicted here (see Figure 2 ). Data were acquired on a 600 MHz spectrometer at 15 °C in NMR buffer of 150 mM NaCl, 2 mM MgCl 2 , 10 mM NaH 2 PO 4 , 0.05 mM EDTA, pH 6.0. Similar spectra were acquired at 25 °C with no differences in the observed patterns (see Figure S4 of Supporting Information ).

    Techniques Used: Nuclear Magnetic Resonance, Labeling

    8) Product Images from "The structural basis of Indisulam-mediated recruitment of RBM39 to the DCAF15-DDB1-DDA1 E3 ligase complex"

    Article Title: The structural basis of Indisulam-mediated recruitment of RBM39 to the DCAF15-DDB1-DDA1 E3 ligase complex

    Journal: bioRxiv

    doi: 10.1101/737510

    Recruitment of RBM39 to DCAF15 is Indisulam-Dependent. 2.5 µM DCAF15-DDB1(ΔBPB)-DDA1 (DCAF15) was incubated with increasing concentrations of His-ZZ-RBM39(Δ150) (RBM39) as listed in the legend. The components were subjected to sedimentation velocity in 50 mM HEPES (7.5), 300 mM NaCl 1 mM TCEP @ 42,000 rpm for 5 h, 20 C. The sedimentation coefficient distribution displays independently migrating His-ZZ- RBM39(Δ150) (2.7 S) DCAF15-DDB1(ΔBPB)-DDA1 (6.8 S). In the absence of Indisulam, concentrations as high as 80 μM His-ZZ-RBM39(Δ150) do not display a dose-dependent increase the integrated area of a putative DCAF15-DDB1(ΔBPB)-DDA1-RBM39 complex peak or migrate with a higher sedimentation coefficient, inconsistent with the formation of a stable complex in the absence of Indisulam.
    Figure Legend Snippet: Recruitment of RBM39 to DCAF15 is Indisulam-Dependent. 2.5 µM DCAF15-DDB1(ΔBPB)-DDA1 (DCAF15) was incubated with increasing concentrations of His-ZZ-RBM39(Δ150) (RBM39) as listed in the legend. The components were subjected to sedimentation velocity in 50 mM HEPES (7.5), 300 mM NaCl 1 mM TCEP @ 42,000 rpm for 5 h, 20 C. The sedimentation coefficient distribution displays independently migrating His-ZZ- RBM39(Δ150) (2.7 S) DCAF15-DDB1(ΔBPB)-DDA1 (6.8 S). In the absence of Indisulam, concentrations as high as 80 μM His-ZZ-RBM39(Δ150) do not display a dose-dependent increase the integrated area of a putative DCAF15-DDB1(ΔBPB)-DDA1-RBM39 complex peak or migrate with a higher sedimentation coefficient, inconsistent with the formation of a stable complex in the absence of Indisulam.

    Techniques Used: Incubation, Sedimentation

    9) Product Images from "The structure of the nucleoprotein of Influenza D shows that all Orthomyxoviridae nucleoproteins have a similar NPCORE, with or without a NPTAIL for nuclear transport"

    Article Title: The structure of the nucleoprotein of Influenza D shows that all Orthomyxoviridae nucleoproteins have a similar NPCORE, with or without a NPTAIL for nuclear transport

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-37306-y

    Interaction of D/NP and D/NP TAIL with importin-α7. ( a ) Size exclusion chromatography profile of a mixture between human importin-α7 and D/NP TAIL . The mixture (molar ratio 1 importin-α7 for 2 D/NP TAIL ) was incubated 1 hour at room temperature and then loaded on a Superdex TM 75 10/300GL column equilibrated with the running buffer 20 mM Tris-HCl pH 7.5, 250 mM NaCl, 5 mM β-mercaptoethanol. ( b ) Thermal stability assay of importin-α7 in absence (green) or in presence (red) of D/NP TAIL using Thermofluor 76 . In presence of D/NP TAIL , the melting temperature of importin-α7 is 5 °C higher. D/NP TAIL alone using Thermofluor did not give any denaturation signal (yellow curve). The upper insert corresponds to the derivative of the fluorescence signal for a precise measure of the melting temperature. ( c ) Affinity of importin-α7 for D/NP TAIL by measured by surface plasmon resonance (SPR). Biotinylated D/NP TAIL (left) and control peptide (right) were captured on a streptavidin-coated sensor chip surface before injections of several importin-α7 concentrations (10 nM in red, 25 nM in orange, 50 nM in green, 75 nM in blue and 100 nM in purple). The sensorgrams of the interaction between D/NP TAIL and importin-α7 were fitted under a Langmuir 1:1 binding model with mass-transfer (black line). ( d ) SEC-MALLS analysis of D/NP in complex with importin-α7. The mixture (molar ratio 1 D/NP for 1.2 importin-α7) was incubated 1 hour at room temperature and then loaded on a Superdex TM 200 increase 10/300 GL. The experimental molecular weight is consistent with the expected mass of four importins-α7 bound per D/NP tetramer. ( e ) Pull-down assays of human importin-α7 by D/NP and the two C-terminal truncated mutants (D/NP-529 and D/NP-511). The his-tags are on D/NP. The mixtures (molar ratio 1 D/NP for 1.2 importin-α7) were incubated 1 hour and the experience was done as described in panel ( a ). The figure shows the coomassie blue-stained SDS-PAGE (12% polyacrylamide) with the Load, FlowThough, Wash and the second fractions (E2).
    Figure Legend Snippet: Interaction of D/NP and D/NP TAIL with importin-α7. ( a ) Size exclusion chromatography profile of a mixture between human importin-α7 and D/NP TAIL . The mixture (molar ratio 1 importin-α7 for 2 D/NP TAIL ) was incubated 1 hour at room temperature and then loaded on a Superdex TM 75 10/300GL column equilibrated with the running buffer 20 mM Tris-HCl pH 7.5, 250 mM NaCl, 5 mM β-mercaptoethanol. ( b ) Thermal stability assay of importin-α7 in absence (green) or in presence (red) of D/NP TAIL using Thermofluor 76 . In presence of D/NP TAIL , the melting temperature of importin-α7 is 5 °C higher. D/NP TAIL alone using Thermofluor did not give any denaturation signal (yellow curve). The upper insert corresponds to the derivative of the fluorescence signal for a precise measure of the melting temperature. ( c ) Affinity of importin-α7 for D/NP TAIL by measured by surface plasmon resonance (SPR). Biotinylated D/NP TAIL (left) and control peptide (right) were captured on a streptavidin-coated sensor chip surface before injections of several importin-α7 concentrations (10 nM in red, 25 nM in orange, 50 nM in green, 75 nM in blue and 100 nM in purple). The sensorgrams of the interaction between D/NP TAIL and importin-α7 were fitted under a Langmuir 1:1 binding model with mass-transfer (black line). ( d ) SEC-MALLS analysis of D/NP in complex with importin-α7. The mixture (molar ratio 1 D/NP for 1.2 importin-α7) was incubated 1 hour at room temperature and then loaded on a Superdex TM 200 increase 10/300 GL. The experimental molecular weight is consistent with the expected mass of four importins-α7 bound per D/NP tetramer. ( e ) Pull-down assays of human importin-α7 by D/NP and the two C-terminal truncated mutants (D/NP-529 and D/NP-511). The his-tags are on D/NP. The mixtures (molar ratio 1 D/NP for 1.2 importin-α7) were incubated 1 hour and the experience was done as described in panel ( a ). The figure shows the coomassie blue-stained SDS-PAGE (12% polyacrylamide) with the Load, FlowThough, Wash and the second fractions (E2).

    Techniques Used: Size-exclusion Chromatography, Incubation, Stability Assay, Fluorescence, SPR Assay, Chromatin Immunoprecipitation, Binding Assay, Molecular Weight, Staining, SDS Page

    Purification and characterized of Influenza D nucleoprotein. ( a ) Size exclusion chromatography profile of wild-type D/NP. The sample was loaded on a Hiload TM 16/600 S200 column equilibrated with the running buffer 20 mM Tris-HCl pH 7.5, 300 mM NaCl and 5 mM β-mercaptoethanol. ( b ) SEC-MALLS-RI analysis of D/NP. SEC was performed with a Superdex TM 200 increase 10/300 GL column equilibrated with 20 mM Tris-HCl pH 7.5, 150 mM NaCl and 5 mM β-ME. The panel shows the theoretical Mw and the measured Mw. ( c ) and ( e ) Electron microscopy images of the elution peak of D/NP and D/NP-511. Samples show different oligomeric states although most oligomers are tetramers. The scale bar corresponds to 100 nm. ( d ) Coomassie blue-stained SDS-PAGE (4–20% gradient polyacrylamide) showing the purified wild-type D/NP and the two C-terminal truncated mutants (D/NP-529 and D/NP-511).
    Figure Legend Snippet: Purification and characterized of Influenza D nucleoprotein. ( a ) Size exclusion chromatography profile of wild-type D/NP. The sample was loaded on a Hiload TM 16/600 S200 column equilibrated with the running buffer 20 mM Tris-HCl pH 7.5, 300 mM NaCl and 5 mM β-mercaptoethanol. ( b ) SEC-MALLS-RI analysis of D/NP. SEC was performed with a Superdex TM 200 increase 10/300 GL column equilibrated with 20 mM Tris-HCl pH 7.5, 150 mM NaCl and 5 mM β-ME. The panel shows the theoretical Mw and the measured Mw. ( c ) and ( e ) Electron microscopy images of the elution peak of D/NP and D/NP-511. Samples show different oligomeric states although most oligomers are tetramers. The scale bar corresponds to 100 nm. ( d ) Coomassie blue-stained SDS-PAGE (4–20% gradient polyacrylamide) showing the purified wild-type D/NP and the two C-terminal truncated mutants (D/NP-529 and D/NP-511).

    Techniques Used: Purification, Size-exclusion Chromatography, Electron Microscopy, Staining, SDS Page

    10) Product Images from "Electrostatic Interaction with the Bacterial Cell Envelope Tunes the Lytic Activity of Two Novel Peptidoglycan Hydrolases"

    Article Title: Electrostatic Interaction with the Bacterial Cell Envelope Tunes the Lytic Activity of Two Novel Peptidoglycan Hydrolases

    Journal: Microbiology Spectrum

    doi: 10.1128/spectrum.00455-22

    Role of charge in enzyme–CW interactions. (A) Comparison of enzymatic lytic activity and net charge on the bacterial cell surface measured in CytC assay. The lytic activity was presented as the reduction of initial OD 600 after 1 h of incubation of bacteria in suspension with 100 nM enzyme in 50 mM glycine buffer pH 8.0, 100 mM NaCl and subtracted to negative control values (bacterial suspension incubated without added enzyme). The values of the measured net charge of the bacterial cell surface is presented as a spectrum of the percentage of the bound CytC fraction. The experiments were performed three times independently, and each bar represents the average ± standard deviation. (B) Comparison of lytic activity of SpM23_B and EAD_B before and upon methylation. SYTOX fluorescence assay performed in 50 mM glycine buffer with pH 8.0 and 100 mM NaCl at room temperature. The mean fluorescence intensity was calculated from the steepest linear region of the obtained lysis curve. The statistical significance of the differences was calculated using Student’s t test. ** * , P
    Figure Legend Snippet: Role of charge in enzyme–CW interactions. (A) Comparison of enzymatic lytic activity and net charge on the bacterial cell surface measured in CytC assay. The lytic activity was presented as the reduction of initial OD 600 after 1 h of incubation of bacteria in suspension with 100 nM enzyme in 50 mM glycine buffer pH 8.0, 100 mM NaCl and subtracted to negative control values (bacterial suspension incubated without added enzyme). The values of the measured net charge of the bacterial cell surface is presented as a spectrum of the percentage of the bound CytC fraction. The experiments were performed three times independently, and each bar represents the average ± standard deviation. (B) Comparison of lytic activity of SpM23_B and EAD_B before and upon methylation. SYTOX fluorescence assay performed in 50 mM glycine buffer with pH 8.0 and 100 mM NaCl at room temperature. The mean fluorescence intensity was calculated from the steepest linear region of the obtained lysis curve. The statistical significance of the differences was calculated using Student’s t test. ** * , P

    Techniques Used: Activity Assay, Incubation, Negative Control, Standard Deviation, Methylation, Fluorescence, Lysis

    11) Product Images from "Monitoring Intracellular pH change with a Genetically Encoded and Ratiometric Luminescence Sensor in Yeast and Mammalian Cells"

    Article Title: Monitoring Intracellular pH change with a Genetically Encoded and Ratiometric Luminescence Sensor in Yeast and Mammalian Cells

    Journal: Methods in molecular biology (Clifton, N.J.)

    doi: 10.1007/978-1-4939-3813-1_9

    pH response of purified pHlash protein in vitro (A) SDS-PAGE gel of purified His-tagged pHlash protein stained with Coomassie Blue dye. Leftmost lane is molecular weight standards with KDa indicated, while the other lanes are the purified pHlash protein loaded at 0.2, 1, and 2 μg per lane. (B ) Construct of the pHlash fusion protein. Rluc8 was linked to cpVenus by the sequence Ala-Glu-Leu. (C ) Raw data (not normalized) of luminescence emission spectra of purified pHlash protein with 10 μM native coelenterazine at different pH values (pH 5.4-9.0) in 50 mM BIS-Tris-propane, 100 mM KCl, and 100 mM NaCl. (D) Normalized luminescence emission spectra of pHlash measured as in panel C. Luminescence intensity was normalized to the peak at 475 nm. (E) The BRET ratio (luminescence at 525nm:475 nm) as a function of pH is shown for pHlash in vitro . Error bars are +/- S.D., but in most cases the error bars are so small that they are obscured by the symbols (n = 3).
    Figure Legend Snippet: pH response of purified pHlash protein in vitro (A) SDS-PAGE gel of purified His-tagged pHlash protein stained with Coomassie Blue dye. Leftmost lane is molecular weight standards with KDa indicated, while the other lanes are the purified pHlash protein loaded at 0.2, 1, and 2 μg per lane. (B ) Construct of the pHlash fusion protein. Rluc8 was linked to cpVenus by the sequence Ala-Glu-Leu. (C ) Raw data (not normalized) of luminescence emission spectra of purified pHlash protein with 10 μM native coelenterazine at different pH values (pH 5.4-9.0) in 50 mM BIS-Tris-propane, 100 mM KCl, and 100 mM NaCl. (D) Normalized luminescence emission spectra of pHlash measured as in panel C. Luminescence intensity was normalized to the peak at 475 nm. (E) The BRET ratio (luminescence at 525nm:475 nm) as a function of pH is shown for pHlash in vitro . Error bars are +/- S.D., but in most cases the error bars are so small that they are obscured by the symbols (n = 3).

    Techniques Used: Purification, In Vitro, SDS Page, Staining, Molecular Weight, Construct, Sequencing, Bioluminescence Resonance Energy Transfer

    12) Product Images from "Expression and one-step purification of active LPL contemplated by biophysical considerations"

    Article Title: Expression and one-step purification of active LPL contemplated by biophysical considerations

    Journal: Journal of Lipid Research

    doi: 10.1016/j.jlr.2021.100149

    Thermal and shelf-life stability of hLPL at different conditions. A: Thermal unfolding profiles of hLPL R297A in the presence of 0.2 M NaCl (red line), 0.4 M NaCl (orange line), 0.8 M NaCl (purple line), 1.2 M NaCl (blue line), 1.2 M NaCl, and 40% glycerol in 10 mM Tris-HCl (pH 7.2) (black line). Shown are the means of the first derivative of the ratio of emission intensity at 350 and 330 nm wavelength as a function of increasing temperature with standard derivation indicated in transparent colors. The apparent T m for the N-terminal α/β-hydrolase domain (NTD 1–312 ) of LPL is highlighted with a colored asterisk. The C-terminal lipid-binding domain of hLPL (CTD 313–448 ) has a T m ). B: The corresponding unfolding profiles are shown for mLPL R297A . C: hLPL R297 was stored at 1 mg/ml at 0.2 M NaCl, pH 7.2 (closed circles), 0.2 M NaCl, 40% glycerol, pH 7.2 (open squares), and 1.2 M NaCl, 40% glycerol, pH 7.2 (closed triangles) at 23°C, (D) 4°C, (E) −20°C, and (F) −80°C. The LPL activity was measured at the indicated time points and normalized to average LPL activity for the first time point at each temperature tested.
    Figure Legend Snippet: Thermal and shelf-life stability of hLPL at different conditions. A: Thermal unfolding profiles of hLPL R297A in the presence of 0.2 M NaCl (red line), 0.4 M NaCl (orange line), 0.8 M NaCl (purple line), 1.2 M NaCl (blue line), 1.2 M NaCl, and 40% glycerol in 10 mM Tris-HCl (pH 7.2) (black line). Shown are the means of the first derivative of the ratio of emission intensity at 350 and 330 nm wavelength as a function of increasing temperature with standard derivation indicated in transparent colors. The apparent T m for the N-terminal α/β-hydrolase domain (NTD 1–312 ) of LPL is highlighted with a colored asterisk. The C-terminal lipid-binding domain of hLPL (CTD 313–448 ) has a T m ). B: The corresponding unfolding profiles are shown for mLPL R297A . C: hLPL R297 was stored at 1 mg/ml at 0.2 M NaCl, pH 7.2 (closed circles), 0.2 M NaCl, 40% glycerol, pH 7.2 (open squares), and 1.2 M NaCl, 40% glycerol, pH 7.2 (closed triangles) at 23°C, (D) 4°C, (E) −20°C, and (F) −80°C. The LPL activity was measured at the indicated time points and normalized to average LPL activity for the first time point at each temperature tested.

