tris hcl  (Millipore)

 
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    Name:
    Trizma hydrochloride
    Description:

    Catalog Number:
    T5941
    Price:
    None
    Applications:
    The pH values of all buffers are temperature and concentration dependent. For Tris buffers, pH increases about 0.03 unit per C decrease in temperature, and decreases 0.03-0.05 unit per ten-fold dilution.For precise applications, use a carefully calibrated pH meter with a glass/calomel combination electrode.
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    Structured Review

    Millipore tris hcl
    Trizma hydrochloride

    https://www.bioz.com/result/tris hcl/product/Millipore
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    tris hcl - by Bioz Stars, 2021-06
    97/100 stars

    Images

    1) Product Images from "Structural and functional insight into the mechanism of an alkaline exonuclease from Laribacter hongkongensis"

    Article Title: Structural and functional insight into the mechanism of an alkaline exonuclease from Laribacter hongkongensis

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr660

    Qualitative analysis of ssDNA and dsDNA hydrolysis activities of LHK-Exo. ( A ) dsDNA exonuclease activities. Agarose gel showing aliquots taken (0–15 min) from an incubation of LHK-Exo (30 µg, 0.41 nmol of trimers) and BamHI-linearized pET28a (1.8 µg, 0.54 pmol) in Tris–HCl (pH 8.0, 50 mM), 50 mM NaCl, 7.5 mM MgCl 2 at 37°C. ( B ) Polarity of dsDNA digestion. A total of 6 µg of LHK-Exo (82 pmol of trimers, lanes 2–5) or λ-exonuclease (74 pmol of trimers, lanes 6–9) protein was incubated with 0.1 µg (0.23 pmol) of a 712-bp linear 5′-phosphorylated dsDNA substrate (‘unmodified’; lanes 2, 3, 6 and 7), or an analogous 5′-phosphorylated linear dsDNA substrate containing three consecutive ‘nuclease-resistant’ phosphorothioate linkages at its 5′-termini (‘PT-modified’; lanes 4, 5, 8, 9). Assays were quenched immediately (0 min) or incubated at 37°C for 20 min, before analysis of digestion products on 1% agarose gels. ( C ) Digestion of 5′-phosphorylated ssDNA. Reaction mixtures (80 µl) containing LHK-Exo (4.5 µg, 61.4 pmol of trimers) and 5′-PO 4 -(dT) 50 (0.4 nmol) in 25 mM Tris–HCl (pH 8.0), 7.5 mM MgCl 2 , 1 mM DTT were incubated at 37°C. Aliquots (20 µl) were withdrawn after 0, 0.5, 5 and 20 min, and immediately quenched. Reaction products were analyzed by denaturing gel electrophoresis. ( D ) Digestion of non-phosphorylated ssDNA. Analogous sets of assays were performed using non-phosphorylated 50-mers of oligothymidine [5′-OH-(dT) 50 ]. Fluorescent gel images were scanned after SYBR Gold staining. A ssDNA ladder [Oligo Length Standards 20/100 Ladder (IDT)] is included in lane 1.
    Figure Legend Snippet: Qualitative analysis of ssDNA and dsDNA hydrolysis activities of LHK-Exo. ( A ) dsDNA exonuclease activities. Agarose gel showing aliquots taken (0–15 min) from an incubation of LHK-Exo (30 µg, 0.41 nmol of trimers) and BamHI-linearized pET28a (1.8 µg, 0.54 pmol) in Tris–HCl (pH 8.0, 50 mM), 50 mM NaCl, 7.5 mM MgCl 2 at 37°C. ( B ) Polarity of dsDNA digestion. A total of 6 µg of LHK-Exo (82 pmol of trimers, lanes 2–5) or λ-exonuclease (74 pmol of trimers, lanes 6–9) protein was incubated with 0.1 µg (0.23 pmol) of a 712-bp linear 5′-phosphorylated dsDNA substrate (‘unmodified’; lanes 2, 3, 6 and 7), or an analogous 5′-phosphorylated linear dsDNA substrate containing three consecutive ‘nuclease-resistant’ phosphorothioate linkages at its 5′-termini (‘PT-modified’; lanes 4, 5, 8, 9). Assays were quenched immediately (0 min) or incubated at 37°C for 20 min, before analysis of digestion products on 1% agarose gels. ( C ) Digestion of 5′-phosphorylated ssDNA. Reaction mixtures (80 µl) containing LHK-Exo (4.5 µg, 61.4 pmol of trimers) and 5′-PO 4 -(dT) 50 (0.4 nmol) in 25 mM Tris–HCl (pH 8.0), 7.5 mM MgCl 2 , 1 mM DTT were incubated at 37°C. Aliquots (20 µl) were withdrawn after 0, 0.5, 5 and 20 min, and immediately quenched. Reaction products were analyzed by denaturing gel electrophoresis. ( D ) Digestion of non-phosphorylated ssDNA. Analogous sets of assays were performed using non-phosphorylated 50-mers of oligothymidine [5′-OH-(dT) 50 ]. Fluorescent gel images were scanned after SYBR Gold staining. A ssDNA ladder [Oligo Length Standards 20/100 Ladder (IDT)] is included in lane 1.

    Techniques Used: Agarose Gel Electrophoresis, Incubation, Modification, Nucleic Acid Electrophoresis, Staining

    Determination of optimum conditions for LHK-Exo dsDNA digestion activities. PicoGreen fluorescence assays were performed to quantify the amounts of a representative 5′-phosphorylatd dsDNA substrate (PstI-linearized pUC18 that were digested by LHK-Exo under various conditions (reported as a percentage of the initial quantities of DNA). ( A and B ) Optimal concentrations of Mg 2+ and Mn 2+ ions. Amounts of PstI-linearized pUC18 (30 ng, 0.018 pmol) digested by LHK-Exo (8 ng, 0.11 pmol of trimers) in Tris–HCl (25 mM, pH 8.0) containing varying concentrations of Mg 2+ ions (1–15 mM, A) or Mn 2+ ions (0.1–1.7 mM, B), respectively, upon incubation at 25°C for 20 min. ( C ) Optimal pH. Amounts of PstI-linearized pUC18 (30 ng, 0.018 pmol) digested by LHK-Exo (8 ng, 0.11 pmol of trimers) in 25 mM Tris–HCl, 7.5 mM MgCl 2 ; adjusted to the appropriate pH value (pH 7.0–9.0); upon incubation at 25°C for 20 min. ( D ) Optimal temperature. Amounts of PstI-linearized pUC18 (30 ng, 0.018 pmol) digested by LHK-Exo (40 ng, 0.55 pmol of trimers) in Tris–HCl (25 mM, pH 8.0), 7.5 mM MgCl 2 , after incubation for 1 min at the indicated temperature (34–54°C). Four to six independent replicates of each experimental condition were performed, and data are reported as the mean ± standard deviation.
    Figure Legend Snippet: Determination of optimum conditions for LHK-Exo dsDNA digestion activities. PicoGreen fluorescence assays were performed to quantify the amounts of a representative 5′-phosphorylatd dsDNA substrate (PstI-linearized pUC18 that were digested by LHK-Exo under various conditions (reported as a percentage of the initial quantities of DNA). ( A and B ) Optimal concentrations of Mg 2+ and Mn 2+ ions. Amounts of PstI-linearized pUC18 (30 ng, 0.018 pmol) digested by LHK-Exo (8 ng, 0.11 pmol of trimers) in Tris–HCl (25 mM, pH 8.0) containing varying concentrations of Mg 2+ ions (1–15 mM, A) or Mn 2+ ions (0.1–1.7 mM, B), respectively, upon incubation at 25°C for 20 min. ( C ) Optimal pH. Amounts of PstI-linearized pUC18 (30 ng, 0.018 pmol) digested by LHK-Exo (8 ng, 0.11 pmol of trimers) in 25 mM Tris–HCl, 7.5 mM MgCl 2 ; adjusted to the appropriate pH value (pH 7.0–9.0); upon incubation at 25°C for 20 min. ( D ) Optimal temperature. Amounts of PstI-linearized pUC18 (30 ng, 0.018 pmol) digested by LHK-Exo (40 ng, 0.55 pmol of trimers) in Tris–HCl (25 mM, pH 8.0), 7.5 mM MgCl 2 , after incubation for 1 min at the indicated temperature (34–54°C). Four to six independent replicates of each experimental condition were performed, and data are reported as the mean ± standard deviation.

    Techniques Used: Fluorescence, Incubation, Standard Deviation

    Processivity of double strand DNA digestion by wild-type LHK-Exo and Arg12Ala mutant. Time course analysis of the digestion of 5′-phosphorylated double strand DNA (5′-PO 4 -dsDNA: EcoRV-linearized pMal-c2) and 5′-dephosphorylated double strand DNA (5′-OH-dsDNA: 5′dephosphorylated EcoRV-linearized pMal-c2) substrates by wild-type LHK-Exo and the Arg12Ala mutant form using a ‘heparin trap’ approach. A total of 6 µg (82 pmol of trimers) of LHK-Exo or Arg12Ala mutant protein was incubated at 25°C with 60 ng (0.015 pmol) of 5′-PO 4 -dsDNA or 5′-OH-dsDNA in Tris–HCl (25 mM, pH 8.0), 1 mM DTT, 7.5 mM MgCl 2 . After 15 s, excess heparin was added to sequester all unbound protein, and to prevent disassociated protein from re-binding. Aliquots were removed at various time points (0–20 min), and dsDNA levels were determined using fluorescent PicoGreen assays, to enable the extent of DNA digestion to be calculated. In one set of assays, heparin was added to LHK-Exo prior to the addition of dsDNA substrate, to confirm the efficacy of the heparin trap method (filled black squares, green line). Graphs show the mean number of nucleotides digested from each terminus (±SD; y -axis) plotted against the time of analysis (in minutes; x -axis).
    Figure Legend Snippet: Processivity of double strand DNA digestion by wild-type LHK-Exo and Arg12Ala mutant. Time course analysis of the digestion of 5′-phosphorylated double strand DNA (5′-PO 4 -dsDNA: EcoRV-linearized pMal-c2) and 5′-dephosphorylated double strand DNA (5′-OH-dsDNA: 5′dephosphorylated EcoRV-linearized pMal-c2) substrates by wild-type LHK-Exo and the Arg12Ala mutant form using a ‘heparin trap’ approach. A total of 6 µg (82 pmol of trimers) of LHK-Exo or Arg12Ala mutant protein was incubated at 25°C with 60 ng (0.015 pmol) of 5′-PO 4 -dsDNA or 5′-OH-dsDNA in Tris–HCl (25 mM, pH 8.0), 1 mM DTT, 7.5 mM MgCl 2 . After 15 s, excess heparin was added to sequester all unbound protein, and to prevent disassociated protein from re-binding. Aliquots were removed at various time points (0–20 min), and dsDNA levels were determined using fluorescent PicoGreen assays, to enable the extent of DNA digestion to be calculated. In one set of assays, heparin was added to LHK-Exo prior to the addition of dsDNA substrate, to confirm the efficacy of the heparin trap method (filled black squares, green line). Graphs show the mean number of nucleotides digested from each terminus (±SD; y -axis) plotted against the time of analysis (in minutes; x -axis).

    Techniques Used: Mutagenesis, Incubation, Binding Assay

    Qualitative analysis of ssDNA and dsDNA hydrolysis activities of LHK-Exo. ( A ) dsDNA exonuclease activities. Agarose gel showing aliquots taken (0–15 min) from an incubation of LHK-Exo (30 µg, 0.41 nmol of trimers) and BamHI-linearized pET28a (1.8 µg, 0.54 pmol) in Tris–HCl (pH 8.0, 50 mM), 50 mM NaCl, 7.5 mM MgCl 2 at 37°C. ( B ) Polarity of dsDNA digestion. A total of 6 µg of LHK-Exo (82 pmol of trimers, lanes 2–5) or λ-exonuclease (74 pmol of trimers, lanes 6–9) protein was incubated with 0.1 µg (0.23 pmol) of a 712-bp linear 5′-phosphorylated dsDNA substrate (‘unmodified’; lanes 2, 3, 6 and 7), or an analogous 5′-phosphorylated linear dsDNA substrate containing three consecutive ‘nuclease-resistant’ phosphorothioate linkages at its 5′-termini (‘PT-modified’; lanes 4, 5, 8, 9). Assays were quenched immediately (0 min) or incubated at 37°C for 20 min, before analysis of digestion products on 1% agarose gels. ( C ) Digestion of 5′-phosphorylated ssDNA. Reaction mixtures (80 µl) containing LHK-Exo (4.5 µg, 61.4 pmol of trimers) and 5′-PO 4 -(dT) 50 (0.4 nmol) in 25 mM Tris–HCl (pH 8.0), 7.5 mM MgCl 2 , 1 mM DTT were incubated at 37°C. Aliquots (20 µl) were withdrawn after 0, 0.5, 5 and 20 min, and immediately quenched. Reaction products were analyzed by denaturing gel electrophoresis. ( D ) Digestion of non-phosphorylated ssDNA. Analogous sets of assays were performed using non-phosphorylated 50-mers of oligothymidine [5′-OH-(dT) 50 ]. Fluorescent gel images were scanned after SYBR Gold staining. A ssDNA ladder [Oligo Length Standards 20/100 Ladder (IDT)] is included in lane 1.
    Figure Legend Snippet: Qualitative analysis of ssDNA and dsDNA hydrolysis activities of LHK-Exo. ( A ) dsDNA exonuclease activities. Agarose gel showing aliquots taken (0–15 min) from an incubation of LHK-Exo (30 µg, 0.41 nmol of trimers) and BamHI-linearized pET28a (1.8 µg, 0.54 pmol) in Tris–HCl (pH 8.0, 50 mM), 50 mM NaCl, 7.5 mM MgCl 2 at 37°C. ( B ) Polarity of dsDNA digestion. A total of 6 µg of LHK-Exo (82 pmol of trimers, lanes 2–5) or λ-exonuclease (74 pmol of trimers, lanes 6–9) protein was incubated with 0.1 µg (0.23 pmol) of a 712-bp linear 5′-phosphorylated dsDNA substrate (‘unmodified’; lanes 2, 3, 6 and 7), or an analogous 5′-phosphorylated linear dsDNA substrate containing three consecutive ‘nuclease-resistant’ phosphorothioate linkages at its 5′-termini (‘PT-modified’; lanes 4, 5, 8, 9). Assays were quenched immediately (0 min) or incubated at 37°C for 20 min, before analysis of digestion products on 1% agarose gels. ( C ) Digestion of 5′-phosphorylated ssDNA. Reaction mixtures (80 µl) containing LHK-Exo (4.5 µg, 61.4 pmol of trimers) and 5′-PO 4 -(dT) 50 (0.4 nmol) in 25 mM Tris–HCl (pH 8.0), 7.5 mM MgCl 2 , 1 mM DTT were incubated at 37°C. Aliquots (20 µl) were withdrawn after 0, 0.5, 5 and 20 min, and immediately quenched. Reaction products were analyzed by denaturing gel electrophoresis. ( D ) Digestion of non-phosphorylated ssDNA. Analogous sets of assays were performed using non-phosphorylated 50-mers of oligothymidine [5′-OH-(dT) 50 ]. Fluorescent gel images were scanned after SYBR Gold staining. A ssDNA ladder [Oligo Length Standards 20/100 Ladder (IDT)] is included in lane 1.

    Techniques Used: Agarose Gel Electrophoresis, Incubation, Modification, Nucleic Acid Electrophoresis, Staining

    Determination of optimum conditions for LHK-Exo dsDNA digestion activities. PicoGreen fluorescence assays were performed to quantify the amounts of a representative 5′-phosphorylatd dsDNA substrate (PstI-linearized pUC18 that were digested by LHK-Exo under various conditions (reported as a percentage of the initial quantities of DNA). ( A and B ) Optimal concentrations of Mg 2+ and Mn 2+ ions. Amounts of PstI-linearized pUC18 (30 ng, 0.018 pmol) digested by LHK-Exo (8 ng, 0.11 pmol of trimers) in Tris–HCl (25 mM, pH 8.0) containing varying concentrations of Mg 2+ ions (1–15 mM, A) or Mn 2+ ions (0.1–1.7 mM, B), respectively, upon incubation at 25°C for 20 min. ( C ) Optimal pH. Amounts of PstI-linearized pUC18 (30 ng, 0.018 pmol) digested by LHK-Exo (8 ng, 0.11 pmol of trimers) in 25 mM Tris–HCl, 7.5 mM MgCl 2 ; adjusted to the appropriate pH value (pH 7.0–9.0); upon incubation at 25°C for 20 min. ( D ) Optimal temperature. Amounts of PstI-linearized pUC18 (30 ng, 0.018 pmol) digested by LHK-Exo (40 ng, 0.55 pmol of trimers) in Tris–HCl (25 mM, pH 8.0), 7.5 mM MgCl 2 , after incubation for 1 min at the indicated temperature (34–54°C). Four to six independent replicates of each experimental condition were performed, and data are reported as the mean ± standard deviation.
    Figure Legend Snippet: Determination of optimum conditions for LHK-Exo dsDNA digestion activities. PicoGreen fluorescence assays were performed to quantify the amounts of a representative 5′-phosphorylatd dsDNA substrate (PstI-linearized pUC18 that were digested by LHK-Exo under various conditions (reported as a percentage of the initial quantities of DNA). ( A and B ) Optimal concentrations of Mg 2+ and Mn 2+ ions. Amounts of PstI-linearized pUC18 (30 ng, 0.018 pmol) digested by LHK-Exo (8 ng, 0.11 pmol of trimers) in Tris–HCl (25 mM, pH 8.0) containing varying concentrations of Mg 2+ ions (1–15 mM, A) or Mn 2+ ions (0.1–1.7 mM, B), respectively, upon incubation at 25°C for 20 min. ( C ) Optimal pH. Amounts of PstI-linearized pUC18 (30 ng, 0.018 pmol) digested by LHK-Exo (8 ng, 0.11 pmol of trimers) in 25 mM Tris–HCl, 7.5 mM MgCl 2 ; adjusted to the appropriate pH value (pH 7.0–9.0); upon incubation at 25°C for 20 min. ( D ) Optimal temperature. Amounts of PstI-linearized pUC18 (30 ng, 0.018 pmol) digested by LHK-Exo (40 ng, 0.55 pmol of trimers) in Tris–HCl (25 mM, pH 8.0), 7.5 mM MgCl 2 , after incubation for 1 min at the indicated temperature (34–54°C). Four to six independent replicates of each experimental condition were performed, and data are reported as the mean ± standard deviation.