    Techniques Used: Binding Assay, Activity Assay

    13) Product Images from "Structure of a phleboviral envelope glycoprotein reveals a consolidated model of membrane fusion"

    Article Title: Structure of a phleboviral envelope glycoprotein reveals a consolidated model of membrane fusion

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

    doi: 10.1073/pnas.1603827113

    Size exclusion (SEC) and Western blot analysis of purified SFTSV Gc. SEC was performed on a Superdex Increase 200 10/30 column (Amersham) equilibrated in 150 mM NaCl and either 10 mM Tris·HCl, pH 8.0, or 20 mM citrate–NaOH, pH 5.0 (see
    Figure Legend Snippet: Size exclusion (SEC) and Western blot analysis of purified SFTSV Gc. SEC was performed on a Superdex Increase 200 10/30 column (Amersham) equilibrated in 150 mM NaCl and either 10 mM Tris·HCl, pH 8.0, or 20 mM citrate–NaOH, pH 5.0 (see

    Techniques Used: Size-exclusion Chromatography, Western Blot, Purification

    14) Product Images from "Thermodynamic and Kinetic Characterization of the Protein Z-dependent Protease Inhibitor (ZPI)-Protein Z Interaction Reveals an Unexpected Role for ZPI Lys-239 *"

    Article Title: Thermodynamic and Kinetic Characterization of the Protein Z-dependent Protease Inhibitor (ZPI)-Protein Z Interaction Reveals an Unexpected Role for ZPI Lys-239 *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M114.633479

    Calcium ion effects on the ZPI-PZ interaction. A , equilibrium binding titrations of 25 n m NBD-ZPI with PZ in pH 7. 1, I 0.15 Tris buffer at 25 °C (●) without or with the addition of 15 m m NaCl (○) or 5 m m CaCl 2 (▴). Observed
    Figure Legend Snippet: Calcium ion effects on the ZPI-PZ interaction. A , equilibrium binding titrations of 25 n m NBD-ZPI with PZ in pH 7. 1, I 0.15 Tris buffer at 25 °C (●) without or with the addition of 15 m m NaCl (○) or 5 m m CaCl 2 (▴). Observed

    Techniques Used: Binding Assay

    15) Product Images from "Ultracentrifugation-free chromatography-mediated large-scale purification of recombinant adeno-associated virus serotype 1 (rAAV1)"

    Article Title: Ultracentrifugation-free chromatography-mediated large-scale purification of recombinant adeno-associated virus serotype 1 (rAAV1)

    Journal: Molecular Therapy. Methods & Clinical Development

    doi: 10.1038/mtm.2015.58

    Effects of chromatography-based purification of recombinant adeno-associated virus serotype 1 (rAAV1). ( a ) rAAV1 eluted from the five Mustang QXTs (anion-exchange column) by stepwise NaCl gradient elution was analyzed by 5–20% gradient sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gel (Oriole staining). The black square indicates the fractions loaded on Superdex 200 10/300 GL at next gel-filtration chromatography step. The white triangle indicates the 200 kDa protein impurities. Lane 1: after dialysis; Lanes 2–4: eluted by 100 mmol/l NaCl; Lanes 5–7: eluted by 120 mmol/l NaCl; Lanes 8–10: eluted by 130 mmol/l NaCl; Lanes 11–13: eluted by 140 mmol/l NaCl; Lanes 14–16: eluted by 200 mmol/l NaCl; M: protein size marker. ( b,c ) The samples were subsequently gel-filtrated by the Superdex 200 10/300 GL column and elution fractions were analyzed by 5–20% gradient SDS–PAGE (Oriole staining). (b) Elution pattern of Superdex 200 10/300 GL. The y-axis: 280 nm absorbance; x-axis: fraction number. The black triangle indicates the peak fractions of rAAV1 (corresponding to the black square on (c)) and the gray arrowhead indicates ~65 kDa protein impurities. (c) Elution fractions analyzed by 5–20% gradient SDS–PAGE gel (Oriole staining). Lane 1: after dialysis; Lanes 2–11: Fr 13–22; Lane 12: Fr 27; M: protein size marker. The gray arrowhead shows the same as (b). ( d–f ) Peak fractions (the black square on ( c )) were collected and concentrated by Ultracel 30 K to obtain the final product. ( d ) The final product of rAAV1 was analyzed by 5–20% gradient SDS–PAGE gel (Oriole staining), ( e ) western blotting, and ( f ) electron microscopy (negative staining). M: protein size marker. The three rAAV1 capsid proteins, VP1 (81.4 kDa), VP2 (66.2 kDa), and VP3 (59.6 kDa), were represented.
    Figure Legend Snippet: Effects of chromatography-based purification of recombinant adeno-associated virus serotype 1 (rAAV1). ( a ) rAAV1 eluted from the five Mustang QXTs (anion-exchange column) by stepwise NaCl gradient elution was analyzed by 5–20% gradient sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gel (Oriole staining). The black square indicates the fractions loaded on Superdex 200 10/300 GL at next gel-filtration chromatography step. The white triangle indicates the 200 kDa protein impurities. Lane 1: after dialysis; Lanes 2–4: eluted by 100 mmol/l NaCl; Lanes 5–7: eluted by 120 mmol/l NaCl; Lanes 8–10: eluted by 130 mmol/l NaCl; Lanes 11–13: eluted by 140 mmol/l NaCl; Lanes 14–16: eluted by 200 mmol/l NaCl; M: protein size marker. ( b,c ) The samples were subsequently gel-filtrated by the Superdex 200 10/300 GL column and elution fractions were analyzed by 5–20% gradient SDS–PAGE (Oriole staining). (b) Elution pattern of Superdex 200 10/300 GL. The y-axis: 280 nm absorbance; x-axis: fraction number. The black triangle indicates the peak fractions of rAAV1 (corresponding to the black square on (c)) and the gray arrowhead indicates ~65 kDa protein impurities. (c) Elution fractions analyzed by 5–20% gradient SDS–PAGE gel (Oriole staining). Lane 1: after dialysis; Lanes 2–11: Fr 13–22; Lane 12: Fr 27; M: protein size marker. The gray arrowhead shows the same as (b). ( d–f ) Peak fractions (the black square on ( c )) were collected and concentrated by Ultracel 30 K to obtain the final product. ( d ) The final product of rAAV1 was analyzed by 5–20% gradient SDS–PAGE gel (Oriole staining), ( e ) western blotting, and ( f ) electron microscopy (negative staining). M: protein size marker. The three rAAV1 capsid proteins, VP1 (81.4 kDa), VP2 (66.2 kDa), and VP3 (59.6 kDa), were represented.

    Techniques Used: Chromatography, Purification, Recombinant, Polyacrylamide Gel Electrophoresis, SDS Page, Staining, Filtration, Marker, Western Blot, Electron Microscopy, Negative Staining

    Purification with a buffer containing 50 mmol/l NaCl. ( a ) Recombinant adeno-associated virus serotype 1 (rAAV1) purified by ion-exchange column with a buffer containing 50 mmol/l NaCl was analyzed by 5–20% gradient sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel (Oriole staining) and ( b ) western blotting. M: protein size marker. The 200 kDa protein impurities were still present (white triangle) and the purity was low. The three rAAV1 capsid proteins, VP1 (81.4 kDa), VP2 (66.2 kDa), and VP3 (59.6 kDa), were represented.
    Figure Legend Snippet: Purification with a buffer containing 50 mmol/l NaCl. ( a ) Recombinant adeno-associated virus serotype 1 (rAAV1) purified by ion-exchange column with a buffer containing 50 mmol/l NaCl was analyzed by 5–20% gradient sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel (Oriole staining) and ( b ) western blotting. M: protein size marker. The 200 kDa protein impurities were still present (white triangle) and the purity was low. The three rAAV1 capsid proteins, VP1 (81.4 kDa), VP2 (66.2 kDa), and VP3 (59.6 kDa), were represented.

    Techniques Used: Purification, Recombinant, Polyacrylamide Gel Electrophoresis, SDS Page, Staining, Western Blot, Marker

    16) Product Images from "The structure of the nucleoprotein of Influenza D shows that all Orthomyxoviridae nucleoproteins have a similar NPCORE, with or without a NPTAIL for nuclear transport"

    Article Title: The structure of the nucleoprotein of Influenza D shows that all Orthomyxoviridae nucleoproteins have a similar NPCORE, with or without a NPTAIL for nuclear transport

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-37306-y

    Purification and characterized of Influenza D nucleoprotein. ( a ) Size exclusion chromatography profile of wild-type D/NP. The sample was loaded on a Hiload TM 16/600 S200 column equilibrated with the running buffer 20 mM Tris-HCl pH 7.5, 300 mM NaCl and 5 mM β-mercaptoethanol. ( b ) SEC-MALLS-RI analysis of D/NP. SEC was performed with a Superdex TM 200 increase 10/300 GL column equilibrated with 20 mM Tris-HCl pH 7.5, 150 mM NaCl and 5 mM β-ME. The panel shows the theoretical Mw and the measured Mw. ( c ) and ( e ) Electron microscopy images of the elution peak of D/NP and D/NP-511. Samples show different oligomeric states although most oligomers are tetramers. The scale bar corresponds to 100 nm. ( d ) Coomassie blue-stained SDS-PAGE (4–20% gradient polyacrylamide) showing the purified wild-type D/NP and the two C-terminal truncated mutants (D/NP-529 and D/NP-511).
    Figure Legend Snippet: Purification and characterized of Influenza D nucleoprotein. ( a ) Size exclusion chromatography profile of wild-type D/NP. The sample was loaded on a Hiload TM 16/600 S200 column equilibrated with the running buffer 20 mM Tris-HCl pH 7.5, 300 mM NaCl and 5 mM β-mercaptoethanol. ( b ) SEC-MALLS-RI analysis of D/NP. SEC was performed with a Superdex TM 200 increase 10/300 GL column equilibrated with 20 mM Tris-HCl pH 7.5, 150 mM NaCl and 5 mM β-ME. The panel shows the theoretical Mw and the measured Mw. ( c ) and ( e ) Electron microscopy images of the elution peak of D/NP and D/NP-511. Samples show different oligomeric states although most oligomers are tetramers. The scale bar corresponds to 100 nm. ( d ) Coomassie blue-stained SDS-PAGE (4–20% gradient polyacrylamide) showing the purified wild-type D/NP and the two C-terminal truncated mutants (D/NP-529 and D/NP-511).

    Techniques Used: Purification, Size-exclusion Chromatography, Electron Microscopy, Staining, SDS Page

    Interaction of D/NP and D/NP TAIL with importin-α7. ( a ) Size exclusion chromatography profile of a mixture between human importin-α7 and D/NP TAIL . The mixture (molar ratio 1 importin-α7 for 2 D/NP TAIL ) was incubated 1 hour at room temperature and then loaded on a Superdex TM 75 10/300GL column equilibrated with the running buffer 20 mM Tris-HCl pH 7.5, 250 mM NaCl, 5 mM β-mercaptoethanol. ( b ) Thermal stability assay of importin-α7 in absence (green) or in presence (red) of D/NP TAIL . In presence of D/NP TAIL , the melting temperature of importin-α7 is 5 °C higher. D/NP TAIL alone using Thermofluor did not give any denaturation signal (yellow curve). The upper insert corresponds to the derivative of the fluorescence signal for a precise measure of the melting temperature. ( c ) Affinity of importin-α7 for D/NP TAIL by measured by surface plasmon resonance (SPR). Biotinylated D/NP TAIL (left) and control peptide (right) were captured on a streptavidin-coated sensor chip surface before injections of several importin-α7 concentrations (10 nM in red, 25 nM in orange, 50 nM in green, 75 nM in blue and 100 nM in purple). The sensorgrams of the interaction between D/NP TAIL and importin-α7 were fitted under a Langmuir 1:1 binding model with mass-transfer (black line). ( d ) SEC-MALLS analysis of D/NP in complex with importin-α7. The mixture (molar ratio 1 D/NP for 1.2 importin-α7) was incubated 1 hour at room temperature and then loaded on a Superdex TM 200 increase 10/300 GL. The experimental molecular weight is consistent with the expected mass of four importins-α7 bound per D/NP tetramer. ( e ) Pull-down assays of human importin-α7 by D/NP and the two C-terminal truncated mutants (D/NP-529 and D/NP-511). The his-tags are on D/NP. The mixtures (molar ratio 1 D/NP for 1.2 importin-α7) were incubated 1 hour and the experience was done as described in panel ( a ). The figure shows the coomassie blue-stained SDS-PAGE (12% polyacrylamide) with the Load, FlowThough, Wash and the second fractions (E2).
    Figure Legend Snippet: Interaction of D/NP and D/NP TAIL with importin-α7. ( a ) Size exclusion chromatography profile of a mixture between human importin-α7 and D/NP TAIL . The mixture (molar ratio 1 importin-α7 for 2 D/NP TAIL ) was incubated 1 hour at room temperature and then loaded on a Superdex TM 75 10/300GL column equilibrated with the running buffer 20 mM Tris-HCl pH 7.5, 250 mM NaCl, 5 mM β-mercaptoethanol. ( b ) Thermal stability assay of importin-α7 in absence (green) or in presence (red) of D/NP TAIL . In presence of D/NP TAIL , the melting temperature of importin-α7 is 5 °C higher. D/NP TAIL alone using Thermofluor did not give any denaturation signal (yellow curve). The upper insert corresponds to the derivative of the fluorescence signal for a precise measure of the melting temperature. ( c ) Affinity of importin-α7 for D/NP TAIL by measured by surface plasmon resonance (SPR). Biotinylated D/NP TAIL (left) and control peptide (right) were captured on a streptavidin-coated sensor chip surface before injections of several importin-α7 concentrations (10 nM in red, 25 nM in orange, 50 nM in green, 75 nM in blue and 100 nM in purple). The sensorgrams of the interaction between D/NP TAIL and importin-α7 were fitted under a Langmuir 1:1 binding model with mass-transfer (black line). ( d ) SEC-MALLS analysis of D/NP in complex with importin-α7. The mixture (molar ratio 1 D/NP for 1.2 importin-α7) was incubated 1 hour at room temperature and then loaded on a Superdex TM 200 increase 10/300 GL. The experimental molecular weight is consistent with the expected mass of four importins-α7 bound per D/NP tetramer. ( e ) Pull-down assays of human importin-α7 by D/NP and the two C-terminal truncated mutants (D/NP-529 and D/NP-511). The his-tags are on D/NP. The mixtures (molar ratio 1 D/NP for 1.2 importin-α7) were incubated 1 hour and the experience was done as described in panel ( a ). The figure shows the coomassie blue-stained SDS-PAGE (12% polyacrylamide) with the Load, FlowThough, Wash and the second fractions (E2).

    Techniques Used: Size-exclusion Chromatography, Incubation, Stability Assay, Fluorescence, SPR Assay, Chromatin Immunoprecipitation, Binding Assay, Molecular Weight, Staining, SDS Page

    17) Product Images from "TNF-α induces endothelial–mesenchymal transition promoting stromal development of pancreatic adenocarcinoma"

    Article Title: TNF-α induces endothelial–mesenchymal transition promoting stromal development of pancreatic adenocarcinoma

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-021-03920-4

    In vivo effect of TNF-α and schematic model of its action. TNF-α increases CAFs in pancreatic tumour in vivo ( A , B ) and schematic model of the reversible TNF-α-induced EndMT involving Tie1 downregulation ( C ). A , B . TNF-α induces an increase of fibrosis ( A ) and an increase of CAFs α-SMA positives ( B ). PK4A tumoral cells were orthotopically injected in mice which were treated daily for 27 days with injections of NaCl (Ctrl) or TNF-α ( n = 6 for each condition). Then, pancreases were collected and sections of tissue were subjected to HES staining, Masson’s trichrome (MT) staining and α-SMA staining. Fibrosis and α-SMA staining were analysed and quantified by calopix. Significant differences are indicated by solid lines (** P
    Figure Legend Snippet: In vivo effect of TNF-α and schematic model of its action. TNF-α increases CAFs in pancreatic tumour in vivo ( A , B ) and schematic model of the reversible TNF-α-induced EndMT involving Tie1 downregulation ( C ). A , B . TNF-α induces an increase of fibrosis ( A ) and an increase of CAFs α-SMA positives ( B ). PK4A tumoral cells were orthotopically injected in mice which were treated daily for 27 days with injections of NaCl (Ctrl) or TNF-α ( n = 6 for each condition). Then, pancreases were collected and sections of tissue were subjected to HES staining, Masson’s trichrome (MT) staining and α-SMA staining. Fibrosis and α-SMA staining were analysed and quantified by calopix. Significant differences are indicated by solid lines (** P

    Techniques Used: In Vivo, Injection, Mouse Assay, Staining

    18) Product Images from "Tau protein liquid–liquid phase separation can initiate tau aggregation"

    Article Title: Tau protein liquid–liquid phase separation can initiate tau aggregation

    Journal: The EMBO Journal

    doi: 10.15252/embj.201798049

    Liquid droplet characteristics of p‐tau441 Qualitative distribution of phosphorylation sites in p‐tau441 [pS68/69, pT153, pT175, pT181, pS184, pS199, pS202, pT205, pS210, pT212, pS214, pT217, pT231, pS235, pS262, pS324, pY310, pS316, pS396, pS404, pS422 (Mair et al )]. The charge at pH 7.4 of domains in unphosphorylated tau441 is indicated as well. Liquid–liquid phase separation (LLPS) of p‐tau441 in presence of molecular crowding (12.5% w/v Ficoll‐400). No phase separation is observed without crowding agent or in the absence of p‐tau441 protein. Liquid droplets formed by p‐tau441 in the presence of 10% (w/v) PEG were negative stained with uranyl‐acetate and visualized by transmission electron microscopy (TEM). p‐tau441 droplets are decorated with gold particles after immunogold labeling using anti‐tau antibody K9JA. . p‐tau441 droplets (in buffer with 10% PEG) exhibit glass surface wetting properties characteristics for liquids. Phase diagram of tau LLPS (p‐tau441 concentration (μM) versus PEG concentration (% w/v). In conditions modeling the intraneuronal environment (∼2 μM tau, 10% PEG, pH 7.5), p‐tau441 droplets can form at very high NaCl concentrations (up to 3 M NaCl) in the buffer. Guanidinium hydrochloride (GdmHCl) prevents p‐tau441 LLPS at 3 M; at this concentration, tau aggregates become visible. The chaotropic salt sodium thiocyanate (NaSCN) can inhibit LLPS with increasing concentration, whereas droplets become larger in the presence of the cosmotropic salt ammonium sulfate ((NH 4 ) 2 SO 4 ). Urea, which denatures proteins by unfolding secondary structures, prevents p‐tau441 LLPS. n = 3 per condition, determined after 3 h. Brightfield images for p‐tau441 LLPS under different salt conditions graphed in (G). The addition of 10% 1,6‐hexanediol to p‐tau441 droplets substantially reduced the amount of tau droplets formed. Data information: In (F) and (G), the phase diagrams show the average of three measurements.
    Figure Legend Snippet: Liquid droplet characteristics of p‐tau441 Qualitative distribution of phosphorylation sites in p‐tau441 [pS68/69, pT153, pT175, pT181, pS184, pS199, pS202, pT205, pS210, pT212, pS214, pT217, pT231, pS235, pS262, pS324, pY310, pS316, pS396, pS404, pS422 (Mair et al )]. The charge at pH 7.4 of domains in unphosphorylated tau441 is indicated as well. Liquid–liquid phase separation (LLPS) of p‐tau441 in presence of molecular crowding (12.5% w/v Ficoll‐400). No phase separation is observed without crowding agent or in the absence of p‐tau441 protein. Liquid droplets formed by p‐tau441 in the presence of 10% (w/v) PEG were negative stained with uranyl‐acetate and visualized by transmission electron microscopy (TEM). p‐tau441 droplets are decorated with gold particles after immunogold labeling using anti‐tau antibody K9JA. . p‐tau441 droplets (in buffer with 10% PEG) exhibit glass surface wetting properties characteristics for liquids. Phase diagram of tau LLPS (p‐tau441 concentration (μM) versus PEG concentration (% w/v). In conditions modeling the intraneuronal environment (∼2 μM tau, 10% PEG, pH 7.5), p‐tau441 droplets can form at very high NaCl concentrations (up to 3 M NaCl) in the buffer. Guanidinium hydrochloride (GdmHCl) prevents p‐tau441 LLPS at 3 M; at this concentration, tau aggregates become visible. The chaotropic salt sodium thiocyanate (NaSCN) can inhibit LLPS with increasing concentration, whereas droplets become larger in the presence of the cosmotropic salt ammonium sulfate ((NH 4 ) 2 SO 4 ). Urea, which denatures proteins by unfolding secondary structures, prevents p‐tau441 LLPS. n = 3 per condition, determined after 3 h. Brightfield images for p‐tau441 LLPS under different salt conditions graphed in (G). The addition of 10% 1,6‐hexanediol to p‐tau441 droplets substantially reduced the amount of tau droplets formed. Data information: In (F) and (G), the phase diagrams show the average of three measurements.