    Techniques Used: Fluorescence, Incubation, Standard Deviation

    Processivity of double strand DNA digestion by wild-type LHK-Exo and Arg12Ala mutant. Time course analysis of the digestion of 5′-phosphorylated double strand DNA (5′-PO 4 -dsDNA: EcoRV-linearized pMal-c2) and 5′-dephosphorylated double strand DNA (5′-OH-dsDNA: 5′dephosphorylated EcoRV-linearized pMal-c2) substrates by wild-type LHK-Exo and the Arg12Ala mutant form using a ‘heparin trap’ approach. A total of 6 µg (82 pmol of trimers) of LHK-Exo or Arg12Ala mutant protein was incubated at 25°C with 60 ng (0.015 pmol) of 5′-PO 4 -dsDNA or 5′-OH-dsDNA in Tris–HCl (25 mM, pH 8.0), 1 mM DTT, 7.5 mM MgCl 2 . After 15 s, excess heparin was added to sequester all unbound protein, and to prevent disassociated protein from re-binding. Aliquots were removed at various time points (0–20 min), and dsDNA levels were determined using fluorescent PicoGreen assays, to enable the extent of DNA digestion to be calculated. In one set of assays, heparin was added to LHK-Exo prior to the addition of dsDNA substrate, to confirm the efficacy of the heparin trap method (filled black squares, green line). Graphs show the mean number of nucleotides digested from each terminus (±SD; y -axis) plotted against the time of analysis (in minutes; x -axis).
    Figure Legend Snippet: Processivity of double strand DNA digestion by wild-type LHK-Exo and Arg12Ala mutant. Time course analysis of the digestion of 5′-phosphorylated double strand DNA (5′-PO 4 -dsDNA: EcoRV-linearized pMal-c2) and 5′-dephosphorylated double strand DNA (5′-OH-dsDNA: 5′dephosphorylated EcoRV-linearized pMal-c2) substrates by wild-type LHK-Exo and the Arg12Ala mutant form using a ‘heparin trap’ approach. A total of 6 µg (82 pmol of trimers) of LHK-Exo or Arg12Ala mutant protein was incubated at 25°C with 60 ng (0.015 pmol) of 5′-PO 4 -dsDNA or 5′-OH-dsDNA in Tris–HCl (25 mM, pH 8.0), 1 mM DTT, 7.5 mM MgCl 2 . After 15 s, excess heparin was added to sequester all unbound protein, and to prevent disassociated protein from re-binding. Aliquots were removed at various time points (0–20 min), and dsDNA levels were determined using fluorescent PicoGreen assays, to enable the extent of DNA digestion to be calculated. In one set of assays, heparin was added to LHK-Exo prior to the addition of dsDNA substrate, to confirm the efficacy of the heparin trap method (filled black squares, green line). Graphs show the mean number of nucleotides digested from each terminus (±SD; y -axis) plotted against the time of analysis (in minutes; x -axis).

    Techniques Used: Mutagenesis, Incubation, Binding Assay

    2) Product Images from "Synthesis Characterization and DNA Interaction Studies of a New Zn(II) Complex Containing Different Dinitrogen Aromatic Ligands"

    Article Title: Synthesis Characterization and DNA Interaction Studies of a New Zn(II) Complex Containing Different Dinitrogen Aromatic Ligands

    Journal: Bioinorganic Chemistry and Applications

    doi: 10.1155/2012/571913

    Circular dichroism spectra of CT-DNA (5.9 × 10 −5 M) in Tris buffer (10 mM), in the presence of increasing amounts of the complex.
    Figure Legend Snippet: Circular dichroism spectra of CT-DNA (5.9 × 10 −5 M) in Tris buffer (10 mM), in the presence of increasing amounts of the complex.

    Techniques Used:

    3) Product Images from "CFTR mutations altering CFTR fragmentation"

    Article Title: CFTR mutations altering CFTR fragmentation

    Journal: Biochemical Journal

    doi: 10.1042/BJ20121240

    CFTR fragments are not an artefact of cell lysis or cell type ( A ) Similar NBD2 and NBD1 fractured bands are present when cells are scraped and lysed into a different lysis buffer [0.5% Triton X-100 and 20 mM Tris/HCl (pH 7.5) and 2 mM EDTA, 2 mM EGTA and 150 mM NaCl] or directly lysed by the addition of the loading buffer for SDS/PAGE ‘on plate’. ( B ) Comparing fracture of NBD1 after low CFTR expression in HBE cells. The faint NBD1 fragments (lane 3) are visible. ( C ) The faint NBD1 fragments of CFTR are enriched by immunoprecipitation (lane 5). CFTR was immunoprecipitated using the mixed four antibody approach (0.5 μg of each antibody individually cross-linked to magnetic beads as described in the Material and methods section) at a lysate protein loading of 200 μg for HBE/EV and 100 μg for WT. CFTR content in the input lysate (lanes 1–3: 20 μg for the HBE/EV and 10 μg for WT) was also compared with equal amounts of proteins in the supernatants after the IP (lanes 7–9). IP, immunoprecipitation.
    Figure Legend Snippet: CFTR fragments are not an artefact of cell lysis or cell type ( A ) Similar NBD2 and NBD1 fractured bands are present when cells are scraped and lysed into a different lysis buffer [0.5% Triton X-100 and 20 mM Tris/HCl (pH 7.5) and 2 mM EDTA, 2 mM EGTA and 150 mM NaCl] or directly lysed by the addition of the loading buffer for SDS/PAGE ‘on plate’. ( B ) Comparing fracture of NBD1 after low CFTR expression in HBE cells. The faint NBD1 fragments (lane 3) are visible. ( C ) The faint NBD1 fragments of CFTR are enriched by immunoprecipitation (lane 5). CFTR was immunoprecipitated using the mixed four antibody approach (0.5 μg of each antibody individually cross-linked to magnetic beads as described in the Material and methods section) at a lysate protein loading of 200 μg for HBE/EV and 100 μg for WT. CFTR content in the input lysate (lanes 1–3: 20 μg for the HBE/EV and 10 μg for WT) was also compared with equal amounts of proteins in the supernatants after the IP (lanes 7–9). IP, immunoprecipitation.

    Techniques Used: Lysis, SDS Page, Expressing, Immunoprecipitation, Magnetic Beads

    4) Product Images from "Structure of mycobacterial 3′-to-5′ RNA:DNA helicase Lhr bound to a ssDNA tracking strand highlights distinctive features of a novel family of bacterial helicases"

    Article Title: Structure of mycobacterial 3′-to-5′ RNA:DNA helicase Lhr bound to a ssDNA tracking strand highlights distinctive features of a novel family of bacterial helicases

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx1163

    Mutational analysis of the loading strand interface. ( A ) ATPase activity. Reaction mixtures (10 μl) containing 20 mM Tris–HCl, pH 7.0, 50 mM NaCl, 1 mM CaCl 2 , 1 mM DTT, 1 mM [α 32 P]ATP (10 nmol ATP), 100 ng heat-denatured salmon sperm DNA and Lhr-(1–856) proteins as specified were incubated at 37°C for 30 min. The extents of [ 32 P]ADP formation are plotted as a function of input enzyme. Each datum represents the average of three independent enzyme titration experiments ± SEM. For the sake of clarity, the data are plotted in two separate graphs, with domain 1 mutants in the left panel and domain 2, 3 and 4 mutants in the right panel. ( B ) Helicase activity. Reaction mixtures (10 μl) contained 20 mM Tris–HCl, pH 7.0, 50 mM NaCl, 5 mM CaCl 2 , 1 mM DTT, 1 mM ATP, 50 nM (0.5 pmol) RNA:DNA hybrid substrate (depicted at the top, with the 5′ 32 P label denoted by •) and Lhr-(1–856) proteins as specified. The products were analyzed by native PAGE. The extents of duplex unwinding [(ssRNA)/(ssRNA + tailed RNA:DNA duplex)] are plotted as a function of input enzyme. Each datum represents the average of three separate enzyme titrations ± SEM. ( C ) Translocase activity. Translocation was assayed by displacement of SA from a SA–(biotin)–ssDNA complex (depicted at the top) as described under ‘Materials and Methods’ section. Complete translocase reaction mixtures (10 μl) contained 20 mM Tris–HCl, pH 7.0, 50 mM NaCl, 1 mM CaCl 2 , 1 mM DTT, 1 mM ATP, 100 nM (1 pmol) SA–DNA complex and Lhr-(1–856) proteins as specified. The products were analyzed by native PAGE. Extents of translocation [(free DNA)/(SA–DNA + free DNA)] are plotted as a function of input enzyme. Each datum represents the average of three separate enzyme titrations ± SEM.
    Figure Legend Snippet: Mutational analysis of the loading strand interface. ( A ) ATPase activity. Reaction mixtures (10 μl) containing 20 mM Tris–HCl, pH 7.0, 50 mM NaCl, 1 mM CaCl 2 , 1 mM DTT, 1 mM [α 32 P]ATP (10 nmol ATP), 100 ng heat-denatured salmon sperm DNA and Lhr-(1–856) proteins as specified were incubated at 37°C for 30 min. The extents of [ 32 P]ADP formation are plotted as a function of input enzyme. Each datum represents the average of three independent enzyme titration experiments ± SEM. For the sake of clarity, the data are plotted in two separate graphs, with domain 1 mutants in the left panel and domain 2, 3 and 4 mutants in the right panel. ( B ) Helicase activity. Reaction mixtures (10 μl) contained 20 mM Tris–HCl, pH 7.0, 50 mM NaCl, 5 mM CaCl 2 , 1 mM DTT, 1 mM ATP, 50 nM (0.5 pmol) RNA:DNA hybrid substrate (depicted at the top, with the 5′ 32 P label denoted by •) and Lhr-(1–856) proteins as specified. The products were analyzed by native PAGE. The extents of duplex unwinding [(ssRNA)/(ssRNA + tailed RNA:DNA duplex)] are plotted as a function of input enzyme. Each datum represents the average of three separate enzyme titrations ± SEM. ( C ) Translocase activity. Translocation was assayed by displacement of SA from a SA–(biotin)–ssDNA complex (depicted at the top) as described under ‘Materials and Methods’ section. Complete translocase reaction mixtures (10 μl) contained 20 mM Tris–HCl, pH 7.0, 50 mM NaCl, 1 mM CaCl 2 , 1 mM DTT, 1 mM ATP, 100 nM (1 pmol) SA–DNA complex and Lhr-(1–856) proteins as specified. The products were analyzed by native PAGE. Extents of translocation [(free DNA)/(SA–DNA + free DNA)] are plotted as a function of input enzyme. Each datum represents the average of three separate enzyme titrations ± SEM.

    Techniques Used: Activity Assay, Incubation, Titration, Clear Native PAGE, Translocation Assay

    Mutational analysis of the ATP site. ( A ) ATPase activity was assayed as described in Figure 5 . The extents of [ 32 P]ADP formation are plotted as a function of the indicated input Lhr-(1–856) proteins. Each datum represents the average of three independent enzyme titration experiments ±SEM. ( B ) Helicase activity was assayed as described in Figure 5 . The extents of duplex unwinding are plotted as a function of input enzyme. Each datum represents the average of three separate enzyme titrations ±SEM. ( C ) Translocase activity was assayed as described in Figure 5 . Extents of SA–DNA displacement are plotted as a function of input enzyme. Each datum represents the average of three separate enzyme titrations ±SEM. ( D ) ATP concentration dependence of ATP hydrolysis. Reaction mixtures (40 μl) containing 20 mM Tris–HCl, pH 7.0, 50 mM NaCl, 1 mM CaCl 2 , 1 mM DTT, 100 ng of heat-denatured salmon sperm DNA, 200 nM Lhr-(1–586) (WT, F24A or D369A) and varying concentrations of [α 32 P]ATP were incubated at 37°C. Aliquots (10 μl) were withdrawn at 10, 20 and 30 s (for 31.3, 62.5, 125 and 250 μM ATP) or 15, 30 and 45 s (for 1.0 and 2.0 mM ATP) and quenched immediately with 2 μl of 5 M formic acid. The extents of ATP hydrolysis were plotted as a function of time for each ATP concentration and the initial rates were derived by linear regression analysis in Prism. The initial rates (pmol•s −1 ) were divided by the molar amount of input enzyme (2 pmol) to obtain a turnover number V (s −1 ), which is plotted in the figure as a function of ATP concentration. Each datum is the average of three separate time course experiments ±SEM. A non-linear regression curve fit of the data to the Michaelis–Menten equation (in Prism) is shown.
    Figure Legend Snippet: Mutational analysis of the ATP site. ( A ) ATPase activity was assayed as described in Figure 5 . The extents of [ 32 P]ADP formation are plotted as a function of the indicated input Lhr-(1–856) proteins. Each datum represents the average of three independent enzyme titration experiments ±SEM. ( B ) Helicase activity was assayed as described in Figure 5 . The extents of duplex unwinding are plotted as a function of input enzyme. Each datum represents the average of three separate enzyme titrations ±SEM. ( C ) Translocase activity was assayed as described in Figure 5 . Extents of SA–DNA displacement are plotted as a function of input enzyme. Each datum represents the average of three separate enzyme titrations ±SEM. ( D ) ATP concentration dependence of ATP hydrolysis. Reaction mixtures (40 μl) containing 20 mM Tris–HCl, pH 7.0, 50 mM NaCl, 1 mM CaCl 2 , 1 mM DTT, 100 ng of heat-denatured salmon sperm DNA, 200 nM Lhr-(1–586) (WT, F24A or D369A) and varying concentrations of [α 32 P]ATP were incubated at 37°C. Aliquots (10 μl) were withdrawn at 10, 20 and 30 s (for 31.3, 62.5, 125 and 250 μM ATP) or 15, 30 and 45 s (for 1.0 and 2.0 mM ATP) and quenched immediately with 2 μl of 5 M formic acid. The extents of ATP hydrolysis were plotted as a function of time for each ATP concentration and the initial rates were derived by linear regression analysis in Prism. The initial rates (pmol•s −1 ) were divided by the molar amount of input enzyme (2 pmol) to obtain a turnover number V (s −1 ), which is plotted in the figure as a function of ATP concentration. Each datum is the average of three separate time course experiments ±SEM. A non-linear regression curve fit of the data to the Michaelis–Menten equation (in Prism) is shown.

    Techniques Used: Activity Assay, Titration, Concentration Assay, Incubation, Derivative Assay

    5) Product Images from "USP7S-dependent inactivation of Mule regulates DNA damage signalling and repair"

    Article Title: USP7S-dependent inactivation of Mule regulates DNA damage signalling and repair

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gks1359

    USP7S interacts with Mule and prevents its self-ubiquitylation in vitro. ( A ) Equal amounts (2.5 pmol) of purified recombinant His-tagged USP7S and GST-tagged Mule were incubated in buffer containing 50 mM Tris–HCl, pH 8.0, 50 mM KCl, 1 mM EDTA, 1 mM DTT for 30 min at room temperature with shaking. USP7S and Mule were then pulled down using either magnetic beads as a control (Mock PD, 100%) or using magnetic Ni-NTA agarose beads (His PD, 100%) or GST agarose beads (GST PD, 100%), respectively. The beads were boiled in SDS–PAGE sample buffer for 5 min, and proteins were separated by 10% SDS–PAGE and analysed using USP7S and Mule antibodies (10% of the input is loaded). ( B ) HeLa cells were simultaneously transfected with USP7S and Mule (1 pmol each) expression plasmids, whole-cell extracts were prepared and used to pull down HA-tagged Mule or Flag-tagged USP7S using either magnetic beads as a control (Mock PD, 100%), HA- (HA PD, 100%) or Flag-agarose (Flag PD, 100%) beads. Proteins were separated by 4–20% SDS–PAGE and analysed using USP7S and Mule antibodies (15% of the input is loaded). ( C ) In vitro deubiquitylation of self-ubiquitylated Mule HECT-domain by recombinant wild-type USP7S protein (wtUSP7S) or its inactive mutant (C223S USP7S) analysed by western blotting. Equal loading of the enzyme is demonstrated using USP7S antibodies.
    Figure Legend Snippet: USP7S interacts with Mule and prevents its self-ubiquitylation in vitro. ( A ) Equal amounts (2.5 pmol) of purified recombinant His-tagged USP7S and GST-tagged Mule were incubated in buffer containing 50 mM Tris–HCl, pH 8.0, 50 mM KCl, 1 mM EDTA, 1 mM DTT for 30 min at room temperature with shaking. USP7S and Mule were then pulled down using either magnetic beads as a control (Mock PD, 100%) or using magnetic Ni-NTA agarose beads (His PD, 100%) or GST agarose beads (GST PD, 100%), respectively. The beads were boiled in SDS–PAGE sample buffer for 5 min, and proteins were separated by 10% SDS–PAGE and analysed using USP7S and Mule antibodies (10% of the input is loaded). ( B ) HeLa cells were simultaneously transfected with USP7S and Mule (1 pmol each) expression plasmids, whole-cell extracts were prepared and used to pull down HA-tagged Mule or Flag-tagged USP7S using either magnetic beads as a control (Mock PD, 100%), HA- (HA PD, 100%) or Flag-agarose (Flag PD, 100%) beads. Proteins were separated by 4–20% SDS–PAGE and analysed using USP7S and Mule antibodies (15% of the input is loaded). ( C ) In vitro deubiquitylation of self-ubiquitylated Mule HECT-domain by recombinant wild-type USP7S protein (wtUSP7S) or its inactive mutant (C223S USP7S) analysed by western blotting. Equal loading of the enzyme is demonstrated using USP7S antibodies.

    Techniques Used: In Vitro, Purification, Recombinant, Incubation, Magnetic Beads, SDS Page, Transfection, Expressing, Mutagenesis, Western Blot

    6) Product Images from "DNA Interaction Studies of a New Platinum(II) Complex Containing Different Aromatic Dinitrogen Ligands"

    Article Title: DNA Interaction Studies of a New Platinum(II) Complex Containing Different Aromatic Dinitrogen Ligands

    Journal: Bioinorganic Chemistry and Applications

    doi: 10.1155/2011/429241

    Effect of increasing amounts of Pt(II) complex on the viscosity of CT-DNA (5 × 10 −5 M) in 10 mM Tris-HCl buffer.
    Figure Legend Snippet: Effect of increasing amounts of Pt(II) complex on the viscosity of CT-DNA (5 × 10 −5 M) in 10 mM Tris-HCl buffer.

    Techniques Used:

    7) Product Images from "Structural analysis of the complex between influenza B nucleoprotein and human importin-α"

    Article Title: Structural analysis of the complex between influenza B nucleoprotein and human importin-α

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-17458-z

    ITC determination of the binding thermodynamics of B/NP TAIL to importin-α7. Titration of 150 µM of importin-α7 into a solution of 15 mM of B/NP TAIL . The experiments were performed in 20 mM Tris-HCl pH 7.5; 150 mM NaCl at 25 °C in triplicate.
    Figure Legend Snippet: ITC determination of the binding thermodynamics of B/NP TAIL to importin-α7. Titration of 150 µM of importin-α7 into a solution of 15 mM of B/NP TAIL . The experiments were performed in 20 mM Tris-HCl pH 7.5; 150 mM NaCl at 25 °C in triplicate.

    Techniques Used: Binding Assay, Titration

    8) Product Images from "Efficiency of Incorporation and Chain Termination Determines the Inhibition Potency of 2′-Modified Nucleotide Analogs against Hepatitis C Virus Polymerase"

    Article Title: Efficiency of Incorporation and Chain Termination Determines the Inhibition Potency of 2′-Modified Nucleotide Analogs against Hepatitis C Virus Polymerase

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.02666-14

    Chain termination in the presence of the next correct nucleotide. High-resolution Tris-borate-EDTA–urea gel electrophoresis showing the extension of 10- to 11-mer RNA products with UTP or UTP analogs, followed by the addition of the next correct
    Figure Legend Snippet: Chain termination in the presence of the next correct nucleotide. High-resolution Tris-borate-EDTA–urea gel electrophoresis showing the extension of 10- to 11-mer RNA products with UTP or UTP analogs, followed by the addition of the next correct

    Techniques Used: Nucleic Acid Electrophoresis

    9) Product Images from "High-affinity Anticalins with aggregation-blocking activity directed against the Alzheimer β-amyloid peptide"

    Article Title: High-affinity Anticalins with aggregation-blocking activity directed against the Alzheimer β-amyloid peptide

    Journal: Biochemical Journal

    doi: 10.1042/BCJ20160114

    Effect of Anticalins on Aβ42 fibril formation and neuronal cytotoxicity ( A ) Macromolecular fibril formation was monitored via TEM starting from Aβ42 dissolved at 200 μM (0.9 mg/ml) in 5 mM NaOH. Subsequently, 1 volume of 20 mM Tris/HCl, pH 6.8 was added by vortex-mixing. The solution was then incubated at 4°C for 6 h without agitation, prior to dilution in RPMI-1640 cell culture medium to a final concentration of 10 μM. Aβ42 alone or in combination with equimolar concentrations of wtLcn2 (negative control), MAb 6E10 (positive control) or the Aβ-specific Anticalins S1A4, H1G1 and H1GA were incubated at 37°C for 72 h and then subjected to TEM. ( B , C ) The toxicity of Aβ42 alone or in combination with Anticalins on NGF-β differentiated PC12 cells was analysed in an MTT reduction assay. ( B ) Aβ42 was preincubated at 4°C for 6 h in a mixture of 1 volume 5 mM NaOH and 1 volume 20 mM Tris/HCl, pH 6.8 and then added at a concentration of 10 μM alone or in combination with equimolar concentrations of the Lcn2 variants to the cells. wtLcn2, S1A4 and H1G1 alone (without Aβ42) showed only minor cytotoxicity (see Figure S5C in the supplementary data section). ( C ) Anticalins with promising effects (H1GA not shown) were further analysed for their potential to support cell viability up to stoichiometric ratios in the presence of 10 μM Aβ42 (measured as in B). For each experiment, measurements of replicates were derived from different wells ( n =4–8) of a single 96-well plate from which the median was calculated. Several plates were measured on different days as independent experiments for cell viability ( n ≥ 3) from which the mean was calculated. Error bars represent standard deviations of the means.
    Figure Legend Snippet: Effect of Anticalins on Aβ42 fibril formation and neuronal cytotoxicity ( A ) Macromolecular fibril formation was monitored via TEM starting from Aβ42 dissolved at 200 μM (0.9 mg/ml) in 5 mM NaOH. Subsequently, 1 volume of 20 mM Tris/HCl, pH 6.8 was added by vortex-mixing. The solution was then incubated at 4°C for 6 h without agitation, prior to dilution in RPMI-1640 cell culture medium to a final concentration of 10 μM. Aβ42 alone or in combination with equimolar concentrations of wtLcn2 (negative control), MAb 6E10 (positive control) or the Aβ-specific Anticalins S1A4, H1G1 and H1GA were incubated at 37°C for 72 h and then subjected to TEM. ( B , C ) The toxicity of Aβ42 alone or in combination with Anticalins on NGF-β differentiated PC12 cells was analysed in an MTT reduction assay. ( B ) Aβ42 was preincubated at 4°C for 6 h in a mixture of 1 volume 5 mM NaOH and 1 volume 20 mM Tris/HCl, pH 6.8 and then added at a concentration of 10 μM alone or in combination with equimolar concentrations of the Lcn2 variants to the cells. wtLcn2, S1A4 and H1G1 alone (without Aβ42) showed only minor cytotoxicity (see Figure S5C in the supplementary data section). ( C ) Anticalins with promising effects (H1GA not shown) were further analysed for their potential to support cell viability up to stoichiometric ratios in the presence of 10 μM Aβ42 (measured as in B). For each experiment, measurements of replicates were derived from different wells ( n =4–8) of a single 96-well plate from which the median was calculated. Several plates were measured on different days as independent experiments for cell viability ( n ≥ 3) from which the mean was calculated. Error bars represent standard deviations of the means.

    Techniques Used: Transmission Electron Microscopy, Incubation, Cell Culture, Concentration Assay, Negative Control, Positive Control, MTT Assay, Derivative Assay

    10) Product Images from "Shedding light on disulfide bond formation: engineering a redox switch in green fluorescent protein"

    Article Title: Shedding light on disulfide bond formation: engineering a redox switch in green fluorescent protein

    Journal: The EMBO Journal

    doi: 10.1093/emboj/20.21.5853

    Fig. 1. Cysteine mutants of the yellow fluorescent protein. ( A . ( B ) Spontaneous oxidation of the cysteine mutants as monitored by non-reducing SDS–PAGE. Reduction of the four mutant proteins was performed by overnight incubation with 20 mM DTT. Subsequently they were dialysed against 20 mM Tris–HCl pH 8.0 for 17 h at room temperature to promote oxidation. Trichloroacetic acid (10% v/v) was added to aliquots of the samples taken before (marked Red) and after (marked Ox) dialysis. The protein precipitate was washed twice with acetone and then alkylated with 50 mM N- ethylmaleimide before analysis by non-reducing SDS–PAGE on a 16% gel.
    Figure Legend Snippet: Fig. 1. Cysteine mutants of the yellow fluorescent protein. ( A . ( B ) Spontaneous oxidation of the cysteine mutants as monitored by non-reducing SDS–PAGE. Reduction of the four mutant proteins was performed by overnight incubation with 20 mM DTT. Subsequently they were dialysed against 20 mM Tris–HCl pH 8.0 for 17 h at room temperature to promote oxidation. Trichloroacetic acid (10% v/v) was added to aliquots of the samples taken before (marked Red) and after (marked Ox) dialysis. The protein precipitate was washed twice with acetone and then alkylated with 50 mM N- ethylmaleimide before analysis by non-reducing SDS–PAGE on a 16% gel.

    Techniques Used: SDS Page, Mutagenesis, Incubation

    11) Product Images from "The lytic polysaccharide monooxygenase CbpD promotes Pseudomonas aeruginosa virulence in systemic infection"

    Article Title: The lytic polysaccharide monooxygenase CbpD promotes Pseudomonas aeruginosa virulence in systemic infection

    Journal: Nature Communications

    doi: 10.1038/s41467-021-21473-0

    Multidomain structure of CbpD and evaluation of its activity on a model substrate. a Schematic representation of the domain architecture of CbpD. The full-length protein contains a signal peptide (SP), an N-terminal AA10-type LPMO domain (AA10) followed by a module with unknown function (module X or MX) and a C-terminal CBM domain (CBM73). The residue boundaries for each domain, as well as potential post-translational modification (PTM) sites based on refs. 25 , 35 , 36 and Supplementary Table 1 , are labeled; phosphorylation sites are indicated above and lysine/arginine modifications below the domain illustration. PTMs identified in this study (Supplementary Table 1 ) are colored red. b Homology model of CbpD generated with Raptor-X 92 showing flexible linkers in an extended conformation. The active site is indicated by a yellow partially transparent square. c SAXS model of monomeric CbpD ( χ 2 = 2.35; produced with Pepsi-SAXS 114 ; SASBDB ID: SASDK42), superimposed onto the ab initio SAXS model “envelope” (produced with DAMMIF 111 , 112 based on an average of 20 calculated models). Panels b and c were generated using PyMol. d Product formation by 1 µM of rCbpD EC variants (FL: full-length; AA10: AA10 module only; MX + CBM73: the MX and CBM73 domains only) after a 2 h reaction at 37 °C with 10 mg ml −1 β-chitin in 20 mM Tris-HCL pH 7.0, with 1 mM ascorbate as reducing agent, analyzed by HILIC. The degrees of polymerization (DP) of oxidized chitooligosaccharide aldonic acids in a standard sample are indicated. Control reactions without ascorbate did not show product formation. e HILIC analysis of reaction products emerging from a reaction of 1 µM rCbpD EC with 10 mg ml −1 β-chitin, 250 µM ascorbate in 20 mM Tris-HCl pH 7.0, and serial dilutions of pyocyanin (PCN) for 2 h at 37 °C. The chromatograms have been offset on the y axis to enable visual interpretation of their quantitative magnitude. Chromatograms for reactions containing serial dilutions of PCN and with or without various reductants that were sampled at different time points are shown in Supplementary Fig. 7c . f HILIC analysis of reaction products emerging from a reaction of 1 μM of copper-free full-length rCbpD PA with 10 mg ml −1 β-chitin in 20 mM Tris-HCL pH 7.0, 100 µM pyocyanin (PCN), 250 µM ascorbate, in the presence or absence of 1 µM azurin (Azu) after incubation for 2 h at 37 °C. Control reactions without added ascorbate did not show product formation (Supplementary Fig. 7d ).
    Figure Legend Snippet: Multidomain structure of CbpD and evaluation of its activity on a model substrate. a Schematic representation of the domain architecture of CbpD. The full-length protein contains a signal peptide (SP), an N-terminal AA10-type LPMO domain (AA10) followed by a module with unknown function (module X or MX) and a C-terminal CBM domain (CBM73). The residue boundaries for each domain, as well as potential post-translational modification (PTM) sites based on refs. 25 , 35 , 36 and Supplementary Table 1 , are labeled; phosphorylation sites are indicated above and lysine/arginine modifications below the domain illustration. PTMs identified in this study (Supplementary Table 1 ) are colored red. b Homology model of CbpD generated with Raptor-X 92 showing flexible linkers in an extended conformation. The active site is indicated by a yellow partially transparent square. c SAXS model of monomeric CbpD ( χ 2 = 2.35; produced with Pepsi-SAXS 114 ; SASBDB ID: SASDK42), superimposed onto the ab initio SAXS model “envelope” (produced with DAMMIF 111 , 112 based on an average of 20 calculated models). Panels b and c were generated using PyMol. d Product formation by 1 µM of rCbpD EC variants (FL: full-length; AA10: AA10 module only; MX + CBM73: the MX and CBM73 domains only) after a 2 h reaction at 37 °C with 10 mg ml −1 β-chitin in 20 mM Tris-HCL pH 7.0, with 1 mM ascorbate as reducing agent, analyzed by HILIC. The degrees of polymerization (DP) of oxidized chitooligosaccharide aldonic acids in a standard sample are indicated. Control reactions without ascorbate did not show product formation. e HILIC analysis of reaction products emerging from a reaction of 1 µM rCbpD EC with 10 mg ml −1 β-chitin, 250 µM ascorbate in 20 mM Tris-HCl pH 7.0, and serial dilutions of pyocyanin (PCN) for 2 h at 37 °C. The chromatograms have been offset on the y axis to enable visual interpretation of their quantitative magnitude. Chromatograms for reactions containing serial dilutions of PCN and with or without various reductants that were sampled at different time points are shown in Supplementary Fig. 7c . f HILIC analysis of reaction products emerging from a reaction of 1 μM of copper-free full-length rCbpD PA with 10 mg ml −1 β-chitin in 20 mM Tris-HCL pH 7.0, 100 µM pyocyanin (PCN), 250 µM ascorbate, in the presence or absence of 1 µM azurin (Azu) after incubation for 2 h at 37 °C. Control reactions without added ascorbate did not show product formation (Supplementary Fig. 7d ).

    Techniques Used: Activity Assay, Modification, Labeling, Generated, Produced, Hydrophilic Interaction Liquid Chromatography, Incubation

    12) Product Images from "Biological and Enzymatic Characterization of Proteases from Crude Venom of the Ant Odontomachus bauri"

    Article Title: Biological and Enzymatic Characterization of Proteases from Crude Venom of the Ant Odontomachus bauri

    Journal: Toxins

    doi: 10.3390/toxins7124869

    Acrylamide-gelatin gel zymography of the O. bauri venom. ( A ) Crude venom samples were analyzed in non-reducing conditions. MrS: molecular size markers; ( B ) Effect of different buffers (50 mM Tris-HCl; 50 mM Tris-HCl and 10 mM CaCl 2 ; 50 mM Tris-HCl, 1 mM CaCl 2 and 1 mM SO 4 Zn; 50 mM Tris-HCl, 150 mM NaCl, 10 mM CaCl 2 , 0.002%CHAPS and 10 mM EDTA) on the gelatin proteolysis activity of the O. bauri venom; ( C ) Effect of different ranges of pH (4 to 10) on the gelatin proteolysis activity of the O. bauri venom. ( * ) optimal buffer and pH.
    Figure Legend Snippet: Acrylamide-gelatin gel zymography of the O. bauri venom. ( A ) Crude venom samples were analyzed in non-reducing conditions. MrS: molecular size markers; ( B ) Effect of different buffers (50 mM Tris-HCl; 50 mM Tris-HCl and 10 mM CaCl 2 ; 50 mM Tris-HCl, 1 mM CaCl 2 and 1 mM SO 4 Zn; 50 mM Tris-HCl, 150 mM NaCl, 10 mM CaCl 2 , 0.002%CHAPS and 10 mM EDTA) on the gelatin proteolysis activity of the O. bauri venom; ( C ) Effect of different ranges of pH (4 to 10) on the gelatin proteolysis activity of the O. bauri venom. ( * ) optimal buffer and pH.

    Techniques Used: Zymography, Activity Assay

    Acrylamide-gelatin gel zymography of the O. bauri venom. ( A ) Crude venom samples were analyzed in non-reducing conditions. MrS: molecular size markers; ( B ) Effect of different buffers (50 mM Tris-HCl; 50 mM Tris-HCl and 10 mM CaCl 2 ; 50 mM Tris-HCl, 1 mM CaCl 2 and 1 mM SO 4 Zn; 50 mM Tris-HCl, 150 mM NaCl, 10 mM CaCl 2 , 0.002%CHAPS and 10 mM EDTA) on the gelatin proteolysis activity of the O. bauri venom; ( C ) Effect of different ranges of pH (4 to 10) on the gelatin proteolysis activity of the O. bauri venom. ( * ) optimal buffer and pH.
    Figure Legend Snippet: Acrylamide-gelatin gel zymography of the O. bauri venom. ( A ) Crude venom samples were analyzed in non-reducing conditions. MrS: molecular size markers; ( B ) Effect of different buffers (50 mM Tris-HCl; 50 mM Tris-HCl and 10 mM CaCl 2 ; 50 mM Tris-HCl, 1 mM CaCl 2 and 1 mM SO 4 Zn; 50 mM Tris-HCl, 150 mM NaCl, 10 mM CaCl 2 , 0.002%CHAPS and 10 mM EDTA) on the gelatin proteolysis activity of the O. bauri venom; ( C ) Effect of different ranges of pH (4 to 10) on the gelatin proteolysis activity of the O. bauri venom. ( * ) optimal buffer and pH.

    Techniques Used: Zymography, Activity Assay

    13) Product Images from "Functionally Important Amino Acids in the Arabidopsis Thylakoid Phosphate Transporter: Homology Modeling and Site-Directed Mutagenesis †"

    Article Title: Functionally Important Amino Acids in the Arabidopsis Thylakoid Phosphate Transporter: Homology Modeling and Site-Directed Mutagenesis †

    Journal: Biochemistry

    doi: 10.1021/bi100239j

    Comparative effects of site-directed mutations on P i uptake and protein expression levels. The transport activity and ANTR1 protein abundance were measured in E. coli cells transformed with an empty vector (control) and cells expressing the wild-type ANTR1 (WT) or mutant protein and are shown as a percentage of the WT levels. The transport activity of 100 μM 32 P i was measured for 3 min in 25 mM Tris−succinate (pH 6.5) in the presence of 25 mM NaCl. Protein expression was determined by quantification of the ANTR1 Western blots. The bars are means ± SD. Inset: Representative Western blot of protein lysate (1 A 600 unit per lane) from E. coli cells with an anti-FLAG peptide antibody. The loaded samples correspond to the strains in the plot. The equal loading of the proteins in each well of the gel was visually monitored by Coomassie staining of duplicate gels (data not shown). The mass of ANTR1 is approximately 56 kDa, indicated to the right of the blot.
    Figure Legend Snippet: Comparative effects of site-directed mutations on P i uptake and protein expression levels. The transport activity and ANTR1 protein abundance were measured in E. coli cells transformed with an empty vector (control) and cells expressing the wild-type ANTR1 (WT) or mutant protein and are shown as a percentage of the WT levels. The transport activity of 100 μM 32 P i was measured for 3 min in 25 mM Tris−succinate (pH 6.5) in the presence of 25 mM NaCl. Protein expression was determined by quantification of the ANTR1 Western blots. The bars are means ± SD. Inset: Representative Western blot of protein lysate (1 A 600 unit per lane) from E. coli cells with an anti-FLAG peptide antibody. The loaded samples correspond to the strains in the plot. The equal loading of the proteins in each well of the gel was visually monitored by Coomassie staining of duplicate gels (data not shown). The mass of ANTR1 is approximately 56 kDa, indicated to the right of the blot.

    Techniques Used: Expressing, Activity Assay, Transformation Assay, Plasmid Preparation, Mutagenesis, Western Blot, Staining

    14) Product Images from "Peroxin 5-peroxin 14 association in the protozoan Leishmania donovani involves a novel protein-protein interaction motif"

    Article Title: Peroxin 5-peroxin 14 association in the protozoan Leishmania donovani involves a novel protein-protein interaction motif

    Journal: Biochemical Journal

    doi: 10.1042/BJ20050328

    LdPEX14–LdPEX5 interaction The interaction of His 6 /S–LdPEX14 ( A ) or His 6 /S–ldpex14-(1–120) (His 6 /S–ldpex14-120) ( B ) with either the wild-type LdPEX5 or ldpex5 WXXXY/F site-directed mutants was assessed by pull-down assays using S-protein agarose beads. LdPEX14 proteins were mixed with 10 μg of purified, ldpex5-W53A, ldpex5-W293A, ldpex5-W176,293A, ldpex5-W53,176,293A or wild-type LdPEX5 or with no LdPEX5 and the mixture was added to S-protein agarose. Beads were stringently washed with 1% Triton X-100 and 500 mM NaCl in TBS (Tris-buffered saline; 50 mM Tris/HCl, pH 8.0 and 150 mM NaCl) and bound proteins were analysed by Coomassie Blue-stained SDS/PAGE.
    Figure Legend Snippet: LdPEX14–LdPEX5 interaction The interaction of His 6 /S–LdPEX14 ( A ) or His 6 /S–ldpex14-(1–120) (His 6 /S–ldpex14-120) ( B ) with either the wild-type LdPEX5 or ldpex5 WXXXY/F site-directed mutants was assessed by pull-down assays using S-protein agarose beads. LdPEX14 proteins were mixed with 10 μg of purified, ldpex5-W53A, ldpex5-W293A, ldpex5-W176,293A, ldpex5-W53,176,293A or wild-type LdPEX5 or with no LdPEX5 and the mixture was added to S-protein agarose. Beads were stringently washed with 1% Triton X-100 and 500 mM NaCl in TBS (Tris-buffered saline; 50 mM Tris/HCl, pH 8.0 and 150 mM NaCl) and bound proteins were analysed by Coomassie Blue-stained SDS/PAGE.