    Techniques Used: Staining, Transmission Assay, Electron Microscopy, Transmission Electron Microscopy, Labeling, Concentration Assay

    In vitro phase separation of tau initiated by crowding agents LLPS of p‐tau441 and p‐tau256 can also be initialized using crowding agent PEG‐8000 or a combination of PEG‐8000 with bovine serum albumin (BSA), whereas the soluble control protein GFP did not undergo LLPS in the presence of 10% PEG. We estimated the concentration of fluorescently labeled p‐tau441‐Alexa568 (10% PEG, 50 mM NaCl, 5 μM p‐tau441‐a568) in the droplets by confocal imaging ( z = 2 μm) of 30‐min‐old droplets. The measured maximum droplet fluorescence intensity was calibrated against different concentrations of p‐tau441‐Alexa568 in solution without LLPS initiation (no PEG, 50 mM NaCl). Cross‐sectional profile along the white arrow visualizes the intensity levels of droplets. The mean of the detected p‐tau441‐a568 concentration was 34.3 ± 6.4 μM in the droplets and 3.6 ± 0.3 μM in the solution phase. This value might be underestimating the actual tau concentration in the droplets because fluorophore:fluorophore quenching and other artifact resulting from the highly viscous and crowded environment in the droplets are not considered. Absorbance spectra of free and p‐tau441 bound Alexa568 shows differences in the intensity but no shift in the wavelengths. The intensity of free Alexa568 dye imaged at 533 nm in confocal microscopy did not change with the amount of PEG in solution. By confocal imaging, the crowding agent dextran remains excluded from p‐tau441 droplets initiated by adding 9% dextran‐70 kDa and 1% of fluorescent dextrans of different molecular weights. The addition of methylene blue, viscous aqua, and ThioS, all dyes with affinity to hydrophobic protein areas, to freshly prepared p‐tau441 droplets reveals the co‐partitioning and retention of these dyes in the droplets. The fluorescence may be further enhanced by inhibition of free rotation of the dyes due to the higher droplet viscosity. Data information: In right graphs of (B, C), data are represented as mean ± s.d.
    Figure Legend Snippet: In vitro phase separation of tau initiated by crowding agents LLPS of p‐tau441 and p‐tau256 can also be initialized using crowding agent PEG‐8000 or a combination of PEG‐8000 with bovine serum albumin (BSA), whereas the soluble control protein GFP did not undergo LLPS in the presence of 10% PEG. We estimated the concentration of fluorescently labeled p‐tau441‐Alexa568 (10% PEG, 50 mM NaCl, 5 μM p‐tau441‐a568) in the droplets by confocal imaging ( z = 2 μm) of 30‐min‐old droplets. The measured maximum droplet fluorescence intensity was calibrated against different concentrations of p‐tau441‐Alexa568 in solution without LLPS initiation (no PEG, 50 mM NaCl). Cross‐sectional profile along the white arrow visualizes the intensity levels of droplets. The mean of the detected p‐tau441‐a568 concentration was 34.3 ± 6.4 μM in the droplets and 3.6 ± 0.3 μM in the solution phase. This value might be underestimating the actual tau concentration in the droplets because fluorophore:fluorophore quenching and other artifact resulting from the highly viscous and crowded environment in the droplets are not considered. Absorbance spectra of free and p‐tau441 bound Alexa568 shows differences in the intensity but no shift in the wavelengths. The intensity of free Alexa568 dye imaged at 533 nm in confocal microscopy did not change with the amount of PEG in solution. By confocal imaging, the crowding agent dextran remains excluded from p‐tau441 droplets initiated by adding 9% dextran‐70 kDa and 1% of fluorescent dextrans of different molecular weights. The addition of methylene blue, viscous aqua, and ThioS, all dyes with affinity to hydrophobic protein areas, to freshly prepared p‐tau441 droplets reveals the co‐partitioning and retention of these dyes in the droplets. The fluorescence may be further enhanced by inhibition of free rotation of the dyes due to the higher droplet viscosity. Data information: In right graphs of (B, C), data are represented as mean ± s.d.

    Techniques Used: In Vitro, Concentration Assay, Labeling, Imaging, Fluorescence, Confocal Microscopy, Inhibition

    19) Product Images from "The P2Y6 Receptor Inhibits Effector T Cell Activation in Allergic Pulmonary Inflammation 1"

    Article Title: The P2Y6 Receptor Inhibits Effector T Cell Activation in Allergic Pulmonary Inflammation 1

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.1003669

    Df -induced pulmonary inflammation in NaCl- and Df -treated +/+ and p2ry6 (flox/flox); cre/+ mice
    Figure Legend Snippet: Df -induced pulmonary inflammation in NaCl- and Df -treated +/+ and p2ry6 (flox/flox); cre/+ mice

    Techniques Used: Mouse Assay

    20) Product Images from "Electrolytes induce long-range orientational order and free energy changes in the H-bond network of bulk water"

    Article Title: Electrolytes induce long-range orientational order and free energy changes in the H-bond network of bulk water

    Journal: Science Advances

    doi: 10.1126/sciadv.1501891

    Macroscopic manifestation of orientational order in the H-bond network of aqueous electrolyte solutions. ( A ) An illustration of the concept of surface tension. ( B ) Normalized resonant I − surface second harmonic response of NaI and KI (| χ (2) | 2 ; black and red data) ( 52 ) and normalized fs-ESHS (bulk) intensity change originating from constraints in the orientational order of bulk water (blue and green data). Ion-induced changes in the H-bond network of bulk water occur at lower concentrations (55 μM) than the surface adsorption (4 mM). The saturation of the bulk structural changes coincides with the minimum in Δγ (dashed line). The top panel shows an illustration of resonant surface SHG. VIS, visible; UV, ultraviolet; GS, ground state. ( C ). Measured surface tension difference (Δγ) for NaCl solutions of H 2 O (blue data) and D 2 O (brown data). Above 20 mM, Δγ increases, as indicated by the dashed line [see Jarvis and Scheiman ( 53 )]. A cartoon illustrating the structural changes in the electrolyte solution is shown on top. The numbers correspond to the different regimes of ionic strength and are also indicated in (B).
    Figure Legend Snippet: Macroscopic manifestation of orientational order in the H-bond network of aqueous electrolyte solutions. ( A ) An illustration of the concept of surface tension. ( B ) Normalized resonant I − surface second harmonic response of NaI and KI (| χ (2) | 2 ; black and red data) ( 52 ) and normalized fs-ESHS (bulk) intensity change originating from constraints in the orientational order of bulk water (blue and green data). Ion-induced changes in the H-bond network of bulk water occur at lower concentrations (55 μM) than the surface adsorption (4 mM). The saturation of the bulk structural changes coincides with the minimum in Δγ (dashed line). The top panel shows an illustration of resonant surface SHG. VIS, visible; UV, ultraviolet; GS, ground state. ( C ). Measured surface tension difference (Δγ) for NaCl solutions of H 2 O (blue data) and D 2 O (brown data). Above 20 mM, Δγ increases, as indicated by the dashed line [see Jarvis and Scheiman ( 53 )]. A cartoon illustrating the structural changes in the electrolyte solution is shown on top. The numbers correspond to the different regimes of ionic strength and are also indicated in (B).

    Techniques Used: Adsorption

    Nonspecific long-range changes in the H-bond network of water. ( A ) fs-ESHS intensities, relative to that of pure water, of 21 different electrolyte solutions obtained at a scattering angle of 90 o (P out P in P in polarization combination). The relative intensities of all electrolyte solutions can be fitted with the same equation. The dashed line indicates the concentration of half saturation. fs-ESHS intensities for CCl 4 are also plotted [the x axis should be read here as “Concentration (μM)”]. ( B ) fs-ESHS intensities of NaCl in H 2 O and D 2 O (P out P in P in polarization combination). ( C ) Water-water orientational correlations (left) and corresponding changes in average tilt angles (right) obtained from a molecular dynamics simulation of pure water. ( D ) Ion-induced change in the orientational order for an 8 mM NaCl solution. Changes in the distance-weighed water-water orientational correlations are shown (left axis), as well as the corresponding changes in the distance-weighted average tilt angle per water molecule (right axis).
    Figure Legend Snippet: Nonspecific long-range changes in the H-bond network of water. ( A ) fs-ESHS intensities, relative to that of pure water, of 21 different electrolyte solutions obtained at a scattering angle of 90 o (P out P in P in polarization combination). The relative intensities of all electrolyte solutions can be fitted with the same equation. The dashed line indicates the concentration of half saturation. fs-ESHS intensities for CCl 4 are also plotted [the x axis should be read here as “Concentration (μM)”]. ( B ) fs-ESHS intensities of NaCl in H 2 O and D 2 O (P out P in P in polarization combination). ( C ) Water-water orientational correlations (left) and corresponding changes in average tilt angles (right) obtained from a molecular dynamics simulation of pure water. ( D ) Ion-induced change in the orientational order for an 8 mM NaCl solution. Changes in the distance-weighed water-water orientational correlations are shown (left axis), as well as the corresponding changes in the distance-weighted average tilt angle per water molecule (right axis).

    Techniques Used: Concentration Assay

    Snapshots of long-range perturbations in aqueous NaCl solutions. ( A ) Illustration of two H-bonded water molecules that are orientationally correlated. The black arrows represent different axes along which H-bonds can be broken. fs-ESHS is only sensitive to the breaking of this H-bond via rotation (black curved arrow). Nuclear quantum effects predict that the H-bond bending mode is stronger, whereas the H-bond stretching mode is weaker, for D 2 O compared to H 2 O. ( B ) Sketch of the fs-ESHS experiment. fs-ESHS intensities are recorded at a scattering angle (θ) of 90 o . P(S) refers to a polarization direction parallel (perpendicular) to the scattering plane. ( C ) Top: Illustration of the different regimes probed in the experiment. At low ionic strengths (1), each ion induces long-range structural water correlations in its vicinity, forming a water-ordered domain. At higher ionic strengths, more domains appear. These domains (2) start to overlap and (3) interfere with one another, leading to a saturation of the observed signal. Bottom: fs-ESHS intensities, relative to that of H 2 O, measured from NaCl solutions. Four different polarization combinations were measured: P out P in P in , P out S in S in , S out P in P in , and S out S in S in . Only the P out P in P in and P out S in S in intensities change with increasing NaCl concentrations.
    Figure Legend Snippet: Snapshots of long-range perturbations in aqueous NaCl solutions. ( A ) Illustration of two H-bonded water molecules that are orientationally correlated. The black arrows represent different axes along which H-bonds can be broken. fs-ESHS is only sensitive to the breaking of this H-bond via rotation (black curved arrow). Nuclear quantum effects predict that the H-bond bending mode is stronger, whereas the H-bond stretching mode is weaker, for D 2 O compared to H 2 O. ( B ) Sketch of the fs-ESHS experiment. fs-ESHS intensities are recorded at a scattering angle (θ) of 90 o . P(S) refers to a polarization direction parallel (perpendicular) to the scattering plane. ( C ) Top: Illustration of the different regimes probed in the experiment. At low ionic strengths (1), each ion induces long-range structural water correlations in its vicinity, forming a water-ordered domain. At higher ionic strengths, more domains appear. These domains (2) start to overlap and (3) interfere with one another, leading to a saturation of the observed signal. Bottom: fs-ESHS intensities, relative to that of H 2 O, measured from NaCl solutions. Four different polarization combinations were measured: P out P in P in , P out S in S in , S out P in P in , and S out S in S in . Only the P out P in P in and P out S in S in intensities change with increasing NaCl concentrations.

    Techniques Used:

    21) Product Images from "Increased Intracellular Cyclic di-AMP Levels Sensitize Streptococcus gallolyticus subsp. gallolyticus to Osmotic Stress and Reduce Biofilm Formation and Adherence on Intestinal Cells"

    Article Title: Increased Intracellular Cyclic di-AMP Levels Sensitize Streptococcus gallolyticus subsp. gallolyticus to Osmotic Stress and Reduce Biofilm Formation and Adherence on Intestinal Cells

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00597-18

    Phenotypic changes associated with an increased intracellular c-di-AMP levels resulted from the deletion of gdpP . (A) Representative anaerobic growth kinetics of S. gallolyticus subsp. gallolyticus UCN34, the Δ gdpP mutant, and the Δ gdpP /p gdpP complemented strain. Initial inoculum was prepared from log-phase culture adjusted to approximately 3 × 10 7 CFU/ml. Growth, reflected in optical density, was measured at 600 nm (OD 600 ) at the indicated time point. The arrow indicates the sample collection time point for biofilm assay and the RNA-seq experiment. Error bars represent the standard deviation of the measurements from three samples. (B) Representative phase-contrast microscopy images on the stationary-phase culture of the S. gallolyticus subsp. gallolyticus UCN34, the Δ gdpP mutant, and the Δ gdpP /p gdpP complemented strain. Images were acquired with Carl Zeiss Axio Observer.Z1 inverted wide-field microscope fitted with 100×/1.3-numerical-aperture (NA) objective oil lens. Images were processed using Imaris version 8.2. Scale bars = 3 μm. (C) Cell area measurement of 300 imaged cells from three independent experiments using ImageJ software. Error bars represent the standard deviation of the 300 measurements. Kruskal-Wallis test: ****, P ≤ 0.0001; ns, P > 0.05. (D) Representative images of 5 μl of S. gallolyticus subsp. gallolyticus log-phase culture adjusted to approximately 3 × 10 7 CFU/ml spotted onto BHI agar and BHI agar supplemented with 0.4 M NaCl. (E) MICs of ampicillin and penicillin G against S. gallolyticus subsp. gallolyticus UCN34, the Δ gdpP mutant, and the Δ gdpP /p gdpP complemented strain. MIC was determined based on the optical density reading at 600 nm on a Tecan microplate reader, Infinite M200Pro.
    Figure Legend Snippet: Phenotypic changes associated with an increased intracellular c-di-AMP levels resulted from the deletion of gdpP . (A) Representative anaerobic growth kinetics of S. gallolyticus subsp. gallolyticus UCN34, the Δ gdpP mutant, and the Δ gdpP /p gdpP complemented strain. Initial inoculum was prepared from log-phase culture adjusted to approximately 3 × 10 7 CFU/ml. Growth, reflected in optical density, was measured at 600 nm (OD 600 ) at the indicated time point. The arrow indicates the sample collection time point for biofilm assay and the RNA-seq experiment. Error bars represent the standard deviation of the measurements from three samples. (B) Representative phase-contrast microscopy images on the stationary-phase culture of the S. gallolyticus subsp. gallolyticus UCN34, the Δ gdpP mutant, and the Δ gdpP /p gdpP complemented strain. Images were acquired with Carl Zeiss Axio Observer.Z1 inverted wide-field microscope fitted with 100×/1.3-numerical-aperture (NA) objective oil lens. Images were processed using Imaris version 8.2. Scale bars = 3 μm. (C) Cell area measurement of 300 imaged cells from three independent experiments using ImageJ software. Error bars represent the standard deviation of the 300 measurements. Kruskal-Wallis test: ****, P ≤ 0.0001; ns, P > 0.05. (D) Representative images of 5 μl of S. gallolyticus subsp. gallolyticus log-phase culture adjusted to approximately 3 × 10 7 CFU/ml spotted onto BHI agar and BHI agar supplemented with 0.4 M NaCl. (E) MICs of ampicillin and penicillin G against S. gallolyticus subsp. gallolyticus UCN34, the Δ gdpP mutant, and the Δ gdpP /p gdpP complemented strain. MIC was determined based on the optical density reading at 600 nm on a Tecan microplate reader, Infinite M200Pro.