    Techniques Used: Purification, Staining, SDS Page

    Size-exclusion chromatography analysis of the ldpex5 mutants Wild-type LdPEX5 and site-directed mutant ldpex5-W53A, ldpex5-W293A, ldpex5-W176,293A, ldpex5-W53,176,293A proteins were purified to homogeneity as fusion proteins using the New England Biolabs IMPACT system. Intein fusion proteins were cleaved using DTT, dialysed and concentrated. The oligomeric state of these proteins was determined by loading 25–50 μg of purified protein on to a Bio-Sil 250 column (7.8 mm×600 mm) in 25 mM Tris/HCl (pH 7.5) and 120 mM NaCl at a flow rate of 0.25 ml/min. Column eluate was monitored spectrophotometrically at 280 nm. Arrows indicate the elution positions of the standard proteins.
    Figure Legend Snippet: Size-exclusion chromatography analysis of the ldpex5 mutants Wild-type LdPEX5 and site-directed mutant ldpex5-W53A, ldpex5-W293A, ldpex5-W176,293A, ldpex5-W53,176,293A proteins were purified to homogeneity as fusion proteins using the New England Biolabs IMPACT system. Intein fusion proteins were cleaved using DTT, dialysed and concentrated. The oligomeric state of these proteins was determined by loading 25–50 μg of purified protein on to a Bio-Sil 250 column (7.8 mm×600 mm) in 25 mM Tris/HCl (pH 7.5) and 120 mM NaCl at a flow rate of 0.25 ml/min. Column eluate was monitored spectrophotometrically at 280 nm. Arrows indicate the elution positions of the standard proteins.

    Techniques Used: Size-exclusion Chromatography, Mutagenesis, Purification, Flow Cytometry

    15) Product Images from "Adducin Is an In Vivo Substrate for Protein Kinase C: Phosphorylation in the MARCKS-related Domain Inhibits Activity in Promoting Spectrin-Actin Complexes and Occurs in Many Cells, Including Dendritic Spines of Neurons "

    Article Title: Adducin Is an In Vivo Substrate for Protein Kinase C: Phosphorylation in the MARCKS-related Domain Inhibits Activity in Promoting Spectrin-Actin Complexes and Occurs in Many Cells, Including Dendritic Spines of Neurons

    Journal: The Journal of Cell Biology

    doi:

    Specificity of the antiphosphoadducin and anti–α adducin antibodies and effect of forskolin and PMA on adducin phosphorylation in vivo. Western blot analysis for the antiphosphoadducin antibody (3.5 μg/ ml) ( A ) and the anti–α adducin antibody (1 μg/ml) ( B ). Lanes a–c , human erythrocyte membrane; lanes d–f , human embryonal kidney (HEK 293) cells; lanes g–i , rat hippocampal slices. Lanes a , d , and g , unstimulated conditions; lanes b , e , and h , 50 μM forskolin/50 μM IBMX treatment; lanes c , f , and i , 1 μM PMA treatment. Both α and β adducin in HEK 293 cells and rat hippocampal lysates migrated at the same position of human erythrocyte α adducin (lanes d–f and g–i ). Each lane was loaded with 10 μg total protein. ( C ) Confluent cultures of stable MDCK cells expressing HA epitope–tagged full-length human α adducin and the PKC sites mutant were serum starved overnight and were either untreated or treated with 0.1 μM PMA for 15 min. After washing with ice-cold PBS containing 2 mM PMSF, the cells were lysed with an ice-cold lysis buffer (1 ml/ø10-cm dish) consisting of 20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 2 mM sodium EGTA, 2 mM sodium EDTA, 50 mM NaF, 100 nM caliculin A, 1% Triton X-100, 1% NP-40, 10 μg/ml leupeptin, 10 μg/ml pepstatin A, 1 mM bebzamidine, and 1 mM PMSF using a glass-Teflon pestle homogenizer. The lysate was cleared by centrifugation at 15,600 g for 10 min. The HA epitope–tagged α adducin was immunoprecipitated for 3 h at 4°C from 1 ml of cell lysate using an HA-specific mouse IgG preadsorbed on protein G–Sepharose beads (50 μl; Pharmacia Biotech ). The beads were washed twice with lysis buffer and then twice with the same buffer without Triton and NP-40 before addition of 100 μl SDS-sample buffer. Erythrocyte adducin phosphorylated by PKM was used as a positive control for the RTPS-serine phosphorylation.
    Figure Legend Snippet: Specificity of the antiphosphoadducin and anti–α adducin antibodies and effect of forskolin and PMA on adducin phosphorylation in vivo. Western blot analysis for the antiphosphoadducin antibody (3.5 μg/ ml) ( A ) and the anti–α adducin antibody (1 μg/ml) ( B ). Lanes a–c , human erythrocyte membrane; lanes d–f , human embryonal kidney (HEK 293) cells; lanes g–i , rat hippocampal slices. Lanes a , d , and g , unstimulated conditions; lanes b , e , and h , 50 μM forskolin/50 μM IBMX treatment; lanes c , f , and i , 1 μM PMA treatment. Both α and β adducin in HEK 293 cells and rat hippocampal lysates migrated at the same position of human erythrocyte α adducin (lanes d–f and g–i ). Each lane was loaded with 10 μg total protein. ( C ) Confluent cultures of stable MDCK cells expressing HA epitope–tagged full-length human α adducin and the PKC sites mutant were serum starved overnight and were either untreated or treated with 0.1 μM PMA for 15 min. After washing with ice-cold PBS containing 2 mM PMSF, the cells were lysed with an ice-cold lysis buffer (1 ml/ø10-cm dish) consisting of 20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 2 mM sodium EGTA, 2 mM sodium EDTA, 50 mM NaF, 100 nM caliculin A, 1% Triton X-100, 1% NP-40, 10 μg/ml leupeptin, 10 μg/ml pepstatin A, 1 mM bebzamidine, and 1 mM PMSF using a glass-Teflon pestle homogenizer. The lysate was cleared by centrifugation at 15,600 g for 10 min. The HA epitope–tagged α adducin was immunoprecipitated for 3 h at 4°C from 1 ml of cell lysate using an HA-specific mouse IgG preadsorbed on protein G–Sepharose beads (50 μl; Pharmacia Biotech ). The beads were washed twice with lysis buffer and then twice with the same buffer without Triton and NP-40 before addition of 100 μl SDS-sample buffer. Erythrocyte adducin phosphorylated by PKM was used as a positive control for the RTPS-serine phosphorylation.

    Techniques Used: In Vivo, Western Blot, Expressing, Mutagenesis, Lysis, Centrifugation, Immunoprecipitation, Positive Control

    16) Product Images from "Vibrio harveyi Nitroreductase Is Also a Chromate Reductase"

    Article Title: Vibrio harveyi Nitroreductase Is Also a Chromate Reductase

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.69.8.4390-4395.2003

    NADH consumption. The reaction mixture contained 164 μg of enzyme GST-VhNfsA with 30 μM chromate (▪) or without chromate (□) in 1 ml of 10 mM Tris-HCl (pH 7.0), and NADH (final concentration 100 μM) was then added. The absorbance change was determined at 340 nm after every 1-min interval. The amount of reduced chromate in the reaction buffer containing 30 μM chromate, 100 μM NADH, and GST-VhNfsA was determined by the dye method described in the text at 5-min intervals (•).
    Figure Legend Snippet: NADH consumption. The reaction mixture contained 164 μg of enzyme GST-VhNfsA with 30 μM chromate (▪) or without chromate (□) in 1 ml of 10 mM Tris-HCl (pH 7.0), and NADH (final concentration 100 μM) was then added. The absorbance change was determined at 340 nm after every 1-min interval. The amount of reduced chromate in the reaction buffer containing 30 μM chromate, 100 μM NADH, and GST-VhNfsA was determined by the dye method described in the text at 5-min intervals (•).

    Techniques Used: Concentration Assay

    (A) Nitrofurazone-reducing activity. The reduction of nitrofurazone was determined by measuring the decrease in the absorbance of nitrofurazone at 400 nm (molar extinction coefficient, 12,960 M −1 cm −1 ). A basic enzyme reaction mixture (1.5 ml) containing 10 mM Tris-HCl buffer (pH 7.0), 0.1 mM NADH, 10 μM nitrofurazone, and 10 μg of either GST-EcNfsA (▪), GST-EcNfsB (□), or GST-VhNfsA (•) was incubated at 30°C. The reaction was started by the addition of NADH. One unit was defined as the amount of the enzyme that reduced 1 pmol of nitrofurazone/μg of protein. (B) TNT-reducing activity. The transformation of TNT was determined by measuring the decrease in the absorbance of TNT at 447 nm. The standard assay mixture (1 ml) containing 50 mM Tris-HCl (pH 7.0), 0.1 mM TNT, 1 mM NADH, and either 172 μg of GST-EcNfsA (▪), 166 μg of GST-EcNfsB (□), or 82 μg of GST-VhNfsA (•) was incubated at 30°C. Theenzymatic reaction was initiated by adding NADH. The reaction was quenched by adding 160 μl of 1 M NaOH, resulting in a pH of 12.2. Quantitative measurements were made 5 min after the addition of NADH to the TNT solution. One unit was defined as the amount of the enzyme that reduced 1 pmol of TNT/μg of protein.
    Figure Legend Snippet: (A) Nitrofurazone-reducing activity. The reduction of nitrofurazone was determined by measuring the decrease in the absorbance of nitrofurazone at 400 nm (molar extinction coefficient, 12,960 M −1 cm −1 ). A basic enzyme reaction mixture (1.5 ml) containing 10 mM Tris-HCl buffer (pH 7.0), 0.1 mM NADH, 10 μM nitrofurazone, and 10 μg of either GST-EcNfsA (▪), GST-EcNfsB (□), or GST-VhNfsA (•) was incubated at 30°C. The reaction was started by the addition of NADH. One unit was defined as the amount of the enzyme that reduced 1 pmol of nitrofurazone/μg of protein. (B) TNT-reducing activity. The transformation of TNT was determined by measuring the decrease in the absorbance of TNT at 447 nm. The standard assay mixture (1 ml) containing 50 mM Tris-HCl (pH 7.0), 0.1 mM TNT, 1 mM NADH, and either 172 μg of GST-EcNfsA (▪), 166 μg of GST-EcNfsB (□), or 82 μg of GST-VhNfsA (•) was incubated at 30°C. Theenzymatic reaction was initiated by adding NADH. The reaction was quenched by adding 160 μl of 1 M NaOH, resulting in a pH of 12.2. Quantitative measurements were made 5 min after the addition of NADH to the TNT solution. One unit was defined as the amount of the enzyme that reduced 1 pmol of TNT/μg of protein.

    Techniques Used: Activity Assay, Incubation, Transformation Assay

    NADH-dependent chromate reductase activity. Three kinds of reaction mixtures were prepared: 10 mM Tris-HCl (pH 7.0), 0.1 mM NADH, 20 μM K 2 CrO 4 , and either GST-EcNfsA, GST-EcNfsB, or GST-VhNfsA (▥); 10 mM Tris-HCl (pH 7.0), 0.1 mM NADH, 20 μM K 2 CrO 4 (no enzyme) (▤); and 10 mM Tris-HCl (pH 7.0), 0.05 mM NADH, 20 μM K 2 CrO 4 , and either GST-EcNfsA, GST-EcNfsB, or GST-VhNfsA (▨). They were incubated at 30°C for 1 h, and the chromate reductase activity was determined by the dye method described in the text. One unit of chromate reductase activity was defined as the amount of the enzyme which decreased 1 nmol of Cr(VI) per min at 30°C per mg of protein. The chromate reductase activity of GST-VhNfsA in the reaction buffer (▥) was regarded as 100%.
    Figure Legend Snippet: NADH-dependent chromate reductase activity. Three kinds of reaction mixtures were prepared: 10 mM Tris-HCl (pH 7.0), 0.1 mM NADH, 20 μM K 2 CrO 4 , and either GST-EcNfsA, GST-EcNfsB, or GST-VhNfsA (▥); 10 mM Tris-HCl (pH 7.0), 0.1 mM NADH, 20 μM K 2 CrO 4 (no enzyme) (▤); and 10 mM Tris-HCl (pH 7.0), 0.05 mM NADH, 20 μM K 2 CrO 4 , and either GST-EcNfsA, GST-EcNfsB, or GST-VhNfsA (▨). They were incubated at 30°C for 1 h, and the chromate reductase activity was determined by the dye method described in the text. One unit of chromate reductase activity was defined as the amount of the enzyme which decreased 1 nmol of Cr(VI) per min at 30°C per mg of protein. The chromate reductase activity of GST-VhNfsA in the reaction buffer (▥) was regarded as 100%.

    Techniques Used: Activity Assay, Incubation

    17) Product Images from "Characterization of the quinol-dependent nitric oxide reductase from the pathogen Neisseria meningitidis, an electrogenic enzyme"

    Article Title: Characterization of the quinol-dependent nitric oxide reductase from the pathogen Neisseria meningitidis, an electrogenic enzyme

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-21804-0

    Visible absorption spectra of wild-type N. meningitidis qNOR. The spectra shown are oxidized state (solid curve) and dithionite-reduced state (broken curve). The sample is in 50 mM Tris-HCl pH 8.0, 150 mM NaCl, and 0.1% DM. The reduced qNOR sample was prepared by the addition of an excess amount of dithionite to the oxidized enzyme under N 2 atmosphere.
    Figure Legend Snippet: Visible absorption spectra of wild-type N. meningitidis qNOR. The spectra shown are oxidized state (solid curve) and dithionite-reduced state (broken curve). The sample is in 50 mM Tris-HCl pH 8.0, 150 mM NaCl, and 0.1% DM. The reduced qNOR sample was prepared by the addition of an excess amount of dithionite to the oxidized enzyme under N 2 atmosphere.

    Techniques Used:

    High frequency region of resonance Raman spectra of wild-type qNOR from N. meningitidis . Traces shown are ( a ) oxidized and ( b ) dithionite-reduced qNOR. The spectra were obtained with excitation at 413.1 nm. The qNOR concentration was adjusted to 20–40 µM in 50 mM Tris-HCl pH 8.0, 150 mM NaCl, and 0.1% DM. The reduced form was prepared by addition of an excess amount of dithionite under N 2 atmosphere.
    Figure Legend Snippet: High frequency region of resonance Raman spectra of wild-type qNOR from N. meningitidis . Traces shown are ( a ) oxidized and ( b ) dithionite-reduced qNOR. The spectra were obtained with excitation at 413.1 nm. The qNOR concentration was adjusted to 20–40 µM in 50 mM Tris-HCl pH 8.0, 150 mM NaCl, and 0.1% DM. The reduced form was prepared by addition of an excess amount of dithionite under N 2 atmosphere.

    Techniques Used: Concentration Assay

    18) Product Images from "Inflammatory response of endothelial cells to a human endogenous retrovirus associated with multiple sclerosis is mediated by TLR4"

    Article Title: Inflammatory response of endothelial cells to a human endogenous retrovirus associated with multiple sclerosis is mediated by TLR4

    Journal: International Immunology

    doi: 10.1093/intimm/dxv025

    TLR4 expression in endothelial cells. (A) TLR4 co-localizes with the Golgi apparatus. Fluorescent staining of Golgi apparatus (red), TLR4 (green) and nuclei (blue) on HCMEC/D3 cells (not confluent) after fixation with PFA 2% and permeabilization with saponin 0.2%. Negative control is obtained after staining with the secondary antibodies only. Pearson’s correlation coefficient Rr = 0.67 and Mander’s overlap coefficient R = 0.75. Scale bar: 20 µm. (B) TLR4 is expressed on HCMEC/D3 and on HUVECs. Cells were lysed with the Tris–HCl 50mM, pH 7.5, NaCl 150mM buffer containing 1% NP40. The presence of TLR4 in the whole extracts is then analyzed by western blotting. (C) TLR4 heavy form (150kDa) leads to a light form (120kDa) after treatment with PNGase. Whole extracts, prepared with a lysis buffer containing only NP40, were treated with PNGase, or water as a negative control, for 2h and then analyzed by western blotting. (D) TLR4 expression on HCMEC/D3 cells after double transfection with siRNAs targeting TLR4 or control siRNAs; cells were lysed with the Tris–HCl 50mM, pH 7.5, NaCl 150mM buffer containing 1% NP40. The presence of TLR4 in the whole extracts was then analyzed by western blotting and quantified with ImageJ software. NT: not transfected.
    Figure Legend Snippet: TLR4 expression in endothelial cells. (A) TLR4 co-localizes with the Golgi apparatus. Fluorescent staining of Golgi apparatus (red), TLR4 (green) and nuclei (blue) on HCMEC/D3 cells (not confluent) after fixation with PFA 2% and permeabilization with saponin 0.2%. Negative control is obtained after staining with the secondary antibodies only. Pearson’s correlation coefficient Rr = 0.67 and Mander’s overlap coefficient R = 0.75. Scale bar: 20 µm. (B) TLR4 is expressed on HCMEC/D3 and on HUVECs. Cells were lysed with the Tris–HCl 50mM, pH 7.5, NaCl 150mM buffer containing 1% NP40. The presence of TLR4 in the whole extracts is then analyzed by western blotting. (C) TLR4 heavy form (150kDa) leads to a light form (120kDa) after treatment with PNGase. Whole extracts, prepared with a lysis buffer containing only NP40, were treated with PNGase, or water as a negative control, for 2h and then analyzed by western blotting. (D) TLR4 expression on HCMEC/D3 cells after double transfection with siRNAs targeting TLR4 or control siRNAs; cells were lysed with the Tris–HCl 50mM, pH 7.5, NaCl 150mM buffer containing 1% NP40. The presence of TLR4 in the whole extracts was then analyzed by western blotting and quantified with ImageJ software. NT: not transfected.

    Techniques Used: Expressing, Staining, Negative Control, Western Blot, Lysis, Transfection, Software

    19) Product Images from "The Arabidopsis thaliana K+-Uptake Permease 5 (AtKUP5) Contains a Functional Cytosolic Adenylate Cyclase Essential for K+ Transport"

    Article Title: The Arabidopsis thaliana K+-Uptake Permease 5 (AtKUP5) Contains a Functional Cytosolic Adenylate Cyclase Essential for K+ Transport

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2018.01645

    Detection of cAMP generated by AtKUP5 1-104 by liquid chromatography tandem mass spectrometry. (A) Is a representative ion chromatogram of cAMP showing the parent and daughter ion peaks (see arrows and inset for the structures) and (B) is an HPLC elution profile of cAMP with a calibration curve shown in the inset. The calculated amount of cAMP generated from a reaction mixture containing 10 μg of the purified recombinant AtKUP5 1-104 , 50 mM Tris-HCl pH 8; 2 mM IBMX, 5 mM MnCl 2 , and 1 mM ATP after 25 min is 49 fmol/μg protein.
    Figure Legend Snippet: Detection of cAMP generated by AtKUP5 1-104 by liquid chromatography tandem mass spectrometry. (A) Is a representative ion chromatogram of cAMP showing the parent and daughter ion peaks (see arrows and inset for the structures) and (B) is an HPLC elution profile of cAMP with a calibration curve shown in the inset. The calculated amount of cAMP generated from a reaction mixture containing 10 μg of the purified recombinant AtKUP5 1-104 , 50 mM Tris-HCl pH 8; 2 mM IBMX, 5 mM MnCl 2 , and 1 mM ATP after 25 min is 49 fmol/μg protein.

    Techniques Used: Generated, Liquid Chromatography, Mass Spectrometry, High Performance Liquid Chromatography, Purification, Recombinant

    cAMP generated by recombinant AtKUP5 1-104 . cAMP was generated from a reaction mixture containing 10 μg of the purified recombinant AtKUP5 1-104 , 50 mM Tris-HCl pH 8; 2 mM IBMX, 1 mM ATP and either 5 mM MnCl 2 or MgCl 2 as a cofactor, at different time points, as measured by enzyme immunoassay.
    Figure Legend Snippet: cAMP generated by recombinant AtKUP5 1-104 . cAMP was generated from a reaction mixture containing 10 μg of the purified recombinant AtKUP5 1-104 , 50 mM Tris-HCl pH 8; 2 mM IBMX, 1 mM ATP and either 5 mM MnCl 2 or MgCl 2 as a cofactor, at different time points, as measured by enzyme immunoassay.

    Techniques Used: Generated, Recombinant, Purification, Enzyme-linked Immunosorbent Assay

    20) Product Images from "Dimerization of Flavivirus NS4B Protein"

    Article Title: Dimerization of Flavivirus NS4B Protein

    Journal: Journal of Virology

    doi: 10.1128/JVI.02782-13

    Dimerization of the WNV NS4B protein according to MALS–UV–RI–size-exclusion chromatography (SEC) analysis. (A) SDS-PAGE analysis of recombinant NS4B protein of WNV. Samples represent different fractions derived from gel filtration. Because the protein migrated faster than its theoretical mass of 30 kDa, its identity was confirmed by mass spectrometry (data not shown). (B) Western blot analysis of the purified WNV NS4B protein by use of MAb 44-4-7. (C) MALS-UV-RI-SEC analysis. The NS4B protein (6 mg/ml) was purified through an S200 5/150 GL column by using a buffer containing 20 mM Tris-HCl (pH 8.0), 300 mM NaCl, 2 mM TCEP, 0.03% DDM, and 0.006% cholesterol. The right y axis represents the OD 280 , and the left y axis represents the molecular mass. Colored traces show the measured molecular masses of the protein-detergent conjugate (red), protein (blue), and detergent (green).
    Figure Legend Snippet: Dimerization of the WNV NS4B protein according to MALS–UV–RI–size-exclusion chromatography (SEC) analysis. (A) SDS-PAGE analysis of recombinant NS4B protein of WNV. Samples represent different fractions derived from gel filtration. Because the protein migrated faster than its theoretical mass of 30 kDa, its identity was confirmed by mass spectrometry (data not shown). (B) Western blot analysis of the purified WNV NS4B protein by use of MAb 44-4-7. (C) MALS-UV-RI-SEC analysis. The NS4B protein (6 mg/ml) was purified through an S200 5/150 GL column by using a buffer containing 20 mM Tris-HCl (pH 8.0), 300 mM NaCl, 2 mM TCEP, 0.03% DDM, and 0.006% cholesterol. The right y axis represents the OD 280 , and the left y axis represents the molecular mass. Colored traces show the measured molecular masses of the protein-detergent conjugate (red), protein (blue), and detergent (green).

    Techniques Used: Size-exclusion Chromatography, SDS Page, Recombinant, Derivative Assay, Filtration, Mass Spectrometry, Western Blot, Purification

    21) Product Images from "Biochemical characterization of aminopeptidase N2 from Toxoplasmagondii"

    Article Title: Biochemical characterization of aminopeptidase N2 from Toxoplasmagondii

    Journal: The Journal of Veterinary Medical Science

    doi: 10.1292/jvms.17-0119

    Restoration of r Tg APN2 activity by divalent metal ions. r Tg APN2 was pre-incubated at 37°C for 30 min in 50 mM Tris-HCl supplemented with the specified metal chloride, and then the substrate (H-Ala-MCA, 0.1 mM final concentration) was added. (A) Enzyme activity in the presence of 1 µ M metal ions. (B) Enzyme activity in the presence of 0.1 µ M metal ions. (C) Different concentration of Zn 2+ ions. (D) Enzyme activity in the presence of EDTA. The differences between samples were evaluated by Student’s t -test (n=3). * P
    Figure Legend Snippet: Restoration of r Tg APN2 activity by divalent metal ions. r Tg APN2 was pre-incubated at 37°C for 30 min in 50 mM Tris-HCl supplemented with the specified metal chloride, and then the substrate (H-Ala-MCA, 0.1 mM final concentration) was added. (A) Enzyme activity in the presence of 1 µ M metal ions. (B) Enzyme activity in the presence of 0.1 µ M metal ions. (C) Different concentration of Zn 2+ ions. (D) Enzyme activity in the presence of EDTA. The differences between samples were evaluated by Student’s t -test (n=3). * P

    Techniques Used: Activity Assay, Incubation, Concentration Assay

    22) Product Images from "Physical interactions between DNA and sepiolite nanofibers, and potential application for DNA transfer into mammalian cells"

    Article Title: Physical interactions between DNA and sepiolite nanofibers, and potential application for DNA transfer into mammalian cells

    Journal: Scientific Reports

    doi: 10.1038/srep36341

    FTIR spectra of sepiolite and the Sep/DNA bionanocomposite prepared in 10 mM Tris-HCl pH = 7.5 compared to Sep/DNA bionanocomposites prepared in the presence of monovalent ( A : NaCl, B : KCl), divalent ( C : CaCl 2 , D : MgCl 2 ), trivalent ( E : spermidine) and tetravalent ( F : spermine) cations.
    Figure Legend Snippet: FTIR spectra of sepiolite and the Sep/DNA bionanocomposite prepared in 10 mM Tris-HCl pH = 7.5 compared to Sep/DNA bionanocomposites prepared in the presence of monovalent ( A : NaCl, B : KCl), divalent ( C : CaCl 2 , D : MgCl 2 ), trivalent ( E : spermidine) and tetravalent ( F : spermine) cations.

    Techniques Used:

    ( A , B ) Adsorption isotherms of DNA on sepiolite in the presence of various polyvalent cations. Each point has error bars for 3 different experiments. Reaction conditions: 10 mM Tris-HCl pH = 7.5 and a sepiolite concentration of 1 mg/ml. ( C ) The effect of the presence of cations with different valences on DNA adsorption. Reaction conditions: 10 mM Tris-HCl pH = 7.5, salmon sperm DNA and sepiolite concentrations of 615 ng·μl −1 and 1 mg/ml, respectively. ( D , E ) Adsorption isotherms of different DNA conformations on sepiolite. Reaction conditions: 10 mM Tris-HCl pH = 7.5, 5 mM MgCl 2 , sepiolite concentration of 1 mg/ml; 50 μg of sepiolite was used in each experiment. Adsorption occurred at 25 °C for 24 hours under agitation at 700 rpm using an Eppendorf Thermomixer.
    Figure Legend Snippet: ( A , B ) Adsorption isotherms of DNA on sepiolite in the presence of various polyvalent cations. Each point has error bars for 3 different experiments. Reaction conditions: 10 mM Tris-HCl pH = 7.5 and a sepiolite concentration of 1 mg/ml. ( C ) The effect of the presence of cations with different valences on DNA adsorption. Reaction conditions: 10 mM Tris-HCl pH = 7.5, salmon sperm DNA and sepiolite concentrations of 615 ng·μl −1 and 1 mg/ml, respectively. ( D , E ) Adsorption isotherms of different DNA conformations on sepiolite. Reaction conditions: 10 mM Tris-HCl pH = 7.5, 5 mM MgCl 2 , sepiolite concentration of 1 mg/ml; 50 μg of sepiolite was used in each experiment. Adsorption occurred at 25 °C for 24 hours under agitation at 700 rpm using an Eppendorf Thermomixer.

    Techniques Used: Adsorption, Concentration Assay

    ( A ) Comparison of DNA desorption efficiency at different concentrations of EDTA. Reaction conditions: Initially, 0.5 ml of Sep/DNA samples were prepared using DNA at 640 ng·μl −1 in a sepiolite dispersion (1 mg/ml) in 10 mM Tris-HCl pH = 7.5 and 5 mM MgCl 2 . Resuspension in 0.1 ml solution of 10 mM Tris-HCl pH = 7.5, and EDTA at 5, 10 and 50 mM. After 15 min of incubation at room temperature, all samples were centrifuged at 5000 rpm for 5 min, and the supernatant was measured using the UV-vis. ( B ) Comparison of DNA desorption efficiency for Sep/DNA prepared in the presence of Mg 2+ , Ca 2+ , spermidine (Spd) or spermine (Spm). ( C ) Characterization with EMSA of desorbed DNA using the “chelation” method. 1) plasmid (5.7 kbp) control; 2) plasmid DNA in the supernatant after synthesis of the bionanocomposite prepared with 5 mM MgCl 2 and before re- suspending the pellet in Tris-HCl pH = 7.5 and EDTA; 3) plasmid DNA desorbed from the bionanocomposite obtained with 5 mM MgCl 2 ; 4) plasmid DNA desorbed from bionanocomposite obtained with 5 mM CaCl 2 .
    Figure Legend Snippet: ( A ) Comparison of DNA desorption efficiency at different concentrations of EDTA. Reaction conditions: Initially, 0.5 ml of Sep/DNA samples were prepared using DNA at 640 ng·μl −1 in a sepiolite dispersion (1 mg/ml) in 10 mM Tris-HCl pH = 7.5 and 5 mM MgCl 2 . Resuspension in 0.1 ml solution of 10 mM Tris-HCl pH = 7.5, and EDTA at 5, 10 and 50 mM. After 15 min of incubation at room temperature, all samples were centrifuged at 5000 rpm for 5 min, and the supernatant was measured using the UV-vis. ( B ) Comparison of DNA desorption efficiency for Sep/DNA prepared in the presence of Mg 2+ , Ca 2+ , spermidine (Spd) or spermine (Spm). ( C ) Characterization with EMSA of desorbed DNA using the “chelation” method. 1) plasmid (5.7 kbp) control; 2) plasmid DNA in the supernatant after synthesis of the bionanocomposite prepared with 5 mM MgCl 2 and before re- suspending the pellet in Tris-HCl pH = 7.5 and EDTA; 3) plasmid DNA desorbed from the bionanocomposite obtained with 5 mM MgCl 2 ; 4) plasmid DNA desorbed from bionanocomposite obtained with 5 mM CaCl 2 .

    Techniques Used: Incubation, Plasmid Preparation

    TEM characterization of the Sep/DNA bionanocomposite ( A–C ). ( D,E ) TEM images of a single molecule of Sep/DNA. The DNA bound to the sepiolite at the edge of the nanofiber and on its external surface. Nanofibers can be completely coated with DNA, but some DNA molecules are bound at one of their extremities, and two different nanofibers can be linked by a DNA plasmid. Reaction conditions: 1 mg/ml sepiolite, 10 mM Tris-HCl, pH = 7.5, 5 mM MgCl 2 , and 50 ng·μl −1 plasmid DNA (5.7 kb). ( G ) Scheme illustrating the DNA-sepiolite bionanocomposite resulting from the sepiolite interaction with linear DNA. It is also show the silanol groups (Si-OH) present at the external surface of the sepiolite fibers which are in hydrogen bonding interaction with nitrogenated bases at the DNA chains.
    Figure Legend Snippet: TEM characterization of the Sep/DNA bionanocomposite ( A–C ). ( D,E ) TEM images of a single molecule of Sep/DNA. The DNA bound to the sepiolite at the edge of the nanofiber and on its external surface. Nanofibers can be completely coated with DNA, but some DNA molecules are bound at one of their extremities, and two different nanofibers can be linked by a DNA plasmid. Reaction conditions: 1 mg/ml sepiolite, 10 mM Tris-HCl, pH = 7.5, 5 mM MgCl 2 , and 50 ng·μl −1 plasmid DNA (5.7 kb). ( G ) Scheme illustrating the DNA-sepiolite bionanocomposite resulting from the sepiolite interaction with linear DNA. It is also show the silanol groups (Si-OH) present at the external surface of the sepiolite fibers which are in hydrogen bonding interaction with nitrogenated bases at the DNA chains.

    Techniques Used: Transmission Electron Microscopy, Plasmid Preparation

    23) Product Images from "Ordering Transitions in Micrometer-Thick Films of Nematic Liquid Crystals Driven by Self-Assembly of Ganglioside GM1"

    Article Title: Ordering Transitions in Micrometer-Thick Films of Nematic Liquid Crystals Driven by Self-Assembly of Ganglioside GM1

    Journal: Journal of colloid and interface science

    doi: 10.1016/j.jcis.2009.03.068

    (A) Surface pressure-area isotherm measured at 25°C for a Langmuir film of GM 1 formed on an aqueous subphase comprising pH 7.5 TRIS buffer. Squares depict surface pressure-area conditions under which Langmuir-Schaefer transfer to the LC interface
    Figure Legend Snippet: (A) Surface pressure-area isotherm measured at 25°C for a Langmuir film of GM 1 formed on an aqueous subphase comprising pH 7.5 TRIS buffer. Squares depict surface pressure-area conditions under which Langmuir-Schaefer transfer to the LC interface

    Techniques Used:

    Quantification of BODIPY FL-GM 1 /GM 1 adsorbed at aqueous-5CB interfaces by fluorescence measurements at 25 °C (10µM GM 1 in pH 7.5 TRIS buffer). (A) Plot of epifluorescence intensity generated by mixed BODIPY FL-GM 1 /GM 1 assemblies adsorbed
    Figure Legend Snippet: Quantification of BODIPY FL-GM 1 /GM 1 adsorbed at aqueous-5CB interfaces by fluorescence measurements at 25 °C (10µM GM 1 in pH 7.5 TRIS buffer). (A) Plot of epifluorescence intensity generated by mixed BODIPY FL-GM 1 /GM 1 assemblies adsorbed

    Techniques Used: Fluorescence, Generated

    (A) Time required for GM 1 adsorption to trigger an ordering transition in nematic 5CB, plotted as a function of the temperature of the system. The GM 1 was adsorbed from a 10 µM GM 1 dispersion in pH 7.5 TRIS buffer. (B) Expanded view of A at the
    Figure Legend Snippet: (A) Time required for GM 1 adsorption to trigger an ordering transition in nematic 5CB, plotted as a function of the temperature of the system. The GM 1 was adsorbed from a 10 µM GM 1 dispersion in pH 7.5 TRIS buffer. (B) Expanded view of A at the

    Techniques Used: Adsorption

    24) Product Images from "Stabilization of a Membrane-Associated Amyloid-β Oligomer for Its Validation in Alzheimer's Disease"

    Article Title: Stabilization of a Membrane-Associated Amyloid-β Oligomer for Its Validation in Alzheimer's Disease

    Journal: Frontiers in Molecular Biosciences

    doi: 10.3389/fmolb.2018.00038

    β PFO Aβ 42 maintains its structural integrity after trapping in NAPols. SDS-PAGE analysis of β PFO Aβ 42 /DPC and β PFO Aβ 42 /NAPol before and after incubation with proteinase K. The β PFO Aβ 42 /DPC sample contains 150 μM nominal Aβ42 concentration in 10 mM Tris, 5.5 mM DPC at pH 9.0 and the β PFO Aβ 42 . Experiments have been repeated at least three times and the data shown is representative of them.
    Figure Legend Snippet: β PFO Aβ 42 maintains its structural integrity after trapping in NAPols. SDS-PAGE analysis of β PFO Aβ 42 /DPC and β PFO Aβ 42 /NAPol before and after incubation with proteinase K. The β PFO Aβ 42 /DPC sample contains 150 μM nominal Aβ42 concentration in 10 mM Tris, 5.5 mM DPC at pH 9.0 and the β PFO Aβ 42 . Experiments have been repeated at least three times and the data shown is representative of them.

    Techniques Used: SDS Page, Incubation, Concentration Assay

    The β PFO Aβ 42 / NAPol complex is stable under high dilution conditions. WB analysis of β PFO Aβ 42 /DPC complex after dilution at 1/32 in buffer (10 mM Tris at pH 7.4 and pH 9.0) containing or not 1.5 mM DPC and of β PFO Aβ 42 / NAPol complex after dilution at 1/32 in buffer (10 mM Tris at pH 7.4 and pH 9.0). The β PFO Aβ 42 /DPC sample contains 150 μM nominal Aβ42 concentration in 10 mM Tris, 5.5 mM DPC at pH 9.0 and the sample of β PFO Aβ 42 . Experiments have been repeated at least three times and the data shown is representative of them.
    Figure Legend Snippet: The β PFO Aβ 42 / NAPol complex is stable under high dilution conditions. WB analysis of β PFO Aβ 42 /DPC complex after dilution at 1/32 in buffer (10 mM Tris at pH 7.4 and pH 9.0) containing or not 1.5 mM DPC and of β PFO Aβ 42 / NAPol complex after dilution at 1/32 in buffer (10 mM Tris at pH 7.4 and pH 9.0). The β PFO Aβ 42 /DPC sample contains 150 μM nominal Aβ42 concentration in 10 mM Tris, 5.5 mM DPC at pH 9.0 and the sample of β PFO Aβ 42 . Experiments have been repeated at least three times and the data shown is representative of them.

    Techniques Used: Western Blot, Concentration Assay

    Optimization of trapping conditions of β PFO Aβ 42 in NAPols. SEC of β PFO Aβ 42 trapped in NAPols at 1:2 (blue line), 1:4 (orange line) and 1:8 (green line) Aβ42/NAPol ratios in a column equilibrated with a detergent-free buffer. After DPC removal, samples were analyzed (A) immediately and (B) after 24 h incubation at 37°C. β PFO Aβ 42 /NAPol contains 150 μM nominal Aβ42 concentration trapped at the indicated Aβ42/NAPol mass ratio in 10 mM Tris at pH 9.0. Experiments have been repeated at least three times and the data shown is representative of them.
    Figure Legend Snippet: Optimization of trapping conditions of β PFO Aβ 42 in NAPols. SEC of β PFO Aβ 42 trapped in NAPols at 1:2 (blue line), 1:4 (orange line) and 1:8 (green line) Aβ42/NAPol ratios in a column equilibrated with a detergent-free buffer. After DPC removal, samples were analyzed (A) immediately and (B) after 24 h incubation at 37°C. β PFO Aβ 42 /NAPol contains 150 μM nominal Aβ42 concentration trapped at the indicated Aβ42/NAPol mass ratio in 10 mM Tris at pH 9.0. Experiments have been repeated at least three times and the data shown is representative of them.