    Techniques Used: Mutagenesis, Biofilm Production Assay, RNA Sequencing Assay, Standard Deviation, Microscopy, Software

    22) Product Images from "Salicylate 5-Hydroxylase: Intermediates in Aromatic Hydroxylation by a Rieske Monooxygenase"

    Article Title: Salicylate 5-Hydroxylase: Intermediates in Aromatic Hydroxylation by a Rieske Monooxygenase

    Journal: Biochemistry

    doi: 10.1021/acs.biochem.9b00292

    Steady-state and optical parameters of S5H. A buffered solution of S5HR (0.09 μM), S5HF (1.84 μM), and S5HH (0.23 μM), was reacted with a solution of NADH (742 μM) and salicylate (1–50 μM) using a stopped-flow spectrophotometer (2 mm pathlength, concentrations after mixing). Solutions were prepared in air-saturated 50 mM HEPES, 20 mM NaCl, 5 % glycerol, pH 8.0 buffer (~250 μM O 2 ) at 23 °C. The slope of the initial portion of the time course was determined and converted into an initial velocity using the extinction of NADH at 340 nm, pH 8 corrected for the contribution of the gentisate product at this wavelength and pH (difference ε = 4300 mM −1 cm −1 ). The solid line is a fit to a hyperbolic expression. Inset: UV-visible spectra of oxidized S5HH (black) (178 μM) and reduced S5HH (red) (178 μM, 5.5 mM sodium dithionite) in standard buffer at 23 °C. Spectra were recorded under anaerobic conditions. The calculated Δε 700 is 198 M −1 cm −1 , a value used in data analysis below.
    Figure Legend Snippet: Steady-state and optical parameters of S5H. A buffered solution of S5HR (0.09 μM), S5HF (1.84 μM), and S5HH (0.23 μM), was reacted with a solution of NADH (742 μM) and salicylate (1–50 μM) using a stopped-flow spectrophotometer (2 mm pathlength, concentrations after mixing). Solutions were prepared in air-saturated 50 mM HEPES, 20 mM NaCl, 5 % glycerol, pH 8.0 buffer (~250 μM O 2 ) at 23 °C. The slope of the initial portion of the time course was determined and converted into an initial velocity using the extinction of NADH at 340 nm, pH 8 corrected for the contribution of the gentisate product at this wavelength and pH (difference ε = 4300 mM −1 cm −1 ). The solid line is a fit to a hyperbolic expression. Inset: UV-visible spectra of oxidized S5HH (black) (178 μM) and reduced S5HH (red) (178 μM, 5.5 mM sodium dithionite) in standard buffer at 23 °C. Spectra were recorded under anaerobic conditions. The calculated Δε 700 is 198 M −1 cm −1 , a value used in data analysis below.

    Techniques Used: Spectrophotometry, Expressing

    Stopped-flow diode array spectra of a single-turnover reaction. Fully reduced S5HH (50 μM) with salicylate (1 mM) was reacted with O 2 (900 μM) and salicylate (1 mM) (concentrations after mixing). (A) Period from 0 to 3 s. (B) Period from 3 s to 1000 s. Inset: Spectrum of the 700 nm intermediate determined by subtracting the spectral contribution of the Rieske cluster from 3 s spectrum. Conditions: 50 mM HEPES, 100 mM NaCl, 5% glycerol, pH 8, 4 °C.
    Figure Legend Snippet: Stopped-flow diode array spectra of a single-turnover reaction. Fully reduced S5HH (50 μM) with salicylate (1 mM) was reacted with O 2 (900 μM) and salicylate (1 mM) (concentrations after mixing). (A) Period from 0 to 3 s. (B) Period from 3 s to 1000 s. Inset: Spectrum of the 700 nm intermediate determined by subtracting the spectral contribution of the Rieske cluster from 3 s spectrum. Conditions: 50 mM HEPES, 100 mM NaCl, 5% glycerol, pH 8, 4 °C.

    Techniques Used:

    23) Product Images from "Study of the Assembly of Vesicular Stomatitis Virus N Protein: Role of the P Protein"

    Article Title: Study of the Assembly of Vesicular Stomatitis Virus N Protein: Role of the P Protein

    Journal: Journal of Virology

    doi:

    Gel filtration profiles of the N/P protein complexes at pH 7.5 (A), 6.0 (C), 5.0 (E), and 4.0 (G). The N and P proteins were coexpressed in E. coli harboring plasmid pET-N/P. Following protein expression, the N/P protein complexes were isolated by Ni affinity chromatography. These partially purified protein complexes were dialyzed in either 0.05 M Tris (pH 7.5) containing 300 mM NaCl or 0.1 M citrate (pH 6.0, 5.0, or 4.0) containing 0.25 M NaCl. Following dialysis, the complexes were chromatographed on a Sephacryl S-300 size exclusion column (16/60; Pharmacia) at a flow rate of 1 ml/min. In each case, the elution buffer was the same as the dialysis buffer. The horizontal axis shows the elution time in minutes; the vertical axis shows A 280 . The proteins contained in the peaks from each purification were analyzed by SDS-polyacrylamide gel electrophoresis (gel B for panel A, gel D for panel C, gel F for panel E, and gel H for panel G). (B, D, F, and H). The proteins were electrophoresed on 10% gels and stained with Coomassie brilliant blue R-250. Lane numbers correspond to the peak numbers on the corresponding chromatogram to the left. Lanes containing molecular weight standards are labeled MW; positions are indicated in kilodaltons.
    Figure Legend Snippet: Gel filtration profiles of the N/P protein complexes at pH 7.5 (A), 6.0 (C), 5.0 (E), and 4.0 (G). The N and P proteins were coexpressed in E. coli harboring plasmid pET-N/P. Following protein expression, the N/P protein complexes were isolated by Ni affinity chromatography. These partially purified protein complexes were dialyzed in either 0.05 M Tris (pH 7.5) containing 300 mM NaCl or 0.1 M citrate (pH 6.0, 5.0, or 4.0) containing 0.25 M NaCl. Following dialysis, the complexes were chromatographed on a Sephacryl S-300 size exclusion column (16/60; Pharmacia) at a flow rate of 1 ml/min. In each case, the elution buffer was the same as the dialysis buffer. The horizontal axis shows the elution time in minutes; the vertical axis shows A 280 . The proteins contained in the peaks from each purification were analyzed by SDS-polyacrylamide gel electrophoresis (gel B for panel A, gel D for panel C, gel F for panel E, and gel H for panel G). (B, D, F, and H). The proteins were electrophoresed on 10% gels and stained with Coomassie brilliant blue R-250. Lane numbers correspond to the peak numbers on the corresponding chromatogram to the left. Lanes containing molecular weight standards are labeled MW; positions are indicated in kilodaltons.

    Techniques Used: Filtration, Plasmid Preparation, Positron Emission Tomography, Expressing, Isolation, Affinity Chromatography, Purification, Flow Cytometry, Polyacrylamide Gel Electrophoresis, Staining, Molecular Weight, Labeling

    24) Product Images from "Bordetella pertussis FbpA Binds Both Unchelated Iron and Iron Siderophore Complexes"

    Article Title: Bordetella pertussis FbpA Binds Both Unchelated Iron and Iron Siderophore Complexes

    Journal: Biochemistry

    doi: 10.1021/bi5002823

    (a) UV–vis spectra with charge transfer (CT) band at 493 nm for Fe-ENT (15.4 μM) before and after addition of FbpA Bp (7.5 μM) in 50 mM MES, 100 mM NaCl at pH 6.5; (b) UV–vis spectra for Fe 2 (ALC) 3 (9 μM) before and after addition of apoFbpA Bp (18 μM) showing a shift in λ max from 428 nm (CT band for Fe 2 ALC 3 ) to 410 nm. The mixture of Fe 2 (ALC) 3 and FbpA Bp was monitored for 3 h, and the spectrum displayed here is from the end of that incubation.
    Figure Legend Snippet: (a) UV–vis spectra with charge transfer (CT) band at 493 nm for Fe-ENT (15.4 μM) before and after addition of FbpA Bp (7.5 μM) in 50 mM MES, 100 mM NaCl at pH 6.5; (b) UV–vis spectra for Fe 2 (ALC) 3 (9 μM) before and after addition of apoFbpA Bp (18 μM) showing a shift in λ max from 428 nm (CT band for Fe 2 ALC 3 ) to 410 nm. The mixture of Fe 2 (ALC) 3 and FbpA Bp was monitored for 3 h, and the spectrum displayed here is from the end of that incubation.

    Techniques Used: Incubation

    Visible region spectra of Fe-FbpA Bp -X (X = citrate, carbonate, oxalate, or NTA) in 50 mM MES, 100 mM NaCl at pH 6.5. Protein concentrations are indicated.
    Figure Legend Snippet: Visible region spectra of Fe-FbpA Bp -X (X = citrate, carbonate, oxalate, or NTA) in 50 mM MES, 100 mM NaCl at pH 6.5. Protein concentrations are indicated.

    Techniques Used:

    25) Product Images from "Structural Stability of Human Protein Tyrosine Phosphatase ? Catalytic Domain: Effect of Point Mutations"

    Article Title: Structural Stability of Human Protein Tyrosine Phosphatase ? Catalytic Domain: Effect of Point Mutations

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0032555

    Intrinsic fluorescence emission spectra of PTPρ wild-type and mutants. Fluorescence spectra of PTPρ wild-type and mutants in 0 M (continuous lines), 8.30 M (dotted lines), 3.95 M (D927G and N1128I, dashed lines) and 4.45 M urea (wild-type and Q987K, dashed lines) were recorded at 0.04 mg/ml protein concentration (295 nm excitation wavelength) at 10°C in 20 mM Tris/HCl, pH 7.5 containing 0.2 M NaCl and 200 µM DTT.
    Figure Legend Snippet: Intrinsic fluorescence emission spectra of PTPρ wild-type and mutants. Fluorescence spectra of PTPρ wild-type and mutants in 0 M (continuous lines), 8.30 M (dotted lines), 3.95 M (D927G and N1128I, dashed lines) and 4.45 M urea (wild-type and Q987K, dashed lines) were recorded at 0.04 mg/ml protein concentration (295 nm excitation wavelength) at 10°C in 20 mM Tris/HCl, pH 7.5 containing 0.2 M NaCl and 200 µM DTT.

    Techniques Used: Fluorescence, Protein Concentration

    Effect of urea on near-UV CD spectra of PTPρ wild-type and D927G. ( A ) Near-UV CD spectra of wild-type and D927G in 0 M, 4.28 M and 7.31 M urea were recorded in a 1-cm quartz cuvette at 2.4 mg/ml protein concentration at 10°C in 20 mM Tris/HCl, pH 7.5 containing 0.2 M NaCl and 2 mM DTT. ( B ) and ( C ) Near-UV CD changes of wild-type and D927G at increasing urea concentrations reported as the first (V1, B ) and the second (V2, C ) column of the V matrix. V1 and V2 were obtained by SVD of the near-UV CD spectral data as described in the text.
    Figure Legend Snippet: Effect of urea on near-UV CD spectra of PTPρ wild-type and D927G. ( A ) Near-UV CD spectra of wild-type and D927G in 0 M, 4.28 M and 7.31 M urea were recorded in a 1-cm quartz cuvette at 2.4 mg/ml protein concentration at 10°C in 20 mM Tris/HCl, pH 7.5 containing 0.2 M NaCl and 2 mM DTT. ( B ) and ( C ) Near-UV CD changes of wild-type and D927G at increasing urea concentrations reported as the first (V1, B ) and the second (V2, C ) column of the V matrix. V1 and V2 were obtained by SVD of the near-UV CD spectral data as described in the text.

    Techniques Used: Protein Concentration

    Spectroscopic properties of PTPρ wild-type and mutants. ( A ) Near-UV CD spectra were recorded in a 1-cm quartz cuvette at 1.0 mg/ml protein concentration in 20 mM Tris/HCl, pH 7.5 containing 0.2 M NaCl and 2 mM DTT. ( B ) Intrinsic fluorescence emission spectra were recorded at 0.04 mg/ml protein concentration (295 nm excitation wavelength) in 20 mM Tris/HCl, pH 7.5 containing 0.2 M NaCl and 200 µM DTT. ( C ) Far-UV CD spectra were recorded in a 0.1-cm quartz cuvette at 0.2 mg/ml in 20 mM Tris/HCl, pH 7.5 containing 0.2 M NaCl and 0.4 mM DTT.
    Figure Legend Snippet: Spectroscopic properties of PTPρ wild-type and mutants. ( A ) Near-UV CD spectra were recorded in a 1-cm quartz cuvette at 1.0 mg/ml protein concentration in 20 mM Tris/HCl, pH 7.5 containing 0.2 M NaCl and 2 mM DTT. ( B ) Intrinsic fluorescence emission spectra were recorded at 0.04 mg/ml protein concentration (295 nm excitation wavelength) in 20 mM Tris/HCl, pH 7.5 containing 0.2 M NaCl and 200 µM DTT. ( C ) Far-UV CD spectra were recorded in a 0.1-cm quartz cuvette at 0.2 mg/ml in 20 mM Tris/HCl, pH 7.5 containing 0.2 M NaCl and 0.4 mM DTT.

    Techniques Used: Protein Concentration, Fluorescence

    Thermal transition of PTPρ wild-type and mutants. ( A ) PTPρ wild-type, N1128I, Q987K and D927G were heated from 10°C to 72°C in a 0.1-cm quartz cuvette at 0.2 mg/ml in 20 mM Tris/HCl, pH 7.5 containing 0.2 M NaCl and 0.4 mM DTT. The dichroic activity at 209 nm was monitored continuously every 0.5°C. The inset shows the first derivative of the same data. ( B ) PMTV data recorded in the same experiments shown in ( A ).
    Figure Legend Snippet: Thermal transition of PTPρ wild-type and mutants. ( A ) PTPρ wild-type, N1128I, Q987K and D927G were heated from 10°C to 72°C in a 0.1-cm quartz cuvette at 0.2 mg/ml in 20 mM Tris/HCl, pH 7.5 containing 0.2 M NaCl and 0.4 mM DTT. The dichroic activity at 209 nm was monitored continuously every 0.5°C. The inset shows the first derivative of the same data. ( B ) PMTV data recorded in the same experiments shown in ( A ).

    Techniques Used: Activity Assay

    26) Product Images from "RNase H1 directs origin-specific initiation of DNA replication in human mitochondria"

    Article Title: RNase H1 directs origin-specific initiation of DNA replication in human mitochondria

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1007781

    Factors affecting R-loop formation in vitro. A. Purified, recombinant proteins used in the present study visualized by Stain Free SDS-PAGE (Bio-Rad). B. In vitro transcription from LSP with POLRMT (20 nM), TFAM (200 nM) and TFB2M (60 nM). R-loops were formed and detected as described in panel C. TEFM (40 nM) was added to the indicated reactions. Products formed are labeled as followed: PT: transcripts prematurely terminated at CSBII; RC: longer transcripts formed by rolling circle transcription; and R-loops: transcripts unaffected by RNase A treatment (lane 6). The RNA was labeled by [ 32 P]UTP incorporation. C. Reaction scheme for R-loop formation. A pUC18 plasmid containing an LSP insert, including the CSB region (pUC-LSP, S1 Table ) was used. When indicated, the template was treated with topoisomerase I to relax supercoils. In vitro transcription was performed in the presence or absence of TEFM followed by the addition of 300 mM NaCl and RNase A to remove free RNA. D. Effects of mtSSB on in vitro transcription and R-loop formation. Templates used were supercoiled pUC-LSP (lanes 1-6) and as a control, linear pUC-HSP (lanes 7-10, see S1 Table for template sequence). mtSSB concentrations are indicated in nM. HSP RO: Run-off product of HSP transcription; PT: transcripts prematurely terminated at CSBII; and R-loops: transcripts unaffected by RNase A treatment. The ratio of R-loops/CSBII pre-terminated transcripts for each mtSSB concentration is indicated (see Materials and methods ). E. R-loop formation was as in 1C, but without RNase A treatment. Increasing RNase H1 concentrations were added (0, 1, 2, 4, 8, 16 and 32 nM in lanes 1-7). PT indicates transcripts prematurely terminated at CSBII.
    Figure Legend Snippet: Factors affecting R-loop formation in vitro. A. Purified, recombinant proteins used in the present study visualized by Stain Free SDS-PAGE (Bio-Rad). B. In vitro transcription from LSP with POLRMT (20 nM), TFAM (200 nM) and TFB2M (60 nM). R-loops were formed and detected as described in panel C. TEFM (40 nM) was added to the indicated reactions. Products formed are labeled as followed: PT: transcripts prematurely terminated at CSBII; RC: longer transcripts formed by rolling circle transcription; and R-loops: transcripts unaffected by RNase A treatment (lane 6). The RNA was labeled by [ 32 P]UTP incorporation. C. Reaction scheme for R-loop formation. A pUC18 plasmid containing an LSP insert, including the CSB region (pUC-LSP, S1 Table ) was used. When indicated, the template was treated with topoisomerase I to relax supercoils. In vitro transcription was performed in the presence or absence of TEFM followed by the addition of 300 mM NaCl and RNase A to remove free RNA. D. Effects of mtSSB on in vitro transcription and R-loop formation. Templates used were supercoiled pUC-LSP (lanes 1-6) and as a control, linear pUC-HSP (lanes 7-10, see S1 Table for template sequence). mtSSB concentrations are indicated in nM. HSP RO: Run-off product of HSP transcription; PT: transcripts prematurely terminated at CSBII; and R-loops: transcripts unaffected by RNase A treatment. The ratio of R-loops/CSBII pre-terminated transcripts for each mtSSB concentration is indicated (see Materials and methods ). E. R-loop formation was as in 1C, but without RNase A treatment. Increasing RNase H1 concentrations were added (0, 1, 2, 4, 8, 16 and 32 nM in lanes 1-7). PT indicates transcripts prematurely terminated at CSBII.