    Techniques Used: Size-exclusion Chromatography, Incubation, Concentration Assay

    β PFO Aβ 42 maintains its structural integrity after trapping in NAPols. 1 H- 13 C HMQC NMR spectrum of (A) a 150 μM Met 35 -[ 13 CH 3 ] Aβ42 sample dissolved in 9 mM Tris·DCl-d 12 , 1 mM Tris·DCl buffer in 100 % D 2 O at pH* 8.6, (B) a 150 μM Met 35 -[ 13 CH 3 ] Aβ42 sample dissolved in 9 mM Tris·DCl-d 12 , 1 mM Tris·DCl buffer in 100% D 2 O containing 46.4 mM SDS-d 25 at pH* 8.6, (C,D) a Met 35 -[ 13 CH 3 ] β PFO Aβ 42 /DPC complex (C) after immediate sample preparation and (D) after 24 h incubation at 37°C. The β PFO Aβ 42 /DPC sample contains 150 μM nominal Aβ42 concentration 10 mM Tris, 5.5 mM DPC at pH 9.0, and (E) a Met 35 -[ 13 CH 3 ] β PFO Aβ 42 /NAPol sample. The β PFO Aβ 42 /NAPol sample contains 150 μM nominal Aβ42 concentration, trapped with an Aβ42/NAPol mass ratio of 1:8 in 10 mM Tris at pH 9.0. Experiments have been repeated at least three times and the data shown is representative of them.
    Figure Legend Snippet: β PFO Aβ 42 maintains its structural integrity after trapping in NAPols. 1 H- 13 C HMQC NMR spectrum of (A) a 150 μM Met 35 -[ 13 CH 3 ] Aβ42 sample dissolved in 9 mM Tris·DCl-d 12 , 1 mM Tris·DCl buffer in 100 % D 2 O at pH* 8.6, (B) a 150 μM Met 35 -[ 13 CH 3 ] Aβ42 sample dissolved in 9 mM Tris·DCl-d 12 , 1 mM Tris·DCl buffer in 100% D 2 O containing 46.4 mM SDS-d 25 at pH* 8.6, (C,D) a Met 35 -[ 13 CH 3 ] β PFO Aβ 42 /DPC complex (C) after immediate sample preparation and (D) after 24 h incubation at 37°C. The β PFO Aβ 42 /DPC sample contains 150 μM nominal Aβ42 concentration 10 mM Tris, 5.5 mM DPC at pH 9.0, and (E) a Met 35 -[ 13 CH 3 ] β PFO Aβ 42 /NAPol sample. The β PFO Aβ 42 /NAPol sample contains 150 μM nominal Aβ42 concentration, trapped with an Aβ42/NAPol mass ratio of 1:8 in 10 mM Tris at pH 9.0. Experiments have been repeated at least three times and the data shown is representative of them.

    Techniques Used: Nuclear Magnetic Resonance, Sample Prep, Incubation, Concentration Assay

    β PFO Aβ 42 are stable only when trapped in NAPols. (A) SEC of β PFO Aβ 42 /DPC complex eluted in a column equilibrated with (top) and without (bottom) the presence of detergent in the elution buffer. SEC of β PFO Aβ 42 trapped in different types of APols: (B) A8-35, (C) SAPol, and (D) NAPol at 1:0.5 (gray line), 1:1 (cyan line) and 1:2 (blue line) Aβ42/APol ratios. After removal of DPC, samples were analyzed immediately (top) and after 24 h of incubation at 37°C (bottom) in a column equilibrated with a detergent- and APol-free buffer. β PFO Aβ 42 /DPC contains 150 μM nominal Aβ42 concentration in 10 mM Tris, 5.5 mM DPC at pH 9.0 and β PFO Aβ 42 /APol contains 150 μM nominal Aβ42 concentration trapped at the indicated Aβ42/APol mass ratio in 10 mM Tris at pH 9.0. Experiments have been repeated at least two times and the data shown is representative of them.
    Figure Legend Snippet: β PFO Aβ 42 are stable only when trapped in NAPols. (A) SEC of β PFO Aβ 42 /DPC complex eluted in a column equilibrated with (top) and without (bottom) the presence of detergent in the elution buffer. SEC of β PFO Aβ 42 trapped in different types of APols: (B) A8-35, (C) SAPol, and (D) NAPol at 1:0.5 (gray line), 1:1 (cyan line) and 1:2 (blue line) Aβ42/APol ratios. After removal of DPC, samples were analyzed immediately (top) and after 24 h of incubation at 37°C (bottom) in a column equilibrated with a detergent- and APol-free buffer. β PFO Aβ 42 /DPC contains 150 μM nominal Aβ42 concentration in 10 mM Tris, 5.5 mM DPC at pH 9.0 and β PFO Aβ 42 /APol contains 150 μM nominal Aβ42 concentration trapped at the indicated Aβ42/APol mass ratio in 10 mM Tris at pH 9.0. Experiments have been repeated at least two times and the data shown is representative of them.

    Techniques Used: Size-exclusion Chromatography, Incubation, Concentration Assay

    25) Product Images from "Refined Immunochemical Characterization in Healthy Dog Skin of the Epidermal Cornification Proteins, Filaggrin, and Corneodesmosin"

    Article Title: Refined Immunochemical Characterization in Healthy Dog Skin of the Epidermal Cornification Proteins, Filaggrin, and Corneodesmosin

    Journal: Journal of Histochemistry and Cytochemistry

    doi: 10.1369/0022155418798807

    Western blotting of dog epidermis extracts. Beagle (Bea) and golden (Gol) retriever dog epidermis was sequentially extracted in Tris-EDTA buffer containing either Nonidet-P40 (TE-NP40 extracts, NP) or 8 M urea (TE-U extracts, U). The extracted proteins were separated by SDS-PAGE, Coomassie blue stained, or immunodetected with AHF10 and G36-19 mAbs, as indicated. The immunoblot corresponding to AHF10 reactivity on the extracts of the beagle dog epidermis was exposed for a longer time (NP′ and U′) to highlight the absence of FLGa-corresponding band. The migration of molecular mass markers (m) is indicated on the left in kDa. ProFLG (Pro) and FLG monomers (FLGa and FLGb) are indicated by arrows, as well as the entire CDSN and its proteolytically derived fragments. For comparison, the Western blotting with AHF10 of TE-NP40 and TE-U extracts of human epidermis (Hum) is shown. Abbreviations: AHF, anti-human filaggrin; PAGE, polyacrylamide gel electrophoresis; FLG, Filaggrin; CDSN, corneodesmosin.
    Figure Legend Snippet: Western blotting of dog epidermis extracts. Beagle (Bea) and golden (Gol) retriever dog epidermis was sequentially extracted in Tris-EDTA buffer containing either Nonidet-P40 (TE-NP40 extracts, NP) or 8 M urea (TE-U extracts, U). The extracted proteins were separated by SDS-PAGE, Coomassie blue stained, or immunodetected with AHF10 and G36-19 mAbs, as indicated. The immunoblot corresponding to AHF10 reactivity on the extracts of the beagle dog epidermis was exposed for a longer time (NP′ and U′) to highlight the absence of FLGa-corresponding band. The migration of molecular mass markers (m) is indicated on the left in kDa. ProFLG (Pro) and FLG monomers (FLGa and FLGb) are indicated by arrows, as well as the entire CDSN and its proteolytically derived fragments. For comparison, the Western blotting with AHF10 of TE-NP40 and TE-U extracts of human epidermis (Hum) is shown. Abbreviations: AHF, anti-human filaggrin; PAGE, polyacrylamide gel electrophoresis; FLG, Filaggrin; CDSN, corneodesmosin.

    Techniques Used: Western Blot, SDS Page, Staining, Migration, Derivative Assay, Polyacrylamide Gel Electrophoresis

    26) Product Images from "Stabilization of a Membrane-Associated Amyloid-β Oligomer for Its Validation in Alzheimer's Disease"

    Article Title: Stabilization of a Membrane-Associated Amyloid-β Oligomer for Its Validation in Alzheimer's Disease

    Journal: Frontiers in Molecular Biosciences

    doi: 10.3389/fmolb.2018.00038

    β PFO Aβ 42 maintains its structural integrity after trapping in NAPols. SDS-PAGE analysis of β PFO Aβ 42 /DPC and β PFO Aβ 42 /NAPol before and after incubation with proteinase K. The β PFO Aβ 42 /DPC sample contains 150 μM nominal Aβ42 concentration in 10 mM Tris, 5.5 mM DPC at pH 9.0 and the β PFO Aβ 42 /NApol sample contains 150 μM nominal Aβ42 concentration trapped with a Aβ42/APol mass ratio of 1:8 in 10 mM Tris at pH 9.0. Original version of SDS-PAGE can be found in Figure S3 . Experiments have been repeated at least three times and the data shown is representative of them.
    Figure Legend Snippet: β PFO Aβ 42 maintains its structural integrity after trapping in NAPols. SDS-PAGE analysis of β PFO Aβ 42 /DPC and β PFO Aβ 42 /NAPol before and after incubation with proteinase K. The β PFO Aβ 42 /DPC sample contains 150 μM nominal Aβ42 concentration in 10 mM Tris, 5.5 mM DPC at pH 9.0 and the β PFO Aβ 42 /NApol sample contains 150 μM nominal Aβ42 concentration trapped with a Aβ42/APol mass ratio of 1:8 in 10 mM Tris at pH 9.0. Original version of SDS-PAGE can be found in Figure S3 . Experiments have been repeated at least three times and the data shown is representative of them.

    Techniques Used: SDS Page, Incubation, Concentration Assay

    The β PFO Aβ 42 / NAPol complex is stable under high dilution conditions. WB analysis of β PFO Aβ 42 /DPC complex after dilution at 1/32 in buffer (10 mM Tris at pH 7.4 and pH 9.0) containing or not 1.5 mM DPC and of β PFO Aβ 42 / NAPol complex after dilution at 1/32 in buffer (10 mM Tris at pH 7.4 and pH 9.0). The β PFO Aβ 42 /DPC sample contains 150 μM nominal Aβ42 concentration in 10 mM Tris, 5.5 mM DPC at pH 9.0 and the sample of β PFO Aβ 42 /NAPol contains 150 μM nominal Aβ42 concentration trapped with a Aβ42/NAPol mass ratio of 1:8 in 10 mM Tris at pH 9.0. Original version of WB can be found in Figure S3 . Experiments have been repeated at least three times and the data shown is representative of them.
    Figure Legend Snippet: The β PFO Aβ 42 / NAPol complex is stable under high dilution conditions. WB analysis of β PFO Aβ 42 /DPC complex after dilution at 1/32 in buffer (10 mM Tris at pH 7.4 and pH 9.0) containing or not 1.5 mM DPC and of β PFO Aβ 42 / NAPol complex after dilution at 1/32 in buffer (10 mM Tris at pH 7.4 and pH 9.0). The β PFO Aβ 42 /DPC sample contains 150 μM nominal Aβ42 concentration in 10 mM Tris, 5.5 mM DPC at pH 9.0 and the sample of β PFO Aβ 42 /NAPol contains 150 μM nominal Aβ42 concentration trapped with a Aβ42/NAPol mass ratio of 1:8 in 10 mM Tris at pH 9.0. Original version of WB can be found in Figure S3 . Experiments have been repeated at least three times and the data shown is representative of them.

    Techniques Used: Western Blot, Concentration Assay

    Optimization of trapping conditions of β PFO Aβ 42 in NAPols. SEC of β PFO Aβ 42 trapped in NAPols at 1:2 (blue line), 1:4 (orange line) and 1:8 (green line) Aβ42/NAPol ratios in a column equilibrated with a detergent-free buffer. After DPC removal, samples were analyzed (A) immediately and (B) after 24 h incubation at 37°C. β PFO Aβ 42 /NAPol contains 150 μM nominal Aβ42 concentration trapped at the indicated Aβ42/NAPol mass ratio in 10 mM Tris at pH 9.0. Experiments have been repeated at least three times and the data shown is representative of them.
    Figure Legend Snippet: Optimization of trapping conditions of β PFO Aβ 42 in NAPols. SEC of β PFO Aβ 42 trapped in NAPols at 1:2 (blue line), 1:4 (orange line) and 1:8 (green line) Aβ42/NAPol ratios in a column equilibrated with a detergent-free buffer. After DPC removal, samples were analyzed (A) immediately and (B) after 24 h incubation at 37°C. β PFO Aβ 42 /NAPol contains 150 μM nominal Aβ42 concentration trapped at the indicated Aβ42/NAPol mass ratio in 10 mM Tris at pH 9.0. Experiments have been repeated at least three times and the data shown is representative of them.

    Techniques Used: Size-exclusion Chromatography, Incubation, Concentration Assay

    β PFO Aβ 42 maintains its structural integrity after trapping in NAPols. 1 H- 13 C HMQC NMR spectrum of (A) a 150 μM Met 35 -[ 13 CH 3 ] Aβ42 sample dissolved in 9 mM Tris·DCl-d 12 , 1 mM Tris·DCl buffer in 100 % D 2 O at pH* 8.6, (B) a 150 μM Met 35 -[ 13 CH 3 ] Aβ42 sample dissolved in 9 mM Tris·DCl-d 12 , 1 mM Tris·DCl buffer in 100% D 2 O containing 46.4 mM SDS-d 25 at pH* 8.6, (C,D) a Met 35 -[ 13 CH 3 ] β PFO Aβ 42 /DPC complex (C) after immediate sample preparation and (D) after 24 h incubation at 37°C. The β PFO Aβ 42 /DPC sample contains 150 μM nominal Aβ42 concentration 10 mM Tris, 5.5 mM DPC at pH 9.0, and (E) a Met 35 -[ 13 CH 3 ] β PFO Aβ 42 /NAPol sample. The β PFO Aβ 42 /NAPol sample contains 150 μM nominal Aβ42 concentration, trapped with an Aβ42/NAPol mass ratio of 1:8 in 10 mM Tris at pH 9.0. Experiments have been repeated at least three times and the data shown is representative of them.
    Figure Legend Snippet: β PFO Aβ 42 maintains its structural integrity after trapping in NAPols. 1 H- 13 C HMQC NMR spectrum of (A) a 150 μM Met 35 -[ 13 CH 3 ] Aβ42 sample dissolved in 9 mM Tris·DCl-d 12 , 1 mM Tris·DCl buffer in 100 % D 2 O at pH* 8.6, (B) a 150 μM Met 35 -[ 13 CH 3 ] Aβ42 sample dissolved in 9 mM Tris·DCl-d 12 , 1 mM Tris·DCl buffer in 100% D 2 O containing 46.4 mM SDS-d 25 at pH* 8.6, (C,D) a Met 35 -[ 13 CH 3 ] β PFO Aβ 42 /DPC complex (C) after immediate sample preparation and (D) after 24 h incubation at 37°C. The β PFO Aβ 42 /DPC sample contains 150 μM nominal Aβ42 concentration 10 mM Tris, 5.5 mM DPC at pH 9.0, and (E) a Met 35 -[ 13 CH 3 ] β PFO Aβ 42 /NAPol sample. The β PFO Aβ 42 /NAPol sample contains 150 μM nominal Aβ42 concentration, trapped with an Aβ42/NAPol mass ratio of 1:8 in 10 mM Tris at pH 9.0. Experiments have been repeated at least three times and the data shown is representative of them.

    Techniques Used: Nuclear Magnetic Resonance, Sample Prep, Incubation, Concentration Assay

    β PFO Aβ 42 are stable only when trapped in NAPols. (A) SEC of β PFO Aβ 42 /DPC complex eluted in a column equilibrated with (top) and without (bottom) the presence of detergent in the elution buffer. SEC of β PFO Aβ 42 trapped in different types of APols: (B) A8-35, (C) SAPol, and (D) NAPol at 1:0.5 (gray line), 1:1 (cyan line) and 1:2 (blue line) Aβ42/APol ratios. After removal of DPC, samples were analyzed immediately (top) and after 24 h of incubation at 37°C (bottom) in a column equilibrated with a detergent- and APol-free buffer. β PFO Aβ 42 /DPC contains 150 μM nominal Aβ42 concentration in 10 mM Tris, 5.5 mM DPC at pH 9.0 and β PFO Aβ 42 /APol contains 150 μM nominal Aβ42 concentration trapped at the indicated Aβ42/APol mass ratio in 10 mM Tris at pH 9.0. Experiments have been repeated at least two times and the data shown is representative of them.
    Figure Legend Snippet: β PFO Aβ 42 are stable only when trapped in NAPols. (A) SEC of β PFO Aβ 42 /DPC complex eluted in a column equilibrated with (top) and without (bottom) the presence of detergent in the elution buffer. SEC of β PFO Aβ 42 trapped in different types of APols: (B) A8-35, (C) SAPol, and (D) NAPol at 1:0.5 (gray line), 1:1 (cyan line) and 1:2 (blue line) Aβ42/APol ratios. After removal of DPC, samples were analyzed immediately (top) and after 24 h of incubation at 37°C (bottom) in a column equilibrated with a detergent- and APol-free buffer. β PFO Aβ 42 /DPC contains 150 μM nominal Aβ42 concentration in 10 mM Tris, 5.5 mM DPC at pH 9.0 and β PFO Aβ 42 /APol contains 150 μM nominal Aβ42 concentration trapped at the indicated Aβ42/APol mass ratio in 10 mM Tris at pH 9.0. Experiments have been repeated at least two times and the data shown is representative of them.

    Techniques Used: Size-exclusion Chromatography, Incubation, Concentration Assay

    27) Product Images from "Study on the Interaction between Isatin-β-Thiosemicarbazone and Calf Thymus DNA by Spectroscopic Techniques"

    Article Title: Study on the Interaction between Isatin-β-Thiosemicarbazone and Calf Thymus DNA by Spectroscopic Techniques

    Journal: Iranian Journal of Pharmaceutical Research : IJPR

    doi:

    Circular dichroism spectra of DNA (8.0×10 -5 M) in 10 mM Tris-HCl buffer, in the presence of increasing amounts of IBT (ri = [IBT]/[DNA] = 0.0, 0.05, 0.4, and 0.7).
    Figure Legend Snippet: Circular dichroism spectra of DNA (8.0×10 -5 M) in 10 mM Tris-HCl buffer, in the presence of increasing amounts of IBT (ri = [IBT]/[DNA] = 0.0, 0.05, 0.4, and 0.7).

    Techniques Used:

    Absorption spectra of the IBT in 0.01 M Tris-HCl buffer (pH 7.4) at room temperature in the presence of increasing amounts of CT-DNA. [IBT] = 50 µM, [DNA] = 0–80 µM from top to the bottom. Arrows indicate the change in absorbance upon increasing the DNA concentration. Inset: plot of [DNA]/εa-εb vs. [DNA]
    Figure Legend Snippet: Absorption spectra of the IBT in 0.01 M Tris-HCl buffer (pH 7.4) at room temperature in the presence of increasing amounts of CT-DNA. [IBT] = 50 µM, [DNA] = 0–80 µM from top to the bottom. Arrows indicate the change in absorbance upon increasing the DNA concentration. Inset: plot of [DNA]/εa-εb vs. [DNA]

    Techniques Used: Concentration Assay

    Effect of increasing amounts of IBT on the viscosity of calf thymus DNA (5× 10 -5 M) in 10 mM Tris–HCl buffer (pH 7.4) at 298K (ri = [IBT]/[DNA] = 0.0, 0.5, 1, 1.2, 1.5, 1.8, and 2).
    Figure Legend Snippet: Effect of increasing amounts of IBT on the viscosity of calf thymus DNA (5× 10 -5 M) in 10 mM Tris–HCl buffer (pH 7.4) at 298K (ri = [IBT]/[DNA] = 0.0, 0.5, 1, 1.2, 1.5, 1.8, and 2).

    Techniques Used:

    28) Product Images from "Structural insight into toxin secretion by contact-dependent growth inhibition transporters"

    Article Title: Structural insight into toxin secretion by contact-dependent growth inhibition transporters

    Journal: eLife

    doi: 10.7554/eLife.58100

    Expression of CdiB Ab double cysteine variant. ( A ) Western blot of isolated membrane fraction from CdiB D225C/F554C double cysteine variant (represented by bold letters A to E). Fraction A, bacterial pellet fraction (‘P ‘lane from Figure 5A ). Fraction B, bacterial lysate fraction homogenized three times. Fraction C, supernatant fraction after centrifugation at 7000 g (where inclusion bodies and unlysed cells are removed). Fraction D, pellet from ultracentrifugation at 160,000 g to isolate membrane fractions after a 2% Triton wash (which removes soluble and inner membrane proteins). Membrane fractions are resuspended in 50 mM Tris-HCl, pH7.4, 200 mM NaCl, and solubilized by constant stirring in 5% Elugent. Fraction E, supernatant of a second ultracentrifugation at 220,000 g containing solubilized membrane proteins. As seen in Figure 5 , β1–β16 are mostly crosslinked in the D225C-F554C mutant where ‘ox’ and ‘red’ indicate the oxidized and reduced form, respectively. Protein ladder bands indicate 70 and 50 kDa. ( B ) Western blot analysis of bacterial pellet from CdiB Ab D225C/F554C variant expressed in MC4100 E. coli parent cell (dsbA ‘+’) and MC4100 E. coli dsbA:ChloroR strain (dsbA ‘-‘), in absence (second lane) or presence of TCEP (‘+TCEP’).
    Figure Legend Snippet: Expression of CdiB Ab double cysteine variant. ( A ) Western blot of isolated membrane fraction from CdiB D225C/F554C double cysteine variant (represented by bold letters A to E). Fraction A, bacterial pellet fraction (‘P ‘lane from Figure 5A ). Fraction B, bacterial lysate fraction homogenized three times. Fraction C, supernatant fraction after centrifugation at 7000 g (where inclusion bodies and unlysed cells are removed). Fraction D, pellet from ultracentrifugation at 160,000 g to isolate membrane fractions after a 2% Triton wash (which removes soluble and inner membrane proteins). Membrane fractions are resuspended in 50 mM Tris-HCl, pH7.4, 200 mM NaCl, and solubilized by constant stirring in 5% Elugent. Fraction E, supernatant of a second ultracentrifugation at 220,000 g containing solubilized membrane proteins. As seen in Figure 5 , β1–β16 are mostly crosslinked in the D225C-F554C mutant where ‘ox’ and ‘red’ indicate the oxidized and reduced form, respectively. Protein ladder bands indicate 70 and 50 kDa. ( B ) Western blot analysis of bacterial pellet from CdiB Ab D225C/F554C variant expressed in MC4100 E. coli parent cell (dsbA ‘+’) and MC4100 E. coli dsbA:ChloroR strain (dsbA ‘-‘), in absence (second lane) or presence of TCEP (‘+TCEP’).

    Techniques Used: Expressing, Variant Assay, Western Blot, Isolation, Centrifugation, Mutagenesis

    29) Product Images from "Estrogen receptor α dependent regulation of estrogen related receptor β and its role in cell cycle in breast cancer"

    Article Title: Estrogen receptor α dependent regulation of estrogen related receptor β and its role in cell cycle in breast cancer

    Journal: BMC Cancer

    doi: 10.1186/s12885-018-4528-x

    ERα interacts to ERRβ promoter in-vitro . a Schematic representation of two functional half ERE sites present in ERRβ promoter. Half ERE sites were situated from − 877 to − 872 and − 810 to − 805 respectively in the upstream region of ERRβ promoter. b Electrophoretic mobility shift assay (EMSA) representing the binding of ERα on both the half ERE sites in ERRβ promoter region. Oligonucleotides including half ERE site were labeled with [γ − 32 P] ATP and were incubated for 20 min with nuclear lysate extracted from MCF7 cells. An unlabeled ERE consensus oligonucleotide sequences were used as cold probe for competition at 50, 100 and 500 folds molar excess. Oligonucleotides were separated in 6% polyacrylamide gel using 0.5X TBE (Tris/Borate/Ethylenediaminetetraacetic acid) for 1 h at 180 V. The gel was dried and was autoradiographed
    Figure Legend Snippet: ERα interacts to ERRβ promoter in-vitro . a Schematic representation of two functional half ERE sites present in ERRβ promoter. Half ERE sites were situated from − 877 to − 872 and − 810 to − 805 respectively in the upstream region of ERRβ promoter. b Electrophoretic mobility shift assay (EMSA) representing the binding of ERα on both the half ERE sites in ERRβ promoter region. Oligonucleotides including half ERE site were labeled with [γ − 32 P] ATP and were incubated for 20 min with nuclear lysate extracted from MCF7 cells. An unlabeled ERE consensus oligonucleotide sequences were used as cold probe for competition at 50, 100 and 500 folds molar excess. Oligonucleotides were separated in 6% polyacrylamide gel using 0.5X TBE (Tris/Borate/Ethylenediaminetetraacetic acid) for 1 h at 180 V. The gel was dried and was autoradiographed

    Techniques Used: In Vitro, Functional Assay, Electrophoretic Mobility Shift Assay, Binding Assay, Labeling, Incubation

    30) Product Images from "Cyclic oligoadenylate signalling mediates Mycobacterium tuberculosis CRISPR defence"

    Article Title: Cyclic oligoadenylate signalling mediates Mycobacterium tuberculosis CRISPR defence

    Journal: bioRxiv

    doi: 10.1101/667758

    Re-programming the Mtb Csm system for cA 4 -responsive immunity. ( A ) In vitro activity of TsuCsx1. The reaction contained 0.5 µM TsuCsx1 dimer, 100 nM 5’- 32 P-labeled RNA A1, 20 mM Tris, 150 mM NaCl, 1 mM DTT, pH 7.5 and was conducted at 35 °C for 1, 5, 15, 30 min. Activators were added as indicated; Csm-derived cOAs were from a 2 h reaction, otherwise as described for Figure 3 . TsuCsx1 was activated by cA 4 but not cA 6 , and Csm-derived cOAs are able to induce Csx1 ribonuclease activity in vitro . ( B ) Plasmid immunity assay using pUC19 lacZα -targeting Csm effector complex in E. coli C43 (pCsm1-5_Csm6/tsuCsx1 and pCRISPR, Figure S4 ). The ribonuclease was either the cognate Csm6 or TsuCsx1. Cells were transformed with pRAT (control plasmid) or pRAT-Target (target plasmid) and 10-fold serial dilutions were plated on selective plates containing arabinose for induction of target transcription. TsuCsx1 confers the same level of plasmid immunity as the cognate Mtb Csm6.
    Figure Legend Snippet: Re-programming the Mtb Csm system for cA 4 -responsive immunity. ( A ) In vitro activity of TsuCsx1. The reaction contained 0.5 µM TsuCsx1 dimer, 100 nM 5’- 32 P-labeled RNA A1, 20 mM Tris, 150 mM NaCl, 1 mM DTT, pH 7.5 and was conducted at 35 °C for 1, 5, 15, 30 min. Activators were added as indicated; Csm-derived cOAs were from a 2 h reaction, otherwise as described for Figure 3 . TsuCsx1 was activated by cA 4 but not cA 6 , and Csm-derived cOAs are able to induce Csx1 ribonuclease activity in vitro . ( B ) Plasmid immunity assay using pUC19 lacZα -targeting Csm effector complex in E. coli C43 (pCsm1-5_Csm6/tsuCsx1 and pCRISPR, Figure S4 ). The ribonuclease was either the cognate Csm6 or TsuCsx1. Cells were transformed with pRAT (control plasmid) or pRAT-Target (target plasmid) and 10-fold serial dilutions were plated on selective plates containing arabinose for induction of target transcription. TsuCsx1 confers the same level of plasmid immunity as the cognate Mtb Csm6.

    Techniques Used: In Vitro, Activity Assay, Labeling, Derivative Assay, Plasmid Preparation, Transformation Assay

    31) Product Images from "Structural and Physiological Analyses of the Alkanesulphonate-Binding Protein (SsuA) of the Citrus Pathogen Xanthomonas citri"

    Article Title: Structural and Physiological Analyses of the Alkanesulphonate-Binding Protein (SsuA) of the Citrus Pathogen Xanthomonas citri

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0080083

    Crystallisation, X-ray diffraction pattern and determination of the tertiary structure of X. citri SsuA. (A) SsuA crystals grown in 0.1 M HEPES, pH 7.3, 1.5 M ammonium sulphate and 0.1 NaCl using 6 mg/ml of protein in 20 mM Tris buffer, pH 7.0, containing 50 mM NaCl. (B) Diffraction pattern of SsuA crystal at 2.0 Å resolution. Data were collected at the D03B-MX1 beam line Brazilian Synchrotron Light Laboratory (LNLS) using 1.433 Å radiation and recorded on a MARCCD 165 detector (oscillation data with Δφ = 1.0 o ). (C) Cartoon illustration of the overall structure of SsuA bound to HEPES, MOPS and MES (stick) showing the alpha-beta structures of domains I (deep blue and cyan) and II (orange and yellow). The N-terminus is shown in domain I.
    Figure Legend Snippet: Crystallisation, X-ray diffraction pattern and determination of the tertiary structure of X. citri SsuA. (A) SsuA crystals grown in 0.1 M HEPES, pH 7.3, 1.5 M ammonium sulphate and 0.1 NaCl using 6 mg/ml of protein in 20 mM Tris buffer, pH 7.0, containing 50 mM NaCl. (B) Diffraction pattern of SsuA crystal at 2.0 Å resolution. Data were collected at the D03B-MX1 beam line Brazilian Synchrotron Light Laboratory (LNLS) using 1.433 Å radiation and recorded on a MARCCD 165 detector (oscillation data with Δφ = 1.0 o ). (C) Cartoon illustration of the overall structure of SsuA bound to HEPES, MOPS and MES (stick) showing the alpha-beta structures of domains I (deep blue and cyan) and II (orange and yellow). The N-terminus is shown in domain I.

    Techniques Used:

    32) Product Images from "DNA Aptamers against Exon v10 of CD44 Inhibit Breast Cancer Cell Migration"

    Article Title: DNA Aptamers against Exon v10 of CD44 Inhibit Breast Cancer Cell Migration

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0088712

    CD44 associated with EphA2 on breast cancer cells. (A) HCC38 cells were lysed with 100 mM Tris-HCl (pH 7.5) containing 1% Brij35, 0.14 M NaCl, 1 mM CaCl 2 , 1 mM MnCl 2 , and a protease inhibitor cocktail by scraping and pipetting. The cell lysates were precleared and immunoprecipitated with anti-CD44 antibody clone (clone 156-3C11)-, anti-EphA2- or control IgG-protein G beads for 4 hours at 4°C. The bound proteins were released by boiling at 95°C in SDS-sample buffer under reducing conditions and separated on SDS-PAGE followed by western blotting with anti-EphA2 antibody followed by HRP-secondary antibody. The proteins are visualized with enhanced chemiluminescence (ECL) reaction. (B) Cells were harvested using 5 mM EDTA in PBS and were lysed in 100 mM Tris-HCL (pH 7.5) containing 1% Brij35, 0.14 M NaCl, 1 mM CaCl 2 , 1 mM MnCl 2 , and a protease inhibitor cocktail. The lysates were precleared with anti-FLAG (M2) agarose beads for 4 hours by shaking at 4°C. The cleared lysates were incubated with recombinant protein CD44v10 P-FLAG-anti-FLAG (M2) antibody-agarose beads (CD44v10) or anti-FLAG (M2) antibody-agarose beads (Con.) in the presence or absence of aptamers (Apt#4 and Apt#7) by shaking at 4°C for 4 hours. The beads were washed extensively with the lysis buffer. The bound proteins were released by boiling at 95°C in SDS-sample buffer under reducing conditions and separated on SDS-PAGE followed by western blotting with anti-EphA2 followed by HRP-secondary antibody or HRP-conjugated anti-FLAG (M2) antibody. The proteins are visualized with enhanced chemiluminescence (ECL) reaction. CD44 exon v10 peptide was used as a loading control for ensuring the same amount of proteins was loaded in each lane.
    Figure Legend Snippet: CD44 associated with EphA2 on breast cancer cells. (A) HCC38 cells were lysed with 100 mM Tris-HCl (pH 7.5) containing 1% Brij35, 0.14 M NaCl, 1 mM CaCl 2 , 1 mM MnCl 2 , and a protease inhibitor cocktail by scraping and pipetting. The cell lysates were precleared and immunoprecipitated with anti-CD44 antibody clone (clone 156-3C11)-, anti-EphA2- or control IgG-protein G beads for 4 hours at 4°C. The bound proteins were released by boiling at 95°C in SDS-sample buffer under reducing conditions and separated on SDS-PAGE followed by western blotting with anti-EphA2 antibody followed by HRP-secondary antibody. The proteins are visualized with enhanced chemiluminescence (ECL) reaction. (B) Cells were harvested using 5 mM EDTA in PBS and were lysed in 100 mM Tris-HCL (pH 7.5) containing 1% Brij35, 0.14 M NaCl, 1 mM CaCl 2 , 1 mM MnCl 2 , and a protease inhibitor cocktail. The lysates were precleared with anti-FLAG (M2) agarose beads for 4 hours by shaking at 4°C. The cleared lysates were incubated with recombinant protein CD44v10 P-FLAG-anti-FLAG (M2) antibody-agarose beads (CD44v10) or anti-FLAG (M2) antibody-agarose beads (Con.) in the presence or absence of aptamers (Apt#4 and Apt#7) by shaking at 4°C for 4 hours. The beads were washed extensively with the lysis buffer. The bound proteins were released by boiling at 95°C in SDS-sample buffer under reducing conditions and separated on SDS-PAGE followed by western blotting with anti-EphA2 followed by HRP-secondary antibody or HRP-conjugated anti-FLAG (M2) antibody. The proteins are visualized with enhanced chemiluminescence (ECL) reaction. CD44 exon v10 peptide was used as a loading control for ensuring the same amount of proteins was loaded in each lane.

    Techniques Used: Protease Inhibitor, Immunoprecipitation, SDS Page, Western Blot, Incubation, Recombinant, Lysis

    33) Product Images from "Structural and functional insight into the mechanism of an alkaline exonuclease from Laribacter hongkongensis"

    Article Title: Structural and functional insight into the mechanism of an alkaline exonuclease from Laribacter hongkongensis

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr660

    Qualitative analysis of ssDNA and dsDNA hydrolysis activities of LHK-Exo. ( A ) dsDNA exonuclease activities. Agarose gel showing aliquots taken (0–15 min) from an incubation of LHK-Exo (30 µg, 0.41 nmol of trimers) and BamHI-linearized pET28a (1.8 µg, 0.54 pmol) in Tris–HCl (pH 8.0, 50 mM), 50 mM NaCl, 7.5 mM MgCl 2 at 37°C. ( B ) Polarity of dsDNA digestion. A total of 6 µg of LHK-Exo (82 pmol of trimers, lanes 2–5) or λ-exonuclease (74 pmol of trimers, lanes 6–9) protein was incubated with 0.1 µg (0.23 pmol) of a 712-bp linear 5′-phosphorylated dsDNA substrate (‘unmodified’; lanes 2, 3, 6 and 7), or an analogous 5′-phosphorylated linear dsDNA substrate containing three consecutive ‘nuclease-resistant’ phosphorothioate linkages at its 5′-termini (‘PT-modified’; lanes 4, 5, 8, 9). Assays were quenched immediately (0 min) or incubated at 37°C for 20 min, before analysis of digestion products on 1% agarose gels. ( C ) Digestion of 5′-phosphorylated ssDNA. Reaction mixtures (80 µl) containing LHK-Exo (4.5 µg, 61.4 pmol of trimers) and 5′-PO 4 -(dT) 50 (0.4 nmol) in 25 mM Tris–HCl (pH 8.0), 7.5 mM MgCl 2 , 1 mM DTT were incubated at 37°C. Aliquots (20 µl) were withdrawn after 0, 0.5, 5 and 20 min, and immediately quenched. Reaction products were analyzed by denaturing gel electrophoresis. ( D ) Digestion of non-phosphorylated ssDNA. Analogous sets of assays were performed using non-phosphorylated 50-mers of oligothymidine [5′-OH-(dT) 50 ]. Fluorescent gel images were scanned after SYBR Gold staining. A ssDNA ladder [Oligo Length Standards 20/100 Ladder (IDT)] is included in lane 1.
    Figure Legend Snippet: Qualitative analysis of ssDNA and dsDNA hydrolysis activities of LHK-Exo. ( A ) dsDNA exonuclease activities. Agarose gel showing aliquots taken (0–15 min) from an incubation of LHK-Exo (30 µg, 0.41 nmol of trimers) and BamHI-linearized pET28a (1.8 µg, 0.54 pmol) in Tris–HCl (pH 8.0, 50 mM), 50 mM NaCl, 7.5 mM MgCl 2 at 37°C. ( B ) Polarity of dsDNA digestion. A total of 6 µg of LHK-Exo (82 pmol of trimers, lanes 2–5) or λ-exonuclease (74 pmol of trimers, lanes 6–9) protein was incubated with 0.1 µg (0.23 pmol) of a 712-bp linear 5′-phosphorylated dsDNA substrate (‘unmodified’; lanes 2, 3, 6 and 7), or an analogous 5′-phosphorylated linear dsDNA substrate containing three consecutive ‘nuclease-resistant’ phosphorothioate linkages at its 5′-termini (‘PT-modified’; lanes 4, 5, 8, 9). Assays were quenched immediately (0 min) or incubated at 37°C for 20 min, before analysis of digestion products on 1% agarose gels. ( C ) Digestion of 5′-phosphorylated ssDNA. Reaction mixtures (80 µl) containing LHK-Exo (4.5 µg, 61.4 pmol of trimers) and 5′-PO 4 -(dT) 50 (0.4 nmol) in 25 mM Tris–HCl (pH 8.0), 7.5 mM MgCl 2 , 1 mM DTT were incubated at 37°C. Aliquots (20 µl) were withdrawn after 0, 0.5, 5 and 20 min, and immediately quenched. Reaction products were analyzed by denaturing gel electrophoresis. ( D ) Digestion of non-phosphorylated ssDNA. Analogous sets of assays were performed using non-phosphorylated 50-mers of oligothymidine [5′-OH-(dT) 50 ]. Fluorescent gel images were scanned after SYBR Gold staining. A ssDNA ladder [Oligo Length Standards 20/100 Ladder (IDT)] is included in lane 1.