    Techniques Used: In Vitro, Purification, Recombinant, Staining, SDS Page, Labeling, Plasmid Preparation, Sequencing, Concentration Assay

    27) Product Images from "A monovalent ion in the DNA binding interface of the eukaryotic junction-resolving enzyme GEN1"

    Article Title: A monovalent ion in the DNA binding interface of the eukaryotic junction-resolving enzyme GEN1

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky863

    Cleavage of branched DNA substrates by C tGEN1 as a function of the monovalent metal ion present. Substrates radioactively [5′- 32 P]-labeled on the x strand were incubated with Ct GEN1 in buffer containing 10 mM cacodylate (pH 6.5), 1 mM MgCl 2, 50 mM NaCl or KCl, 0.1% BSA for 3 min at 37°C. The products separated by polyacrylamide gel electrophoresis followed by phosphorimaging. ( A ) Denaturing gel electrophoresis. The major product of cleavage is arrowed. Note that each substrate is cleaved at the same position. The scheme on the right shows the sequence of the core of the junction, and the nomenclature of the arms and strands. The position of the radioactive label is indicated and the cleavage sites are arrowed. The major cleavages are made in the B and R arms, 1 nt 3′ to the point of strand exchange on the h and x strands. In this experiment the x strand is radioactively [5′- 32 P]-labeled, and thus only cleavage in this strand is detected. ( B ) Native gel electrophoresis. The products of Ct GEN1 cleavage are arrowed, with the relevant tracks indicated; e.g. the arrow labeled 1–3 indicates the product of four-way junction cleavage. In A and B tracks 1–3 contain the four-way junction (4H), tracks 4–6 contain the nicked three-way junction, tracks 7–9 contain the three-way junction (3H) and tracks 10–12 contain the splayed helix junction. For each is shown no cleavage with Ct GEN1 i.e. intact junction, tracks 1, 4, 7 and 10, cleavage with Ct GEN1 in the presence of Na + ions, tracks 2, 5, 8 and 11, and cleavage with Ct GEN1 in the presence of K + ions, tracks 3, 6, 9 and 12. ( C ) Progress curve for cleavage of a nicked three-way junction. ( D ) Progress curve for cleavage of a splayed helix junction. In both C and D cleavage in K + and Na + .
    Figure Legend Snippet: Cleavage of branched DNA substrates by C tGEN1 as a function of the monovalent metal ion present. Substrates radioactively [5′- 32 P]-labeled on the x strand were incubated with Ct GEN1 in buffer containing 10 mM cacodylate (pH 6.5), 1 mM MgCl 2, 50 mM NaCl or KCl, 0.1% BSA for 3 min at 37°C. The products separated by polyacrylamide gel electrophoresis followed by phosphorimaging. ( A ) Denaturing gel electrophoresis. The major product of cleavage is arrowed. Note that each substrate is cleaved at the same position. The scheme on the right shows the sequence of the core of the junction, and the nomenclature of the arms and strands. The position of the radioactive label is indicated and the cleavage sites are arrowed. The major cleavages are made in the B and R arms, 1 nt 3′ to the point of strand exchange on the h and x strands. In this experiment the x strand is radioactively [5′- 32 P]-labeled, and thus only cleavage in this strand is detected. ( B ) Native gel electrophoresis. The products of Ct GEN1 cleavage are arrowed, with the relevant tracks indicated; e.g. the arrow labeled 1–3 indicates the product of four-way junction cleavage. In A and B tracks 1–3 contain the four-way junction (4H), tracks 4–6 contain the nicked three-way junction, tracks 7–9 contain the three-way junction (3H) and tracks 10–12 contain the splayed helix junction. For each is shown no cleavage with Ct GEN1 i.e. intact junction, tracks 1, 4, 7 and 10, cleavage with Ct GEN1 in the presence of Na + ions, tracks 2, 5, 8 and 11, and cleavage with Ct GEN1 in the presence of K + ions, tracks 3, 6, 9 and 12. ( C ) Progress curve for cleavage of a nicked three-way junction. ( D ) Progress curve for cleavage of a splayed helix junction. In both C and D cleavage in K + and Na + .

    Techniques Used: Labeling, Incubation, Polyacrylamide Gel Electrophoresis, Nucleic Acid Electrophoresis, Sequencing

    28) Product Images from "Input-Output Relationship of CA1 Pyramidal Neurons Reveals Intact Homeostatic Mechanisms in a Mouse Model of Fragile X Syndrome"

    Article Title: Input-Output Relationship of CA1 Pyramidal Neurons Reveals Intact Homeostatic Mechanisms in a Mouse Model of Fragile X Syndrome

    Journal: Cell Reports

    doi: 10.1016/j.celrep.2020.107988

    Short-Term AIS Shortening Is Absent in Acute Slices after Sustained Depolarization (A) Representative flattened confocal stack of AIS labeled in acute hippocampal slices from WT (top) and Fmr1 −/y mice (bottom), following 3-h incubation with 15 mM KCl (right) or NaCl osmotic controls (left). AISs were visualized with ankyrinG (green pseudocolor) and measured in neurons labeled with NeuN (blue pseudocolor). Scale bars: 20 μm. (B) Quantification of AIS length following 3-h application of 15 mM KCl, compared to NaCl osmotic controls in WT mice. Average AIS length of each mouse tested is shown overlaid. (C) The same analysis as in (B), but in Fmr1 −/y mice. (D) AIS measured in primary dissociated hippocampal cell cultures produced from WT (left) and Fmr1 −/y (right) mice following 3 h of 15 mM NaCl or KCl and labeled with ankyrinG (green) and NeuN (blue). Scale bars: 20 μm (top), 10 μm (bottom). (E) Quantification of AIS length under control conditions from WT (black) and Fmr1 −/y (red) single-mouse cultures. The average AIS length per mouse (from 2 coverslips) is shown overlain; the number of mice is indicated in parentheses. (F) AIS lengths plotted for WT mouse cultured neurons following 3 h of 15 mM KCl and NaCl. (G) AIS lengths of Fmr1 −/y neurons following 15 mM KCl and NaCl application. (H) Comparative difference in AIS length (KCl length − NaCl length), plotted for each mouse. ns, p > 0.05, ∗ p
    Figure Legend Snippet: Short-Term AIS Shortening Is Absent in Acute Slices after Sustained Depolarization (A) Representative flattened confocal stack of AIS labeled in acute hippocampal slices from WT (top) and Fmr1 −/y mice (bottom), following 3-h incubation with 15 mM KCl (right) or NaCl osmotic controls (left). AISs were visualized with ankyrinG (green pseudocolor) and measured in neurons labeled with NeuN (blue pseudocolor). Scale bars: 20 μm. (B) Quantification of AIS length following 3-h application of 15 mM KCl, compared to NaCl osmotic controls in WT mice. Average AIS length of each mouse tested is shown overlaid. (C) The same analysis as in (B), but in Fmr1 −/y mice. (D) AIS measured in primary dissociated hippocampal cell cultures produced from WT (left) and Fmr1 −/y (right) mice following 3 h of 15 mM NaCl or KCl and labeled with ankyrinG (green) and NeuN (blue). Scale bars: 20 μm (top), 10 μm (bottom). (E) Quantification of AIS length under control conditions from WT (black) and Fmr1 −/y (red) single-mouse cultures. The average AIS length per mouse (from 2 coverslips) is shown overlain; the number of mice is indicated in parentheses. (F) AIS lengths plotted for WT mouse cultured neurons following 3 h of 15 mM KCl and NaCl. (G) AIS lengths of Fmr1 −/y neurons following 15 mM KCl and NaCl application. (H) Comparative difference in AIS length (KCl length − NaCl length), plotted for each mouse. ns, p > 0.05, ∗ p

    Techniques Used: Labeling, Mouse Assay, Incubation, Produced, Cell Culture

    Live Imaging of the AIS Fails to Reveal Short-Term Structural Plasticity (A) Low-power flattened confocal stack of CA1 of the hippocampus showing β1-NaV-GFP (β1-GFP, green pseudocolor) expression compared to ankyrinG (red pseudocolor), showing an overlapping distribution. Scale bar: 100 μm. (B) High magnification of a β1-NaV-GFP labeled PC and AIS, demonstrating faithful overlap of GFP with ankyrinG labeling. Scale bar: 20 μm. (C) Representative 2-photon images of CA1 showing β1-NaV-GFP labeling under control conditions (0 h) compared to 3 h of treatment with 15 mM NaCl (top) or KCl (bottom). Scale bar: 10 μm. (D) Comparison of AIS length before (x axis) to the AIS length 3 h later for 15 mM NaCl (filled circles) and 15 mM KCl (open circles) in WT CA1 PCs. Data are shown for 99 AISs treated with NaCl and 65 AISs treated with KCl from 7 WT mice and fitted with linear regression (solid line, NaCl; dashed line, KCl). (E) The same data as in (D), but plotted for 44 AISs treated with NaCl and 45 AISs treated with KCl from 5 Fmr1 −/y mice. All of the data are shown as individual cell replicates with, where appropriate, fitted linear relationships.
    Figure Legend Snippet: Live Imaging of the AIS Fails to Reveal Short-Term Structural Plasticity (A) Low-power flattened confocal stack of CA1 of the hippocampus showing β1-NaV-GFP (β1-GFP, green pseudocolor) expression compared to ankyrinG (red pseudocolor), showing an overlapping distribution. Scale bar: 100 μm. (B) High magnification of a β1-NaV-GFP labeled PC and AIS, demonstrating faithful overlap of GFP with ankyrinG labeling. Scale bar: 20 μm. (C) Representative 2-photon images of CA1 showing β1-NaV-GFP labeling under control conditions (0 h) compared to 3 h of treatment with 15 mM NaCl (top) or KCl (bottom). Scale bar: 10 μm. (D) Comparison of AIS length before (x axis) to the AIS length 3 h later for 15 mM NaCl (filled circles) and 15 mM KCl (open circles) in WT CA1 PCs. Data are shown for 99 AISs treated with NaCl and 65 AISs treated with KCl from 7 WT mice and fitted with linear regression (solid line, NaCl; dashed line, KCl). (E) The same data as in (D), but plotted for 44 AISs treated with NaCl and 45 AISs treated with KCl from 5 Fmr1 −/y mice. All of the data are shown as individual cell replicates with, where appropriate, fitted linear relationships.

    Techniques Used: Imaging, Expressing, Labeling, Mouse Assay

    Intrinsic Physiological Plasticity and Homeostatic Responses in WT and Fmr1 −/y Mice (A and B) Representative voltage responses from (A) WT (black) and (B) Fmr1 −/y (red) CA1 PCs to current injections, from −70 mV (0–400 pA, 25 pA steps, 500 ms duration). (C) Current frequency plots for the same CA1 PCs from WT mice, when recorded before (top) and after (bottom) 3-h NaCl (12 cells from 5 mice) or KCl (19 cells from 8 mice) applications. (D) According to the same format as (C), but for Fmr1 −/y mice (NaCl: 7 cells from 4 mice; KCl: 18 cells from 8 mice). (E and F) Pairwise analysis of rheobase current (E) and voltage threshold (F) from the same WT and Fmr1 −/y CA1 PCs. (G) Subtracted AP discharge across the range of injected currents given to CA1 PCs. ns, p > 0.05, ∗ p
    Figure Legend Snippet: Intrinsic Physiological Plasticity and Homeostatic Responses in WT and Fmr1 −/y Mice (A and B) Representative voltage responses from (A) WT (black) and (B) Fmr1 −/y (red) CA1 PCs to current injections, from −70 mV (0–400 pA, 25 pA steps, 500 ms duration). (C) Current frequency plots for the same CA1 PCs from WT mice, when recorded before (top) and after (bottom) 3-h NaCl (12 cells from 5 mice) or KCl (19 cells from 8 mice) applications. (D) According to the same format as (C), but for Fmr1 −/y mice (NaCl: 7 cells from 4 mice; KCl: 18 cells from 8 mice). (E and F) Pairwise analysis of rheobase current (E) and voltage threshold (F) from the same WT and Fmr1 −/y CA1 PCs. (G) Subtracted AP discharge across the range of injected currents given to CA1 PCs. ns, p > 0.05, ∗ p

    Techniques Used: Mouse Assay, Injection

    29) Product Images from "Control of VWF A2 domain stability and ADAMTS13 access to the scissile bond of full-length VWF"

    Article Title: Control of VWF A2 domain stability and ADAMTS13 access to the scissile bond of full-length VWF

    Journal: Blood

    doi: 10.1182/blood-2013-11-538173

    Detection of proteolysis of FL-VWF variants by collagen-binding assay. (A-D) The VWF variants and ADAMTS13 were separately preincubated in 20 mM Tris (pH 7.8), 50 mM NaCl, 5 mM CaCl 2 ± 1.5 M urea at 37°C for 45 minutes. VWF (1 µg/mL)
    Figure Legend Snippet: Detection of proteolysis of FL-VWF variants by collagen-binding assay. (A-D) The VWF variants and ADAMTS13 were separately preincubated in 20 mM Tris (pH 7.8), 50 mM NaCl, 5 mM CaCl 2 ± 1.5 M urea at 37°C for 45 minutes. VWF (1 µg/mL)

    Techniques Used: Binding Assay

    30) Product Images from "DNA-interacting properties of two analogous square-planar cis-chlorido complexes: copper versus palladium"

    Article Title: DNA-interacting properties of two analogous square-planar cis-chlorido complexes: copper versus palladium

    Journal: Journal of Biological Inorganic Chemistry

    doi: 10.1007/s00775-021-01888-2

    UV–Vis spectra of 25 μM solutions of a [Cu(CPYA)Cl 2 ] ( 1 ) and b [Pd(CPYA)Cl 2 ] ( 2 ) in the absence and presence of increasing amounts of ct-DNA (0 − 51.2 μM). The arrows show the intensity changes at the indicated wavelengths upon increase of [ct-DNA]. Measurements carried out in cacodylate–NaCl buffer after incubation at 37 °C for 1 h ( 1 ) and 24 h ( 2 )
    Figure Legend Snippet: UV–Vis spectra of 25 μM solutions of a [Cu(CPYA)Cl 2 ] ( 1 ) and b [Pd(CPYA)Cl 2 ] ( 2 ) in the absence and presence of increasing amounts of ct-DNA (0 − 51.2 μM). The arrows show the intensity changes at the indicated wavelengths upon increase of [ct-DNA]. Measurements carried out in cacodylate–NaCl buffer after incubation at 37 °C for 1 h ( 1 ) and 24 h ( 2 )

    Techniques Used: Incubation

    31) Product Images from "HOS15 Interacts with the Histone Deacetylase HDA9 and the Evening Complex to Epigenetically Regulate the Floral Activator GIGANTEA [OPEN]"

    Article Title: HOS15 Interacts with the Histone Deacetylase HDA9 and the Evening Complex to Epigenetically Regulate the Floral Activator GIGANTEA [OPEN]

    Journal: The Plant Cell

    doi: 10.1105/tpc.18.00721

    HOS15 Associates with EC Components. (A) BiFC analysis of proteins transiently expressed in N. benthamiana leaves. VN and VC indicate the N and C termini, respectively, of Venus (eYFP). Fluorescence (left panels) was detected by confocal microscopy. Right panels are overlay of fluorescence and differential interference contest images. Bars = 100 μm. (B) HOS15 interacts with ELF3. Wild type (10 d old) and hos15-2 plants were cross-linked in 1% formaldehyde to preserve in vivo interaction after harvesting plant material. Total protein extracts were immunoprecipitated with α-HOS15 antibody and resolved by SDS-PAGE. Immunoblots were probed with α-HOS15, or with α-ELF3 to detect ELF3. *Nonspecific bands. (C) HOS15 forms a complex with EC components. N. benthamiana plants were infiltrated with Agrobacterium harboring 35S:HA-LUX, 35S:FLAG-HOS15, and 35S:MYC-ELF3 for transient expression. The proteins were immunoprecipitated with α-HA antibody and resolved by SDS-PAGE. The immunoblots were probed with α-HA to detect LUX, α-HOS15, or α-MYC to detect ELF3. (D) Gel-filtration analysis of HOS15, LUX, and ELF3. Total proteins extracted from 2-week-old wild type plants were fractionated by size-exclusion chromatography using a Superdex 200 10/300GL column equilibrated with elution buffer [50 mM Tris-Cl (pH 7.5), 100 mM NaCl, 0.02% sodium azide]. The samples were separated by SDS-PAGE and subjected to immunoblotting analysis using α-HOS15, α-LUX, or α-ELF3. Aliquots of total protein extracts from wild type, input after ammonium sulfate precipitation of wild type total extracts, and total protein extracts individually isolated from elf3-1 , hos15-2 , or pcl1-1 mutants were used as input controls. Molecular mass markers (ferritin, 440 kD; β-amylase, 200 kD; alcohol dehydrogenase, 150 kD; BSA, 67 kD) were independently eluted using the same equilibrated column. Total, total protein extract; Input, input after ammonium sulfate precipitation.
    Figure Legend Snippet: HOS15 Associates with EC Components. (A) BiFC analysis of proteins transiently expressed in N. benthamiana leaves. VN and VC indicate the N and C termini, respectively, of Venus (eYFP). Fluorescence (left panels) was detected by confocal microscopy. Right panels are overlay of fluorescence and differential interference contest images. Bars = 100 μm. (B) HOS15 interacts with ELF3. Wild type (10 d old) and hos15-2 plants were cross-linked in 1% formaldehyde to preserve in vivo interaction after harvesting plant material. Total protein extracts were immunoprecipitated with α-HOS15 antibody and resolved by SDS-PAGE. Immunoblots were probed with α-HOS15, or with α-ELF3 to detect ELF3. *Nonspecific bands. (C) HOS15 forms a complex with EC components. N. benthamiana plants were infiltrated with Agrobacterium harboring 35S:HA-LUX, 35S:FLAG-HOS15, and 35S:MYC-ELF3 for transient expression. The proteins were immunoprecipitated with α-HA antibody and resolved by SDS-PAGE. The immunoblots were probed with α-HA to detect LUX, α-HOS15, or α-MYC to detect ELF3. (D) Gel-filtration analysis of HOS15, LUX, and ELF3. Total proteins extracted from 2-week-old wild type plants were fractionated by size-exclusion chromatography using a Superdex 200 10/300GL column equilibrated with elution buffer [50 mM Tris-Cl (pH 7.5), 100 mM NaCl, 0.02% sodium azide]. The samples were separated by SDS-PAGE and subjected to immunoblotting analysis using α-HOS15, α-LUX, or α-ELF3. Aliquots of total protein extracts from wild type, input after ammonium sulfate precipitation of wild type total extracts, and total protein extracts individually isolated from elf3-1 , hos15-2 , or pcl1-1 mutants were used as input controls. Molecular mass markers (ferritin, 440 kD; β-amylase, 200 kD; alcohol dehydrogenase, 150 kD; BSA, 67 kD) were independently eluted using the same equilibrated column. Total, total protein extract; Input, input after ammonium sulfate precipitation.

    Techniques Used: Bimolecular Fluorescence Complementation Assay, Fluorescence, Confocal Microscopy, In Vivo, Immunoprecipitation, SDS Page, Western Blot, Expressing, Filtration, Size-exclusion Chromatography, Isolation

    32) Product Images from "Impact of meriolins, a new class of cyclin-dependent kinase inhibitors, on malignant glioma proliferation and neo-angiogenesis"

    Article Title: Impact of meriolins, a new class of cyclin-dependent kinase inhibitors, on malignant glioma proliferation and neo-angiogenesis

    Journal: Neuro-Oncology

    doi: 10.1093/neuonc/nou102

    Inhibitory role of meriolin 15 on U87 and human GBM sc1 xenograft development in nude mice. (A and B) U87 cells (3 × 10 6 cells, 100 µL PBS) were implanted subcutaneously into the right flanks of nude mice. (A) When tumors reached a volume of ∼100–150 mm 3 , mice were treated i.p. every 2 days with meriolin 15 (2 mg/kg/2d, n = 4) or (5 mg/kg/2d, n = 5) or every day (5 mg/kg/d, n = 4) or with vehicle (DMSO in NaCl 0.9%, n = 4) during 22 days. Statistical significance was determined by the Freidman test followed by the Dunn' multiple comparison test. * P
    Figure Legend Snippet: Inhibitory role of meriolin 15 on U87 and human GBM sc1 xenograft development in nude mice. (A and B) U87 cells (3 × 10 6 cells, 100 µL PBS) were implanted subcutaneously into the right flanks of nude mice. (A) When tumors reached a volume of ∼100–150 mm 3 , mice were treated i.p. every 2 days with meriolin 15 (2 mg/kg/2d, n = 4) or (5 mg/kg/2d, n = 5) or every day (5 mg/kg/d, n = 4) or with vehicle (DMSO in NaCl 0.9%, n = 4) during 22 days. Statistical significance was determined by the Freidman test followed by the Dunn' multiple comparison test. * P

    Techniques Used: Mouse Assay

    33) Product Images from "Biochemical phenotype of a common disease-causing mutation and a possible therapeutic approach for the phosphomannomutase 2-associated disorder of glycosylation"

    Article Title: Biochemical phenotype of a common disease-causing mutation and a possible therapeutic approach for the phosphomannomutase 2-associated disorder of glycosylation

    Journal: Molecular Genetics & Genomic Medicine

    doi: 10.1002/mgg3.3

    Ligand binding can affect the thermal stability of PMM2. Heat-induced melting profile of wild-type PMM2 (A and C) and F119L-PMM2 (B and D) were recorded by thermal shift assay and by circular dichroism. For thermal shift assay, the proteins (0.2 mg/mL) were equilibrated in buffer (HEPES 20 mmol/L, NaCl 150 mmol/L, pH 7.5) containing Sypro Orange2.5X and the appropriate ligands: MgCl 2 5 mmol/L, EDTA 5 mmol/L, Glc-6-P 0.5 mmol/L + MgCl 2 5 mmol/L, Glc-6-P 0.5 mmol/L + MgCl 2 5 mmol/L + vanadate 0.5 mmol/L, Glu-1,6-P 0.5 mmol/L + MgCl 2 5 mmol/L. The samples were distributed in 96-well PCR plates, the plates were sealed, and heated from 25 to 80° at 0.5°C/min. The experiment was run on an iCycler iQ Real Time PCR Detection System. An excitation wavelength of 490 nm and an emission wavelength of 575 nm were used to collect the data. When the melting profile was obtained by circular dichroism the proteins (0.2 mg/mL) were equilibrated in the same buffer in the presence of MgCl 2 1 mmol/L or EDTA 5 mmol/L. The signal at 222 nm was recorded while temperature was increased at 0.5°C/min from 20°C. The raw data were corrected by taking into account the slopes of the pre- and post-transition baselines, then they were normalized.
    Figure Legend Snippet: Ligand binding can affect the thermal stability of PMM2. Heat-induced melting profile of wild-type PMM2 (A and C) and F119L-PMM2 (B and D) were recorded by thermal shift assay and by circular dichroism. For thermal shift assay, the proteins (0.2 mg/mL) were equilibrated in buffer (HEPES 20 mmol/L, NaCl 150 mmol/L, pH 7.5) containing Sypro Orange2.5X and the appropriate ligands: MgCl 2 5 mmol/L, EDTA 5 mmol/L, Glc-6-P 0.5 mmol/L + MgCl 2 5 mmol/L, Glc-6-P 0.5 mmol/L + MgCl 2 5 mmol/L + vanadate 0.5 mmol/L, Glu-1,6-P 0.5 mmol/L + MgCl 2 5 mmol/L. The samples were distributed in 96-well PCR plates, the plates were sealed, and heated from 25 to 80° at 0.5°C/min. The experiment was run on an iCycler iQ Real Time PCR Detection System. An excitation wavelength of 490 nm and an emission wavelength of 575 nm were used to collect the data. When the melting profile was obtained by circular dichroism the proteins (0.2 mg/mL) were equilibrated in the same buffer in the presence of MgCl 2 1 mmol/L or EDTA 5 mmol/L. The signal at 222 nm was recorded while temperature was increased at 0.5°C/min from 20°C. The raw data were corrected by taking into account the slopes of the pre- and post-transition baselines, then they were normalized.

    Techniques Used: Ligand Binding Assay, Thermal Shift Assay, Gas Chromatography, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

    Specific activity of PMM2 depends on glucose-1,6-bisphosphate concentration. The assay was performed at 32°C in a reaction mixture containing HEPES 20 mmol/L, pH 7.5, MgCl 2 5 mmol/L, NaCl 150 mmol/L, NADP+ 0.25 mmol/L, Glu-1-P (0.04 or 0.60 mmol/L), and yeast glucose 6-phosphate dehydrogenase 10 μg/mL, while Glc-1,6-P 2 was changed in the range 0–80 μmol/L. Enzymes concentrations were 107 nmol/L for wt-PMM2 and 73 nmol/L for F119L-PMM2. The hyperbolic dependence of velocity on the activator concentration was fitted using Michaelis and Menten equation to evaluate EC50.
    Figure Legend Snippet: Specific activity of PMM2 depends on glucose-1,6-bisphosphate concentration. The assay was performed at 32°C in a reaction mixture containing HEPES 20 mmol/L, pH 7.5, MgCl 2 5 mmol/L, NaCl 150 mmol/L, NADP+ 0.25 mmol/L, Glu-1-P (0.04 or 0.60 mmol/L), and yeast glucose 6-phosphate dehydrogenase 10 μg/mL, while Glc-1,6-P 2 was changed in the range 0–80 μmol/L. Enzymes concentrations were 107 nmol/L for wt-PMM2 and 73 nmol/L for F119L-PMM2. The hyperbolic dependence of velocity on the activator concentration was fitted using Michaelis and Menten equation to evaluate EC50.

    Techniques Used: Activity Assay, Concentration Assay, Gas Chromatography

    Long-term stability of F119L-PMM2. F119L-PMM2 (0.027 mmol/L of monomer equivalents) was equilibrated in HEPES 50 mmol/L pH 7.1 containing NaCl 150 mmol/L. Aliquots containing 1.6 μg of protein were taken at known incubation time and diluted immediately to assay the residual activity with Glc-1-P under standard conditions. (A) Results obtained at 37°C in the presence of EDTA 0.1 mmol/L or MgCl 2 5 mmol/L. (B) Results obtained at 44°C in the presence of MgCl 2 5 mmol/L, MgCl 2 5 mmol/L plus Glc-1-P 0.5 mmol/L, MgCl 2 5 mmol/L plus Glc-1-P 0.5 mmol/L and vanadate 0.5 mmol/L or MgCl 2 5 mmol/L plus Glu-1,6-P 2 0.5 mmol/L
    Figure Legend Snippet: Long-term stability of F119L-PMM2. F119L-PMM2 (0.027 mmol/L of monomer equivalents) was equilibrated in HEPES 50 mmol/L pH 7.1 containing NaCl 150 mmol/L. Aliquots containing 1.6 μg of protein were taken at known incubation time and diluted immediately to assay the residual activity with Glc-1-P under standard conditions. (A) Results obtained at 37°C in the presence of EDTA 0.1 mmol/L or MgCl 2 5 mmol/L. (B) Results obtained at 44°C in the presence of MgCl 2 5 mmol/L, MgCl 2 5 mmol/L plus Glc-1-P 0.5 mmol/L, MgCl 2 5 mmol/L plus Glc-1-P 0.5 mmol/L and vanadate 0.5 mmol/L or MgCl 2 5 mmol/L plus Glu-1,6-P 2 0.5 mmol/L

    Techniques Used: Incubation, Activity Assay, Gas Chromatography

    Ligand binding can increase the resistance to proteases of PMM2. Purified wild-type PMM2 and F119L-PMM2 (A) were incubated (0.5 mg/mL) with thermolysin in HEPES 20 mmol/L, NaCl 150 mmol/L, MgCl 2 0.1 mmol/L, pH 7.5 at the indicated protease substrate ratio (w/w) for 1 or 2 h at 37°C before they were analyzed (5 μg of each sample) by SDS-PAGE. Purified F119L-PMM2 (B) was incubated (0.2 mg/mL) with thermolysin in HEPES 20 mmol/L, NaCl 150 mmol/L, MgCl 2 0.1 mmol/L, pH 7.5 at the indicated protease substrate ratio (w/w), in the presence of no ligands, Glc-1,6-P 2 0.5 mmol/L or Glu-6-P 0.5 mmol/L plus vanadate 0.5 mmol/L, for 2 h at 37°C before they were analyzed (2 μg of each sample) by SDS-PAGE. The protein bands were visualized by Coomassie blue staining and the intensity of the bands quantified. The not-digested protein was quantified and expressed as percentage of the starting material (no protease panel A; time 0 panel B).
    Figure Legend Snippet: Ligand binding can increase the resistance to proteases of PMM2. Purified wild-type PMM2 and F119L-PMM2 (A) were incubated (0.5 mg/mL) with thermolysin in HEPES 20 mmol/L, NaCl 150 mmol/L, MgCl 2 0.1 mmol/L, pH 7.5 at the indicated protease substrate ratio (w/w) for 1 or 2 h at 37°C before they were analyzed (5 μg of each sample) by SDS-PAGE. Purified F119L-PMM2 (B) was incubated (0.2 mg/mL) with thermolysin in HEPES 20 mmol/L, NaCl 150 mmol/L, MgCl 2 0.1 mmol/L, pH 7.5 at the indicated protease substrate ratio (w/w), in the presence of no ligands, Glc-1,6-P 2 0.5 mmol/L or Glu-6-P 0.5 mmol/L plus vanadate 0.5 mmol/L, for 2 h at 37°C before they were analyzed (2 μg of each sample) by SDS-PAGE. The protein bands were visualized by Coomassie blue staining and the intensity of the bands quantified. The not-digested protein was quantified and expressed as percentage of the starting material (no protease panel A; time 0 panel B).

    Techniques Used: Ligand Binding Assay, Purification, Incubation, SDS Page, Gas Chromatography, Staining

    Ligand binding affects the quaternary structure of PMM2. Wild-type PMM2 (0.010 mg) and F119L-PMM2 (0.0065 mg) were subjected to size exclusion chromatography on BioSep-SEC-S3000 column equilibrated in HEPES 20 mmol/L pH 7.5, NaCl 150 mmol/L, MgCl 2 5 mmol/L (long dashed line for the wild-type PMM2 or short dashed line for F119L-PMM2) or in the same buffer containing Glc-6-P 0.5 mmol/L and vanadate 0.1 mmol/L (continuous line for wild-type PMM2 or dotted line for F119L-PMM2). The chromatography was run at room temperature at 0.5 mL/min.
    Figure Legend Snippet: Ligand binding affects the quaternary structure of PMM2. Wild-type PMM2 (0.010 mg) and F119L-PMM2 (0.0065 mg) were subjected to size exclusion chromatography on BioSep-SEC-S3000 column equilibrated in HEPES 20 mmol/L pH 7.5, NaCl 150 mmol/L, MgCl 2 5 mmol/L (long dashed line for the wild-type PMM2 or short dashed line for F119L-PMM2) or in the same buffer containing Glc-6-P 0.5 mmol/L and vanadate 0.1 mmol/L (continuous line for wild-type PMM2 or dotted line for F119L-PMM2). The chromatography was run at room temperature at 0.5 mL/min.

    Techniques Used: Ligand Binding Assay, Size-exclusion Chromatography, Gas Chromatography, Chromatography

    Specific activity of F119L-PMM2 depends on enzyme concentration. The assay was performed at 32°C in a reaction mixture containing HEPES 20 mmol/L, pH 7.5, MgCl 2 5 mmol/L, NaCl 150 mmol/L, NADP+ 0.25 mmol/L, Glc-1,6-P 2 0.030 mmol/L, and yeast glucose 6-phosphate dehydrogenase 10 μg/mL. The reaction mixture also contained BSA at 0.5 mg/mL. Three sets of experiments were carried out in the presence of 0.04, 0.16, or 0.6 mmol/L Glc-1-P and the F119L-PMM2 concentration changed in the range 10–240 nmol/L (monomer equivalents).
    Figure Legend Snippet: Specific activity of F119L-PMM2 depends on enzyme concentration. The assay was performed at 32°C in a reaction mixture containing HEPES 20 mmol/L, pH 7.5, MgCl 2 5 mmol/L, NaCl 150 mmol/L, NADP+ 0.25 mmol/L, Glc-1,6-P 2 0.030 mmol/L, and yeast glucose 6-phosphate dehydrogenase 10 μg/mL. The reaction mixture also contained BSA at 0.5 mg/mL. Three sets of experiments were carried out in the presence of 0.04, 0.16, or 0.6 mmol/L Glc-1-P and the F119L-PMM2 concentration changed in the range 10–240 nmol/L (monomer equivalents).

    Techniques Used: Activity Assay, Concentration Assay, Gas Chromatography

    Specific activity of wt-PMM2 changes as a function of protein concentration. The assay was performed at 32°C in a reaction mixture containing HEPES 20 mmol/L, pH 7.5, MgCl 2 5 mmol/L, NaCl 150 mmol/L, NADP+ 0.25 mmol/L, Glc-1,6-P 2 0.030 mmol/L and yeast glucose 6-phosphate dehydrogenase 0.010 mg/mL. The reaction mixture also contained Glc-1-P 0.020 mmol/L and BSA 0.5 mg/mL. The wt-PMM2 concentration changed in the range 2–110 nmol/L (monomer equivalents).
    Figure Legend Snippet: Specific activity of wt-PMM2 changes as a function of protein concentration. The assay was performed at 32°C in a reaction mixture containing HEPES 20 mmol/L, pH 7.5, MgCl 2 5 mmol/L, NaCl 150 mmol/L, NADP+ 0.25 mmol/L, Glc-1,6-P 2 0.030 mmol/L and yeast glucose 6-phosphate dehydrogenase 0.010 mg/mL. The reaction mixture also contained Glc-1-P 0.020 mmol/L and BSA 0.5 mg/mL. The wt-PMM2 concentration changed in the range 2–110 nmol/L (monomer equivalents).

    Techniques Used: Activity Assay, Protein Concentration, Gas Chromatography, Concentration Assay

    34) Product Images from "D’ domain region Arg782-Cys799 of von Willebrand factor contributes to factor VIII binding"

    Article Title: D’ domain region Arg782-Cys799 of von Willebrand factor contributes to factor VIII binding

    Journal: Haematologica

    doi: 10.3324/haematol.2019.221994

    The FVIII binding efficiency of D’-D3 Leu786Ala. (A) Part of the crystal structure of D’-D3 (PDB entry: 6n29)30 with a zoom-in of the helical region comprising the residues 786-Leu-Glu-Cys-789. (B) Multiple concentrations of D’-D3 Leu786Ala were passed over coagulation factor VIII (FVIII) that was immobilized via antibody EL14 to the surface of a CM5 sensor chip. The binding response is indicated as response units (RU) and was assessed in 20 mM HEPES (pH 7.4), 150 mM NaCl, 5 mM CaCl 2 , 0.05% (v/v) Tween 20 at a flow rate of 30 μL/min at 25°C. (C) FVIII was pre-incubated with increasing concentrations of D’-D3 and D’-D3 Leu786Ala in a buffer comprising 50 mM Tris (pH 7.4), 150 mM NaCl, 5mM CaCl 2 , 2% human serum albumin and 0.1% Tween 20 at 37°C. The protein mixtures were next incubated with immobilized von Willebrand factor (VWF) in the same buffer. Residual FVIII binding to immobilized VWF was assessed employing HRP-conjugated CAg12 antibody as described in the methods. Data represents mean ± standard deviation (SD) of three independent experiments.
    Figure Legend Snippet: The FVIII binding efficiency of D’-D3 Leu786Ala. (A) Part of the crystal structure of D’-D3 (PDB entry: 6n29)30 with a zoom-in of the helical region comprising the residues 786-Leu-Glu-Cys-789. (B) Multiple concentrations of D’-D3 Leu786Ala were passed over coagulation factor VIII (FVIII) that was immobilized via antibody EL14 to the surface of a CM5 sensor chip. The binding response is indicated as response units (RU) and was assessed in 20 mM HEPES (pH 7.4), 150 mM NaCl, 5 mM CaCl 2 , 0.05% (v/v) Tween 20 at a flow rate of 30 μL/min at 25°C. (C) FVIII was pre-incubated with increasing concentrations of D’-D3 and D’-D3 Leu786Ala in a buffer comprising 50 mM Tris (pH 7.4), 150 mM NaCl, 5mM CaCl 2 , 2% human serum albumin and 0.1% Tween 20 at 37°C. The protein mixtures were next incubated with immobilized von Willebrand factor (VWF) in the same buffer. Residual FVIII binding to immobilized VWF was assessed employing HRP-conjugated CAg12 antibody as described in the methods. Data represents mean ± standard deviation (SD) of three independent experiments.

    Techniques Used: Binding Assay, Coagulation, Chromatin Immunoprecipitation, Incubation, Standard Deviation

    D’-D3 variants in competition with immobilized von Willebrand factor for binding FVIII. Coagulation factor VIII (FVIII) was incubated with increasing concentrations of the indicated D’-D3 variants in a buffer comprising 50 mM Tris (pH 7.4), 150 mM NaCl, 5mM CaCl 2 , 2% human serum albumin and 0.1% Tween 20 at 37°C. The protein mixtures were next incubated with immobilized von Willebrand factor (VWF) in the same buffer. Residual FVIII binding to immobilized VWF was assessed employing HRP-conjugated CAg12 antibody as described in the methods. Data represents mean ± standard deviation (SD) of three independent experiments.
    Figure Legend Snippet: D’-D3 variants in competition with immobilized von Willebrand factor for binding FVIII. Coagulation factor VIII (FVIII) was incubated with increasing concentrations of the indicated D’-D3 variants in a buffer comprising 50 mM Tris (pH 7.4), 150 mM NaCl, 5mM CaCl 2 , 2% human serum albumin and 0.1% Tween 20 at 37°C. The protein mixtures were next incubated with immobilized von Willebrand factor (VWF) in the same buffer. Residual FVIII binding to immobilized VWF was assessed employing HRP-conjugated CAg12 antibody as described in the methods. Data represents mean ± standard deviation (SD) of three independent experiments.

    Techniques Used: Binding Assay, Coagulation, Incubation, Standard Deviation

    35) Product Images from "CRISPR-Associated Primase-Polymerases are implicated in prokaryotic CRISPR-Cas adaptation"

    Article Title: CRISPR-Associated Primase-Polymerases are implicated in prokaryotic CRISPR-Cas adaptation

    Journal: Nature Communications

    doi: 10.1038/s41467-021-23535-9

    MpCas1 site-specific integration and disintegration activities. a Schematic representation of the MpCRISPR array, before and after prespacer integration, used in the Cas1-integration assay (panel b ). b 26 nM CRISPR array (PCR-synthetised) was incubated with wild-type Cas1 (WT) in presence of Cas2, increasing concentration of IHF and 200 nM prespacer (Cy5-labelled) in integration buffer (10 mM Bis-Tris Propane; pH 7, 10 mM MgCl 2 , 100 mM NaCl, 0.5 mM TCEP and 0.1 mg/ml BSA) for 90 min at 50 °C. After Proteinase K digestion, the products were resolved on denaturing urea-PAGE. Green signal – Atto550 (CRISPR array), Red signal – Cy5 (prespacer) Red dot – prespacer, Green dot – CRISPR array without any integration, Red and green arrows– products after prespacer integration. c Schematic representation of branched substrates used in Cas1 transesterification assay (panel d). RF – replication fork. d MpCas1 prefers transesterification of the 5′-flap over RF and other tested branched structures. Increasing concentration of Cas1 was incubated with 100 nM branched substrates in buffer containing 10 mM Bis-Tris Propane; pH 7, 10 mM MgCl 2 , 10 mM NaCl and 0.3 mg/ml BSA for 30 min at 50 °C. After Proteinase K digestion the products were resolved by denaturing urea-PAGE. Green signal – Atto550; Red signal – Cy5; Blue signal – FAM, nts – nucleotide length of DNA markers. Results shown are representative of three independent repeats (7b, d).
    Figure Legend Snippet: MpCas1 site-specific integration and disintegration activities. a Schematic representation of the MpCRISPR array, before and after prespacer integration, used in the Cas1-integration assay (panel b ). b 26 nM CRISPR array (PCR-synthetised) was incubated with wild-type Cas1 (WT) in presence of Cas2, increasing concentration of IHF and 200 nM prespacer (Cy5-labelled) in integration buffer (10 mM Bis-Tris Propane; pH 7, 10 mM MgCl 2 , 100 mM NaCl, 0.5 mM TCEP and 0.1 mg/ml BSA) for 90 min at 50 °C. After Proteinase K digestion, the products were resolved on denaturing urea-PAGE. Green signal – Atto550 (CRISPR array), Red signal – Cy5 (prespacer) Red dot – prespacer, Green dot – CRISPR array without any integration, Red and green arrows– products after prespacer integration. c Schematic representation of branched substrates used in Cas1 transesterification assay (panel d). RF – replication fork. d MpCas1 prefers transesterification of the 5′-flap over RF and other tested branched structures. Increasing concentration of Cas1 was incubated with 100 nM branched substrates in buffer containing 10 mM Bis-Tris Propane; pH 7, 10 mM MgCl 2 , 10 mM NaCl and 0.3 mg/ml BSA for 30 min at 50 °C. After Proteinase K digestion the products were resolved by denaturing urea-PAGE. Green signal – Atto550; Red signal – Cy5; Blue signal – FAM, nts – nucleotide length of DNA markers. Results shown are representative of three independent repeats (7b, d).

    Techniques Used: CRISPR, Polymerase Chain Reaction, Incubation, Concentration Assay, Immunohistofluorescence, Polyacrylamide Gel Electrophoresis

    36) Product Images from "Comparison of Uncleaved and Mature Human Immunodeficiency Virus Membrane Envelope Glycoprotein Trimers"

    Article Title: Comparison of Uncleaved and Mature Human Immunodeficiency Virus Membrane Envelope Glycoprotein Trimers

    Journal: Journal of Virology

    doi: 10.1128/JVI.00277-18

    Preparation of cleaved and uncleaved HIV-1 AD8 membrane Envs. (A) HOS cells expressing the HIV-1 AD8 ) (middle and bottom). (B) Purified membranes from HOS cells expressing the HIV-1 AD8 Env(+)Δ712 and Env(−)Δ712 glycoproteins were solubilized and analyzed by Western blotting with serum from an HIV-1-infected individual. Note the efficiency with which the HIV-1 AD8 Env(+)Δ712 glycoprotein is cleaved. (C) Glutaraldehyde-cross-linked HIV-1 AD8 Env(+)Δ712 GA and Env(−)Δ712 GA glycoproteins were purified and analyzed by SDS-PAGE and silver staining. (D) Glutaraldehyde-cross-linked HIV-1 AD8 Env(+)Δ712 GA and Env(−)Δ712 GA glycoproteins were purified from HOS cell membranes and analyzed by size exclusion chromatography. The HIV-1 AD8 gp120 Env was analyzed in parallel. The Envs were analyzed on a Yarra 4000 column equilibrated with 20 mM Tris-HCl, 150 mM NaCl, and 0.1% Cymal-6. O.D., optical density. (E) Fractions of the glutaraldehyde-cross-linked Env(+)Δ712 GA (top) and Env(−)Δ712 GA (bottom) glycoproteins from the Yarra 4000 size exclusion chromatography column were run on SDS-PAGE and Western blotted with serum from an HIV-1-infected individual. (Middle) The HIV-1 AD8 Env(+)Δ712 glycoprotein was purified from a whole-cell lysate and Western blotted as described above.
    Figure Legend Snippet: Preparation of cleaved and uncleaved HIV-1 AD8 membrane Envs. (A) HOS cells expressing the HIV-1 AD8 ) (middle and bottom). (B) Purified membranes from HOS cells expressing the HIV-1 AD8 Env(+)Δ712 and Env(−)Δ712 glycoproteins were solubilized and analyzed by Western blotting with serum from an HIV-1-infected individual. Note the efficiency with which the HIV-1 AD8 Env(+)Δ712 glycoprotein is cleaved. (C) Glutaraldehyde-cross-linked HIV-1 AD8 Env(+)Δ712 GA and Env(−)Δ712 GA glycoproteins were purified and analyzed by SDS-PAGE and silver staining. (D) Glutaraldehyde-cross-linked HIV-1 AD8 Env(+)Δ712 GA and Env(−)Δ712 GA glycoproteins were purified from HOS cell membranes and analyzed by size exclusion chromatography. The HIV-1 AD8 gp120 Env was analyzed in parallel. The Envs were analyzed on a Yarra 4000 column equilibrated with 20 mM Tris-HCl, 150 mM NaCl, and 0.1% Cymal-6. O.D., optical density. (E) Fractions of the glutaraldehyde-cross-linked Env(+)Δ712 GA (top) and Env(−)Δ712 GA (bottom) glycoproteins from the Yarra 4000 size exclusion chromatography column were run on SDS-PAGE and Western blotted with serum from an HIV-1-infected individual. (Middle) The HIV-1 AD8 Env(+)Δ712 glycoprotein was purified from a whole-cell lysate and Western blotted as described above.

    Techniques Used: Expressing, Purification, Western Blot, Infection, SDS Page, Silver Staining, Size-exclusion Chromatography

    37) Product Images from "Molecular Mechanisms of Steric Pressure Generation and Membrane Remodeling by Intrinsically Disordered Proteins"

    Article Title: Molecular Mechanisms of Steric Pressure Generation and Membrane Remodeling by Intrinsically Disordered Proteins

    Journal: bioRxiv

    doi: 10.1101/2022.05.10.491320

    The chain length of AP180CTD and PEG does not significantly impact steric pressure generated at the membrane surface at constant coverage. For AP180CTD crowding experiments, the membrane composition was 72 mol% DOPC, 16 mol% Ni 2+ -DGS-NTA, 1 mol% DP-EG10-biotin, 1 mol% PEG 10K-DSPE-NH 2 , and 10 mol% Texas Red-DHPE. Vesicles were extruded to a diameter of 100 nm. For PEG crowding experiments, the membrane composition was 83-85.75 mol% DOPC, 1 mol% DP-EG10-biotin, 1 mol% PEG 10K-DSPE-NH 2 , 2.25-5 mol% PEG 3.4K-DSPE-SH 2 , PEG 5K-DSPE-SH 2 , or PEG 10K-DSPE-SH 2 and 10 mol% Texas Red-DHPE. (A) Schematic of AP180CTD-1/3 crowding the PEG sensor. (B) Donor fluorescence decay curves upon crowding with increasing concentration of AP180CTD-1/3 (uncrowded black line, 500nM red line, 2 μM blue line). Curves are displayed with a moving average over a 10-point interval to better visualize the shift in decay curves. (C) Decrease in the number of polymer segments needed to fit each donor fluorescence decay curve as the concentration of AP180CTD-1/3 increased. The corresponding free energy was calculated for each effective segment decrease. (D) Measured fractional coverage of the membrane surface by AP180CTD-1/3 domains as protein concentration in solution increased. (E) Approximate steric pressure generated by AP180CTD-1/3 compared to AP180CTD-FL as membrane coverage increased with 150 mM NaCl in solution. (F) Approximate steric pressure generated by AP180CTD-1/3 compared to AP180CTD-FL as membrane coverage increased with 1M NaCl in solution. All data points were grouped together and fit (red line) by a power law, y = Ax 9/4 , where A was a free fitting parameter. (G) Calculated free energy of PEG crowding for different sizes of PEG as the number density of PEG chains on the membrane surface increased. (H) Approximate steric pressure associated with crowding different sizes of PEG as the fractional membrane coverage of PEG chains increased. All data points were grouped together and fit (red line) by a power law, y = Ax 9/4 , where A was a free fitting parameter. Error bars in (C-H) were calculated as standard error of the mean.
    Figure Legend Snippet: The chain length of AP180CTD and PEG does not significantly impact steric pressure generated at the membrane surface at constant coverage. For AP180CTD crowding experiments, the membrane composition was 72 mol% DOPC, 16 mol% Ni 2+ -DGS-NTA, 1 mol% DP-EG10-biotin, 1 mol% PEG 10K-DSPE-NH 2 , and 10 mol% Texas Red-DHPE. Vesicles were extruded to a diameter of 100 nm. For PEG crowding experiments, the membrane composition was 83-85.75 mol% DOPC, 1 mol% DP-EG10-biotin, 1 mol% PEG 10K-DSPE-NH 2 , 2.25-5 mol% PEG 3.4K-DSPE-SH 2 , PEG 5K-DSPE-SH 2 , or PEG 10K-DSPE-SH 2 and 10 mol% Texas Red-DHPE. (A) Schematic of AP180CTD-1/3 crowding the PEG sensor. (B) Donor fluorescence decay curves upon crowding with increasing concentration of AP180CTD-1/3 (uncrowded black line, 500nM red line, 2 μM blue line). Curves are displayed with a moving average over a 10-point interval to better visualize the shift in decay curves. (C) Decrease in the number of polymer segments needed to fit each donor fluorescence decay curve as the concentration of AP180CTD-1/3 increased. The corresponding free energy was calculated for each effective segment decrease. (D) Measured fractional coverage of the membrane surface by AP180CTD-1/3 domains as protein concentration in solution increased. (E) Approximate steric pressure generated by AP180CTD-1/3 compared to AP180CTD-FL as membrane coverage increased with 150 mM NaCl in solution. (F) Approximate steric pressure generated by AP180CTD-1/3 compared to AP180CTD-FL as membrane coverage increased with 1M NaCl in solution. All data points were grouped together and fit (red line) by a power law, y = Ax 9/4 , where A was a free fitting parameter. (G) Calculated free energy of PEG crowding for different sizes of PEG as the number density of PEG chains on the membrane surface increased. (H) Approximate steric pressure associated with crowding different sizes of PEG as the fractional membrane coverage of PEG chains increased. All data points were grouped together and fit (red line) by a power law, y = Ax 9/4 , where A was a free fitting parameter. Error bars in (C-H) were calculated as standard error of the mean.

    Techniques Used: Generated, Fluorescence, Concentration Assay, Protein Concentration

    Ionic strength impacts steric pressure generated by AP180CTD-FL. The membrane composition was 72 mol% DOPC, 16 mol% Ni 2+ -DGS-NTA, 1 mol% DP-EG10-biotin, 1 mol% PEG 10K-DSPE-NH 2 , and 10 mol% Texas Red-DHPE. Vesicles were extruded to a diameter of 100 nm. (A) Schematic of the electrostatic interactions between AP180CTD-FL domains and the impact on crowding the PEG sensor. (B) Donor fluorescence decay curves with decreasing NaCl concentration (1 M NaCl black line, 150 mM NaCl red line, and 10 mM NaCl blue line). All curves shown are at a constant protein concentration of 500 nM AP180CTD-FL. Curves are displayed with a moving average over a 10-point interval to better visualize the shift in decay curves. (C) Increase in number of polymer segments needed to fit each donor fluorescence decay curve at a constant protein concentration (500 nM) as the NaCl concentration in solution increased. (D) Measured fractional coverage of the membrane surface by AP180CTD-FL at a constant protein concentration (500 nM) as the NaCl concentration in solution increased. (E) Approximate steric pressure generated by AP180CTD-FL at the membrane surface with increasing NaCl concentration. Error bars in (C) are calculated as standard deviation of the mean. Error bars in (D) are calculated as standard error of the mean.
    Figure Legend Snippet: Ionic strength impacts steric pressure generated by AP180CTD-FL. The membrane composition was 72 mol% DOPC, 16 mol% Ni 2+ -DGS-NTA, 1 mol% DP-EG10-biotin, 1 mol% PEG 10K-DSPE-NH 2 , and 10 mol% Texas Red-DHPE. Vesicles were extruded to a diameter of 100 nm. (A) Schematic of the electrostatic interactions between AP180CTD-FL domains and the impact on crowding the PEG sensor. (B) Donor fluorescence decay curves with decreasing NaCl concentration (1 M NaCl black line, 150 mM NaCl red line, and 10 mM NaCl blue line). All curves shown are at a constant protein concentration of 500 nM AP180CTD-FL. Curves are displayed with a moving average over a 10-point interval to better visualize the shift in decay curves. (C) Increase in number of polymer segments needed to fit each donor fluorescence decay curve at a constant protein concentration (500 nM) as the NaCl concentration in solution increased. (D) Measured fractional coverage of the membrane surface by AP180CTD-FL at a constant protein concentration (500 nM) as the NaCl concentration in solution increased. (E) Approximate steric pressure generated by AP180CTD-FL at the membrane surface with increasing NaCl concentration. Error bars in (C) are calculated as standard deviation of the mean. Error bars in (D) are calculated as standard error of the mean.

    Techniques Used: Generated, Fluorescence, Concentration Assay, Protein Concentration, Standard Deviation

    38) Product Images from "Ligand-Mediated Folding of the OmpA Periplasmic Domain from Acinetobacter baumannii"

    Article Title: Ligand-Mediated Folding of the OmpA Periplasmic Domain from Acinetobacter baumannii

    Journal: Biophysical Journal

    doi: 10.1016/j.bpj.2017.04.015

    ( a ) Shown here is the 1 H- 15 N HSQC spectrum of 0.6 mM apo-AbOmpA-PD in 50 mM Bis-Tris (pH 6.5), 50 mM NaCl, and 10% (v/v) D 2 O buffer. ( b ) Shown here is the 1 H- 15 N HSQC spectrum of 0.6 mM apo-AbOmpA-PD in 8 M urea, 50 mM Bis-Tris (pH 6.5), 50 mM NaCl, and 10% (v/v) D 2 O buffer. ( c ) Shown here is the 1 H- 15 N HSQC spectrum of 0.5 mM holo-AbOmpA-PD in 1 mM glycine, 50 mM Bis-Tris (pH 6.5), 50 mM NaCl, and 10% (v/v) D 2 O buffer. All three spectra were acquired at 298 K.
    Figure Legend Snippet: ( a ) Shown here is the 1 H- 15 N HSQC spectrum of 0.6 mM apo-AbOmpA-PD in 50 mM Bis-Tris (pH 6.5), 50 mM NaCl, and 10% (v/v) D 2 O buffer. ( b ) Shown here is the 1 H- 15 N HSQC spectrum of 0.6 mM apo-AbOmpA-PD in 8 M urea, 50 mM Bis-Tris (pH 6.5), 50 mM NaCl, and 10% (v/v) D 2 O buffer. ( c ) Shown here is the 1 H- 15 N HSQC spectrum of 0.5 mM holo-AbOmpA-PD in 1 mM glycine, 50 mM Bis-Tris (pH 6.5), 50 mM NaCl, and 10% (v/v) D 2 O buffer. All three spectra were acquired at 298 K.

    Techniques Used:

    39) Product Images from "The role of the N-terminus in determining metal specific responses in the E. coli Ni- and Co-responsive metalloregulator, RcnR"

    Article Title: The role of the N-terminus in determining metal specific responses in the E. coli Ni- and Co-responsive metalloregulator, RcnR

    Journal: Journal of the American Chemical Society

    doi: 10.1021/ja300834b

    Metal complexes of wild-type RcnR in buffer with 20 mM Hepes, 300 mM NaCl, and 10 % glycerol at pH 7.0. Left: Fourier-filtered XAS data (colored lines) and best fits (black lines) from . Right: Unfiltered k 3 -weighted EXAFS spectra and fits.
    Figure Legend Snippet: Metal complexes of wild-type RcnR in buffer with 20 mM Hepes, 300 mM NaCl, and 10 % glycerol at pH 7.0. Left: Fourier-filtered XAS data (colored lines) and best fits (black lines) from . Right: Unfiltered k 3 -weighted EXAFS spectra and fits.

    Techniques Used:

    40) Product Images from "Non-Covalent Associates of siRNAs and AuNPs Enveloped with Lipid Layer and Doped with Amphiphilic Peptide for Efficient siRNA Delivery"

    Article Title: Non-Covalent Associates of siRNAs and AuNPs Enveloped with Lipid Layer and Doped with Amphiphilic Peptide for Efficient siRNA Delivery

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19072096

    Surface density of siRNA in the composition of a core in Samples I–III. Right column shows type of a sample and NaCl concentration (10–40 mM).
    Figure Legend Snippet: Surface density of siRNA in the composition of a core in Samples I–III. Right column shows type of a sample and NaCl concentration (10–40 mM).

    Techniques Used: Concentration Assay

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    Thermo Fisher nacl
    Purification of <t>Cas9</t> recombinant protein. (A) Purification of Cas9 by affinity chromatography using a Nickel-charged HiTrap Chelating HP. Gradient of Imidazole is indicated by the green line. (B) Coomassie-stained gel electrophoresis of peaks 1 and 2 after affinity chromatography (26 μl of each fraction) shows that peak 2 corresponds to Cas9. M: SeeBlue Plus2 Pre-Stained Protein Standard (Life Technologies). (C) Purification of Cas9 by cation-exchange chromatography using a MonoS 5/50 GL column. Gradient of <t>NaCl</t> is indicated by the green line. (D) Coomassie-stained gel electrophoresis of fractions correspondent to different peaks after cation-exchange chromatography (26 μl of each fraction, peaks 1–4: lanes 1–4). All peaks showed a band of the correct Cas9 molecular weight. Only the majoritarian peak (#2) was collected. Lane 5 corresponds to purified Cas9 after dialysis. M: SeeBlue Plus2 Pre-Stained Protein Standard (Life Technologies). (E) Purification of recombinant Cas9 produced in our laboratory showed endonuclease activity comparable to a commercial Cas9 (PNABio). 200 ng of PCR product of target gene was incubated with each individual sgRNA (lanes 1–12; 3.2 μM) in the presence of 3.8 μM Cas9 protein either purchased from PNABio (upper gel) or obtained in our laboratory (lower gel). As negative controls, 200 ng of PCR product alone (lane 13) or in combination with Cas9 protein (lane 14) were included. All samples were run on 2.2% agarose gels and visualized under UV light.
    Nacl, supplied by Thermo Fisher, 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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    Thermo Fisher sodium chloride nacl
    HOS15 Associates with EC Components. (A) BiFC analysis of proteins transiently expressed in N. benthamiana leaves. VN and VC indicate the N and C termini, respectively, of Venus (eYFP). Fluorescence (left panels) was detected by confocal microscopy. Right panels are overlay of fluorescence and differential interference contest images. Bars = 100 μm. (B) HOS15 interacts with ELF3. Wild type (10 d old) and hos15-2 plants were cross-linked in 1% formaldehyde to preserve in vivo interaction after harvesting plant material. Total protein extracts were immunoprecipitated with α-HOS15 antibody and resolved by SDS-PAGE. Immunoblots were probed with α-HOS15, or with α-ELF3 to detect ELF3. *Nonspecific bands. (C) HOS15 forms a complex with EC components. N. benthamiana plants were infiltrated with Agrobacterium harboring 35S:HA-LUX, 35S:FLAG-HOS15, and 35S:MYC-ELF3 for transient expression. The proteins were immunoprecipitated with α-HA antibody and resolved by SDS-PAGE. The immunoblots were probed with α-HA to detect LUX, α-HOS15, or α-MYC to detect ELF3. (D) Gel-filtration analysis of HOS15, LUX, and ELF3. Total proteins extracted from 2-week-old wild type plants were fractionated by size-exclusion chromatography using a Superdex 200 10/300GL column equilibrated with elution buffer [50 mM <t>Tris-Cl</t> (pH 7.5), 100 mM <t>NaCl,</t> 0.02% sodium azide]. The samples were separated by SDS-PAGE and subjected to immunoblotting analysis using α-HOS15, α-LUX, or α-ELF3. Aliquots of total protein extracts from wild type, input after ammonium sulfate precipitation of wild type total extracts, and total protein extracts individually isolated from elf3-1 , hos15-2 , or pcl1-1 mutants were used as input controls. Molecular mass markers (ferritin, 440 kD; β-amylase, 200 kD; alcohol dehydrogenase, 150 kD; BSA, 67 kD) were independently eluted using the same equilibrated column. Total, total protein extract; Input, input after ammonium sulfate precipitation.
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    Purification of Cas9 recombinant protein. (A) Purification of Cas9 by affinity chromatography using a Nickel-charged HiTrap Chelating HP. Gradient of Imidazole is indicated by the green line. (B) Coomassie-stained gel electrophoresis of peaks 1 and 2 after affinity chromatography (26 μl of each fraction) shows that peak 2 corresponds to Cas9. M: SeeBlue Plus2 Pre-Stained Protein Standard (Life Technologies). (C) Purification of Cas9 by cation-exchange chromatography using a MonoS 5/50 GL column. Gradient of NaCl is indicated by the green line. (D) Coomassie-stained gel electrophoresis of fractions correspondent to different peaks after cation-exchange chromatography (26 μl of each fraction, peaks 1–4: lanes 1–4). All peaks showed a band of the correct Cas9 molecular weight. Only the majoritarian peak (#2) was collected. Lane 5 corresponds to purified Cas9 after dialysis. M: SeeBlue Plus2 Pre-Stained Protein Standard (Life Technologies). (E) Purification of recombinant Cas9 produced in our laboratory showed endonuclease activity comparable to a commercial Cas9 (PNABio). 200 ng of PCR product of target gene was incubated with each individual sgRNA (lanes 1–12; 3.2 μM) in the presence of 3.8 μM Cas9 protein either purchased from PNABio (upper gel) or obtained in our laboratory (lower gel). As negative controls, 200 ng of PCR product alone (lane 13) or in combination with Cas9 protein (lane 14) were included. All samples were run on 2.2% agarose gels and visualized under UV light.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Optimization of sand fly embryo microinjection for gene editing by CRISPR/Cas9

    doi: 10.1371/journal.pntd.0006769

    Figure Lengend Snippet: Purification of Cas9 recombinant protein. (A) Purification of Cas9 by affinity chromatography using a Nickel-charged HiTrap Chelating HP. Gradient of Imidazole is indicated by the green line. (B) Coomassie-stained gel electrophoresis of peaks 1 and 2 after affinity chromatography (26 μl of each fraction) shows that peak 2 corresponds to Cas9. M: SeeBlue Plus2 Pre-Stained Protein Standard (Life Technologies). (C) Purification of Cas9 by cation-exchange chromatography using a MonoS 5/50 GL column. Gradient of NaCl is indicated by the green line. (D) Coomassie-stained gel electrophoresis of fractions correspondent to different peaks after cation-exchange chromatography (26 μl of each fraction, peaks 1–4: lanes 1–4). All peaks showed a band of the correct Cas9 molecular weight. Only the majoritarian peak (#2) was collected. Lane 5 corresponds to purified Cas9 after dialysis. M: SeeBlue Plus2 Pre-Stained Protein Standard (Life Technologies). (E) Purification of recombinant Cas9 produced in our laboratory showed endonuclease activity comparable to a commercial Cas9 (PNABio). 200 ng of PCR product of target gene was incubated with each individual sgRNA (lanes 1–12; 3.2 μM) in the presence of 3.8 μM Cas9 protein either purchased from PNABio (upper gel) or obtained in our laboratory (lower gel). As negative controls, 200 ng of PCR product alone (lane 13) or in combination with Cas9 protein (lane 14) were included. All samples were run on 2.2% agarose gels and visualized under UV light.

    Article Snippet: Fractions that contain the recombinant Cas9 were combined and NaCl was removed by dialysis with 25 mM 2-(N-Morpholino) ethanesulfonic acid (MES), pH 6.0 at 4 °C overnight using dialysis cassettes (Thermo Scientific, MWCO 10 kDa).

    Techniques: Purification, Recombinant, Affinity Chromatography, Staining, Nucleic Acid Electrophoresis, Chromatography, Molecular Weight, Produced, Activity Assay, Polymerase Chain Reaction, Incubation

    NMN improved mitochondrial function and cell viability in vitro . ( A ) Evaluation of ultrastructural damage in mitochondria from A10 cells with Ndufc2 knockdown; representative micrographs of mitochondria (left) and graphical representation of the ultrastructural damage in either untreated or treated A10 cells (n = 3). Legend: Nu, nucleus; NM, nuclear membrane, PM, plasma membrane; rER, rough endoplasmic reticulum; Gx, grade of mitochondrial (Mt) damage; IMM, inner mitochondrial membrane; OMM, outer mitochondrial membrane. (B) Fluorescence microscope analysis of mitochondrial membrane potential (ΔΨm) levels through JC1 dye (n = 3); representative images (left) and corresponding quantification (right) are shown. (C) FACS analysis in A10 cells with Ndufc2 knockdown treated with NaCl without or with NMN (n = 3); CTR indicates non-silenced and untreated cells. Results are presented as mean values ± SEM; *p

    Journal: Autophagy

    Article Title: Pharmacological restoration of autophagy reduces hypertension-related stroke occurrence

    doi: 10.1080/15548627.2019.1687215

    Figure Lengend Snippet: NMN improved mitochondrial function and cell viability in vitro . ( A ) Evaluation of ultrastructural damage in mitochondria from A10 cells with Ndufc2 knockdown; representative micrographs of mitochondria (left) and graphical representation of the ultrastructural damage in either untreated or treated A10 cells (n = 3). Legend: Nu, nucleus; NM, nuclear membrane, PM, plasma membrane; rER, rough endoplasmic reticulum; Gx, grade of mitochondrial (Mt) damage; IMM, inner mitochondrial membrane; OMM, outer mitochondrial membrane. (B) Fluorescence microscope analysis of mitochondrial membrane potential (ΔΨm) levels through JC1 dye (n = 3); representative images (left) and corresponding quantification (right) are shown. (C) FACS analysis in A10 cells with Ndufc2 knockdown treated with NaCl without or with NMN (n = 3); CTR indicates non-silenced and untreated cells. Results are presented as mean values ± SEM; *p

    Article Snippet: Mitophagy in vitro Mitophagy in vitro was assessed in siRNA A10 exposed to NaCl by means of the mitochondrial probe MitoTracker green (Invitrogen, M7514).

    Techniques: In Vitro, Fluorescence, Microscopy, FACS

    CD spectra of the artificial WSTF PHD_EL5 RING finger and its five mutants. Spectra of 25 μM samples were collected in 20 mM Tris-HCl (pH 6.9), 50 mM NaCl, 1 mM dithiothreitol, and 50 μM ZnCl 2 at room temperature. (1) K4R, (2) K8R, (3) K9R, (4) K14R, and (5) K23R are denoted by solid lines, and the dotted line displays the wild-type.

    Journal: Scientific Reports

    Article Title: Structural model of ubiquitin transfer onto an artificial RING finger as an E3 ligase

    doi: 10.1038/srep06574

    Figure Lengend Snippet: CD spectra of the artificial WSTF PHD_EL5 RING finger and its five mutants. Spectra of 25 μM samples were collected in 20 mM Tris-HCl (pH 6.9), 50 mM NaCl, 1 mM dithiothreitol, and 50 μM ZnCl 2 at room temperature. (1) K4R, (2) K8R, (3) K9R, (4) K14R, and (5) K23R are denoted by solid lines, and the dotted line displays the wild-type.

    Article Snippet: The peptides dissolved in 1 ml of 8 M guanidine-HCl were dialyzed against degassed Solution A (20 mM Tris-HCl (pH 6.9), 50 mM NaCl, 1 mM dithiothreitol, 50 μM ZnCl2 ) overnight at 4°C using a Slide-A-Lyzer dialysis cassette (Thermo scientific, Rockford, IL, USA) as described previously .

    Techniques:

    HOS15 Associates with EC Components. (A) BiFC analysis of proteins transiently expressed in N. benthamiana leaves. VN and VC indicate the N and C termini, respectively, of Venus (eYFP). Fluorescence (left panels) was detected by confocal microscopy. Right panels are overlay of fluorescence and differential interference contest images. Bars = 100 μm. (B) HOS15 interacts with ELF3. Wild type (10 d old) and hos15-2 plants were cross-linked in 1% formaldehyde to preserve in vivo interaction after harvesting plant material. Total protein extracts were immunoprecipitated with α-HOS15 antibody and resolved by SDS-PAGE. Immunoblots were probed with α-HOS15, or with α-ELF3 to detect ELF3. *Nonspecific bands. (C) HOS15 forms a complex with EC components. N. benthamiana plants were infiltrated with Agrobacterium harboring 35S:HA-LUX, 35S:FLAG-HOS15, and 35S:MYC-ELF3 for transient expression. The proteins were immunoprecipitated with α-HA antibody and resolved by SDS-PAGE. The immunoblots were probed with α-HA to detect LUX, α-HOS15, or α-MYC to detect ELF3. (D) Gel-filtration analysis of HOS15, LUX, and ELF3. Total proteins extracted from 2-week-old wild type plants were fractionated by size-exclusion chromatography using a Superdex 200 10/300GL column equilibrated with elution buffer [50 mM Tris-Cl (pH 7.5), 100 mM NaCl, 0.02% sodium azide]. The samples were separated by SDS-PAGE and subjected to immunoblotting analysis using α-HOS15, α-LUX, or α-ELF3. Aliquots of total protein extracts from wild type, input after ammonium sulfate precipitation of wild type total extracts, and total protein extracts individually isolated from elf3-1 , hos15-2 , or pcl1-1 mutants were used as input controls. Molecular mass markers (ferritin, 440 kD; β-amylase, 200 kD; alcohol dehydrogenase, 150 kD; BSA, 67 kD) were independently eluted using the same equilibrated column. Total, total protein extract; Input, input after ammonium sulfate precipitation.

    Journal: The Plant Cell

    Article Title: HOS15 Interacts with the Histone Deacetylase HDA9 and the Evening Complex to Epigenetically Regulate the Floral Activator GIGANTEA [OPEN]

    doi: 10.1105/tpc.18.00721

    Figure Lengend Snippet: HOS15 Associates with EC Components. (A) BiFC analysis of proteins transiently expressed in N. benthamiana leaves. VN and VC indicate the N and C termini, respectively, of Venus (eYFP). Fluorescence (left panels) was detected by confocal microscopy. Right panels are overlay of fluorescence and differential interference contest images. Bars = 100 μm. (B) HOS15 interacts with ELF3. Wild type (10 d old) and hos15-2 plants were cross-linked in 1% formaldehyde to preserve in vivo interaction after harvesting plant material. Total protein extracts were immunoprecipitated with α-HOS15 antibody and resolved by SDS-PAGE. Immunoblots were probed with α-HOS15, or with α-ELF3 to detect ELF3. *Nonspecific bands. (C) HOS15 forms a complex with EC components. N. benthamiana plants were infiltrated with Agrobacterium harboring 35S:HA-LUX, 35S:FLAG-HOS15, and 35S:MYC-ELF3 for transient expression. The proteins were immunoprecipitated with α-HA antibody and resolved by SDS-PAGE. The immunoblots were probed with α-HA to detect LUX, α-HOS15, or α-MYC to detect ELF3. (D) Gel-filtration analysis of HOS15, LUX, and ELF3. Total proteins extracted from 2-week-old wild type plants were fractionated by size-exclusion chromatography using a Superdex 200 10/300GL column equilibrated with elution buffer [50 mM Tris-Cl (pH 7.5), 100 mM NaCl, 0.02% sodium azide]. The samples were separated by SDS-PAGE and subjected to immunoblotting analysis using α-HOS15, α-LUX, or α-ELF3. Aliquots of total protein extracts from wild type, input after ammonium sulfate precipitation of wild type total extracts, and total protein extracts individually isolated from elf3-1 , hos15-2 , or pcl1-1 mutants were used as input controls. Molecular mass markers (ferritin, 440 kD; β-amylase, 200 kD; alcohol dehydrogenase, 150 kD; BSA, 67 kD) were independently eluted using the same equilibrated column. Total, total protein extract; Input, input after ammonium sulfate precipitation.

    Article Snippet: Total proteins from N. benthamiana ) or 10-d-old Arabidopsis plants harvested at ZT8 were extracted in extraction buffer [100 mM Tris-HCl (pH 7.5), 150 mM sodium chloride (NaCl), 0.5% Nonidet P-40, 1 mM EDTA, 3 mM DTT, and protease inhibitors] and incubated with α-HA cross-linked to Protein G Agarose (Invitrogen), α-GFP cross-linked to protein A agarose (Invitrogen), or protein A agarose fused to α-HOS15 at 4°C for 2 h. For the Co-IP shown in and , wild type and hos15-2 plants were cross-linked in 1% formaldehyde (10 min) to preserve in vivo interactions after harvesting.

    Techniques: Bimolecular Fluorescence Complementation Assay, Fluorescence, Confocal Microscopy, In Vivo, Immunoprecipitation, SDS Page, Western Blot, Expressing, Filtration, Size-exclusion Chromatography, Isolation