    Techniques Used: Agarose Gel Electrophoresis, Incubation, Modification, Nucleic Acid Electrophoresis, Staining

    Determination of optimum conditions for LHK-Exo dsDNA digestion activities. PicoGreen fluorescence assays were performed to quantify the amounts of a representative 5′-phosphorylatd dsDNA substrate (PstI-linearized pUC18 that were digested by LHK-Exo under various conditions (reported as a percentage of the initial quantities of DNA). ( A and B ) Optimal concentrations of Mg 2+ and Mn 2+ ions. Amounts of PstI-linearized pUC18 (30 ng, 0.018 pmol) digested by LHK-Exo (8 ng, 0.11 pmol of trimers) in Tris–HCl (25 mM, pH 8.0) containing varying concentrations of Mg 2+ ions (1–15 mM, A) or Mn 2+ ions (0.1–1.7 mM, B), respectively, upon incubation at 25°C for 20 min. ( C ) Optimal pH. Amounts of PstI-linearized pUC18 (30 ng, 0.018 pmol) digested by LHK-Exo (8 ng, 0.11 pmol of trimers) in 25 mM Tris–HCl, 7.5 mM MgCl 2 ; adjusted to the appropriate pH value (pH 7.0–9.0); upon incubation at 25°C for 20 min. ( D ) Optimal temperature. Amounts of PstI-linearized pUC18 (30 ng, 0.018 pmol) digested by LHK-Exo (40 ng, 0.55 pmol of trimers) in Tris–HCl (25 mM, pH 8.0), 7.5 mM MgCl 2 , after incubation for 1 min at the indicated temperature (34–54°C). Four to six independent replicates of each experimental condition were performed, and data are reported as the mean ± standard deviation.
    Figure Legend Snippet: Determination of optimum conditions for LHK-Exo dsDNA digestion activities. PicoGreen fluorescence assays were performed to quantify the amounts of a representative 5′-phosphorylatd dsDNA substrate (PstI-linearized pUC18 that were digested by LHK-Exo under various conditions (reported as a percentage of the initial quantities of DNA). ( A and B ) Optimal concentrations of Mg 2+ and Mn 2+ ions. Amounts of PstI-linearized pUC18 (30 ng, 0.018 pmol) digested by LHK-Exo (8 ng, 0.11 pmol of trimers) in Tris–HCl (25 mM, pH 8.0) containing varying concentrations of Mg 2+ ions (1–15 mM, A) or Mn 2+ ions (0.1–1.7 mM, B), respectively, upon incubation at 25°C for 20 min. ( C ) Optimal pH. Amounts of PstI-linearized pUC18 (30 ng, 0.018 pmol) digested by LHK-Exo (8 ng, 0.11 pmol of trimers) in 25 mM Tris–HCl, 7.5 mM MgCl 2 ; adjusted to the appropriate pH value (pH 7.0–9.0); upon incubation at 25°C for 20 min. ( D ) Optimal temperature. Amounts of PstI-linearized pUC18 (30 ng, 0.018 pmol) digested by LHK-Exo (40 ng, 0.55 pmol of trimers) in Tris–HCl (25 mM, pH 8.0), 7.5 mM MgCl 2 , after incubation for 1 min at the indicated temperature (34–54°C). Four to six independent replicates of each experimental condition were performed, and data are reported as the mean ± standard deviation.

    Techniques Used: Fluorescence, Incubation, Standard Deviation

    Processivity of double strand DNA digestion by wild-type LHK-Exo and Arg12Ala mutant. Time course analysis of the digestion of 5′-phosphorylated double strand DNA (5′-PO 4 -dsDNA: EcoRV-linearized pMal-c2) and 5′-dephosphorylated double strand DNA (5′-OH-dsDNA: 5′dephosphorylated EcoRV-linearized pMal-c2) substrates by wild-type LHK-Exo and the Arg12Ala mutant form using a ‘heparin trap’ approach. A total of 6 µg (82 pmol of trimers) of LHK-Exo or Arg12Ala mutant protein was incubated at 25°C with 60 ng (0.015 pmol) of 5′-PO 4 -dsDNA or 5′-OH-dsDNA in Tris–HCl (25 mM, pH 8.0), 1 mM DTT, 7.5 mM MgCl 2 . After 15 s, excess heparin was added to sequester all unbound protein, and to prevent disassociated protein from re-binding. Aliquots were removed at various time points (0–20 min), and dsDNA levels were determined using fluorescent PicoGreen assays, to enable the extent of DNA digestion to be calculated. In one set of assays, heparin was added to LHK-Exo prior to the addition of dsDNA substrate, to confirm the efficacy of the heparin trap method (filled black squares, green line). Graphs show the mean number of nucleotides digested from each terminus (±SD; y -axis) plotted against the time of analysis (in minutes; x -axis).
    Figure Legend Snippet: Processivity of double strand DNA digestion by wild-type LHK-Exo and Arg12Ala mutant. Time course analysis of the digestion of 5′-phosphorylated double strand DNA (5′-PO 4 -dsDNA: EcoRV-linearized pMal-c2) and 5′-dephosphorylated double strand DNA (5′-OH-dsDNA: 5′dephosphorylated EcoRV-linearized pMal-c2) substrates by wild-type LHK-Exo and the Arg12Ala mutant form using a ‘heparin trap’ approach. A total of 6 µg (82 pmol of trimers) of LHK-Exo or Arg12Ala mutant protein was incubated at 25°C with 60 ng (0.015 pmol) of 5′-PO 4 -dsDNA or 5′-OH-dsDNA in Tris–HCl (25 mM, pH 8.0), 1 mM DTT, 7.5 mM MgCl 2 . After 15 s, excess heparin was added to sequester all unbound protein, and to prevent disassociated protein from re-binding. Aliquots were removed at various time points (0–20 min), and dsDNA levels were determined using fluorescent PicoGreen assays, to enable the extent of DNA digestion to be calculated. In one set of assays, heparin was added to LHK-Exo prior to the addition of dsDNA substrate, to confirm the efficacy of the heparin trap method (filled black squares, green line). Graphs show the mean number of nucleotides digested from each terminus (±SD; y -axis) plotted against the time of analysis (in minutes; x -axis).

    Techniques Used: Mutagenesis, Incubation, Binding Assay

    Qualitative analysis of ssDNA and dsDNA hydrolysis activities of LHK-Exo. ( A ) dsDNA exonuclease activities. Agarose gel showing aliquots taken (0–15 min) from an incubation of LHK-Exo (30 µg, 0.41 nmol of trimers) and BamHI-linearized pET28a (1.8 µg, 0.54 pmol) in Tris–HCl (pH 8.0, 50 mM), 50 mM NaCl, 7.5 mM MgCl 2 at 37°C. ( B ) Polarity of dsDNA digestion. A total of 6 µg of LHK-Exo (82 pmol of trimers, lanes 2–5) or λ-exonuclease (74 pmol of trimers, lanes 6–9) protein was incubated with 0.1 µg (0.23 pmol) of a 712-bp linear 5′-phosphorylated dsDNA substrate (‘unmodified’; lanes 2, 3, 6 and 7), or an analogous 5′-phosphorylated linear dsDNA substrate containing three consecutive ‘nuclease-resistant’ phosphorothioate linkages at its 5′-termini (‘PT-modified’; lanes 4, 5, 8, 9). Assays were quenched immediately (0 min) or incubated at 37°C for 20 min, before analysis of digestion products on 1% agarose gels. ( C ) Digestion of 5′-phosphorylated ssDNA. Reaction mixtures (80 µl) containing LHK-Exo (4.5 µg, 61.4 pmol of trimers) and 5′-PO 4 -(dT) 50 (0.4 nmol) in 25 mM Tris–HCl (pH 8.0), 7.5 mM MgCl 2 , 1 mM DTT were incubated at 37°C. Aliquots (20 µl) were withdrawn after 0, 0.5, 5 and 20 min, and immediately quenched. Reaction products were analyzed by denaturing gel electrophoresis. ( D ) Digestion of non-phosphorylated ssDNA. Analogous sets of assays were performed using non-phosphorylated 50-mers of oligothymidine [5′-OH-(dT) 50 ]. Fluorescent gel images were scanned after SYBR Gold staining. A ssDNA ladder [Oligo Length Standards 20/100 Ladder (IDT)] is included in lane 1.
    Figure Legend Snippet: Qualitative analysis of ssDNA and dsDNA hydrolysis activities of LHK-Exo. ( A ) dsDNA exonuclease activities. Agarose gel showing aliquots taken (0–15 min) from an incubation of LHK-Exo (30 µg, 0.41 nmol of trimers) and BamHI-linearized pET28a (1.8 µg, 0.54 pmol) in Tris–HCl (pH 8.0, 50 mM), 50 mM NaCl, 7.5 mM MgCl 2 at 37°C. ( B ) Polarity of dsDNA digestion. A total of 6 µg of LHK-Exo (82 pmol of trimers, lanes 2–5) or λ-exonuclease (74 pmol of trimers, lanes 6–9) protein was incubated with 0.1 µg (0.23 pmol) of a 712-bp linear 5′-phosphorylated dsDNA substrate (‘unmodified’; lanes 2, 3, 6 and 7), or an analogous 5′-phosphorylated linear dsDNA substrate containing three consecutive ‘nuclease-resistant’ phosphorothioate linkages at its 5′-termini (‘PT-modified’; lanes 4, 5, 8, 9). Assays were quenched immediately (0 min) or incubated at 37°C for 20 min, before analysis of digestion products on 1% agarose gels. ( C ) Digestion of 5′-phosphorylated ssDNA. Reaction mixtures (80 µl) containing LHK-Exo (4.5 µg, 61.4 pmol of trimers) and 5′-PO 4 -(dT) 50 (0.4 nmol) in 25 mM Tris–HCl (pH 8.0), 7.5 mM MgCl 2 , 1 mM DTT were incubated at 37°C. Aliquots (20 µl) were withdrawn after 0, 0.5, 5 and 20 min, and immediately quenched. Reaction products were analyzed by denaturing gel electrophoresis. ( D ) Digestion of non-phosphorylated ssDNA. Analogous sets of assays were performed using non-phosphorylated 50-mers of oligothymidine [5′-OH-(dT) 50 ]. Fluorescent gel images were scanned after SYBR Gold staining. A ssDNA ladder [Oligo Length Standards 20/100 Ladder (IDT)] is included in lane 1.

    Techniques Used: Agarose Gel Electrophoresis, Incubation, Modification, Nucleic Acid Electrophoresis, Staining

    Determination of optimum conditions for LHK-Exo dsDNA digestion activities. PicoGreen fluorescence assays were performed to quantify the amounts of a representative 5′-phosphorylatd dsDNA substrate (PstI-linearized pUC18 that were digested by LHK-Exo under various conditions (reported as a percentage of the initial quantities of DNA). ( A and B ) Optimal concentrations of Mg 2+ and Mn 2+ ions. Amounts of PstI-linearized pUC18 (30 ng, 0.018 pmol) digested by LHK-Exo (8 ng, 0.11 pmol of trimers) in Tris–HCl (25 mM, pH 8.0) containing varying concentrations of Mg 2+ ions (1–15 mM, A) or Mn 2+ ions (0.1–1.7 mM, B), respectively, upon incubation at 25°C for 20 min. ( C ) Optimal pH. Amounts of PstI-linearized pUC18 (30 ng, 0.018 pmol) digested by LHK-Exo (8 ng, 0.11 pmol of trimers) in 25 mM Tris–HCl, 7.5 mM MgCl 2 ; adjusted to the appropriate pH value (pH 7.0–9.0); upon incubation at 25°C for 20 min. ( D ) Optimal temperature. Amounts of PstI-linearized pUC18 (30 ng, 0.018 pmol) digested by LHK-Exo (40 ng, 0.55 pmol of trimers) in Tris–HCl (25 mM, pH 8.0), 7.5 mM MgCl 2 , after incubation for 1 min at the indicated temperature (34–54°C). Four to six independent replicates of each experimental condition were performed, and data are reported as the mean ± standard deviation.
    Figure Legend Snippet: Determination of optimum conditions for LHK-Exo dsDNA digestion activities. PicoGreen fluorescence assays were performed to quantify the amounts of a representative 5′-phosphorylatd dsDNA substrate (PstI-linearized pUC18 that were digested by LHK-Exo under various conditions (reported as a percentage of the initial quantities of DNA). ( A and B ) Optimal concentrations of Mg 2+ and Mn 2+ ions. Amounts of PstI-linearized pUC18 (30 ng, 0.018 pmol) digested by LHK-Exo (8 ng, 0.11 pmol of trimers) in Tris–HCl (25 mM, pH 8.0) containing varying concentrations of Mg 2+ ions (1–15 mM, A) or Mn 2+ ions (0.1–1.7 mM, B), respectively, upon incubation at 25°C for 20 min. ( C ) Optimal pH. Amounts of PstI-linearized pUC18 (30 ng, 0.018 pmol) digested by LHK-Exo (8 ng, 0.11 pmol of trimers) in 25 mM Tris–HCl, 7.5 mM MgCl 2 ; adjusted to the appropriate pH value (pH 7.0–9.0); upon incubation at 25°C for 20 min. ( D ) Optimal temperature. Amounts of PstI-linearized pUC18 (30 ng, 0.018 pmol) digested by LHK-Exo (40 ng, 0.55 pmol of trimers) in Tris–HCl (25 mM, pH 8.0), 7.5 mM MgCl 2 , after incubation for 1 min at the indicated temperature (34–54°C). Four to six independent replicates of each experimental condition were performed, and data are reported as the mean ± standard deviation.

    Techniques Used: Fluorescence, Incubation, Standard Deviation

    Processivity of double strand DNA digestion by wild-type LHK-Exo and Arg12Ala mutant. Time course analysis of the digestion of 5′-phosphorylated double strand DNA (5′-PO 4 -dsDNA: EcoRV-linearized pMal-c2) and 5′-dephosphorylated double strand DNA (5′-OH-dsDNA: 5′dephosphorylated EcoRV-linearized pMal-c2) substrates by wild-type LHK-Exo and the Arg12Ala mutant form using a ‘heparin trap’ approach. A total of 6 µg (82 pmol of trimers) of LHK-Exo or Arg12Ala mutant protein was incubated at 25°C with 60 ng (0.015 pmol) of 5′-PO 4 -dsDNA or 5′-OH-dsDNA in Tris–HCl (25 mM, pH 8.0), 1 mM DTT, 7.5 mM MgCl 2 . After 15 s, excess heparin was added to sequester all unbound protein, and to prevent disassociated protein from re-binding. Aliquots were removed at various time points (0–20 min), and dsDNA levels were determined using fluorescent PicoGreen assays, to enable the extent of DNA digestion to be calculated. In one set of assays, heparin was added to LHK-Exo prior to the addition of dsDNA substrate, to confirm the efficacy of the heparin trap method (filled black squares, green line). Graphs show the mean number of nucleotides digested from each terminus (±SD; y -axis) plotted against the time of analysis (in minutes; x -axis).
    Figure Legend Snippet: Processivity of double strand DNA digestion by wild-type LHK-Exo and Arg12Ala mutant. Time course analysis of the digestion of 5′-phosphorylated double strand DNA (5′-PO 4 -dsDNA: EcoRV-linearized pMal-c2) and 5′-dephosphorylated double strand DNA (5′-OH-dsDNA: 5′dephosphorylated EcoRV-linearized pMal-c2) substrates by wild-type LHK-Exo and the Arg12Ala mutant form using a ‘heparin trap’ approach. A total of 6 µg (82 pmol of trimers) of LHK-Exo or Arg12Ala mutant protein was incubated at 25°C with 60 ng (0.015 pmol) of 5′-PO 4 -dsDNA or 5′-OH-dsDNA in Tris–HCl (25 mM, pH 8.0), 1 mM DTT, 7.5 mM MgCl 2 . After 15 s, excess heparin was added to sequester all unbound protein, and to prevent disassociated protein from re-binding. Aliquots were removed at various time points (0–20 min), and dsDNA levels were determined using fluorescent PicoGreen assays, to enable the extent of DNA digestion to be calculated. In one set of assays, heparin was added to LHK-Exo prior to the addition of dsDNA substrate, to confirm the efficacy of the heparin trap method (filled black squares, green line). Graphs show the mean number of nucleotides digested from each terminus (±SD; y -axis) plotted against the time of analysis (in minutes; x -axis).

    Techniques Used: Mutagenesis, Incubation, Binding Assay

    34) Product Images from "Functional Genomics Approach to Identifying Genes Required for Biofilm Development by Streptococcus mutans"

    Article Title: Functional Genomics Approach to Identifying Genes Required for Biofilm Development by Streptococcus mutans

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.68.3.1196-1203.2002

    Autolysis of the whole cells of S. mutans UA159 (WT) and the brpA -deficient strain (brpA) grown in BHI medium. Mid-log-phase cultures (OD 600 ≅ 0.7) were harvested by centrifugation, washed twice with ice-cold water, resuspended in 0.05 M Tris-HCl (pH 8.0) containing 0.2% Triton X-100, and incubated at 37°C with agitation (200 rpm). The changes of absorbance at 600 nm were determined as described in Materials and Methods. Data represent the average of three separate experiments, and the error bars indicate standard deviations.
    Figure Legend Snippet: Autolysis of the whole cells of S. mutans UA159 (WT) and the brpA -deficient strain (brpA) grown in BHI medium. Mid-log-phase cultures (OD 600 ≅ 0.7) were harvested by centrifugation, washed twice with ice-cold water, resuspended in 0.05 M Tris-HCl (pH 8.0) containing 0.2% Triton X-100, and incubated at 37°C with agitation (200 rpm). The changes of absorbance at 600 nm were determined as described in Materials and Methods. Data represent the average of three separate experiments, and the error bars indicate standard deviations.

    Techniques Used: Centrifugation, Incubation

    35) Product Images from "sNASP, a Histone H1-Specific Eukaryotic Chaperone Dimer that Facilitates Chromatin Assembly"

    Article Title: sNASP, a Histone H1-Specific Eukaryotic Chaperone Dimer that Facilitates Chromatin Assembly

    Journal: Biophysical Journal

    doi: 10.1529/biophysj.108.130021

    Analytical ultracentrifuge analysis of recombinant human sNASP. ( A ) Integral distribution of the sedimentation coefficient of sNASP at different ionic strengths: 25 mM NaCl ( black triangles ); 150 mM NaCl ( black circles ); 500 mM NaCl, 20 mM Tris-HCl (pH
    Figure Legend Snippet: Analytical ultracentrifuge analysis of recombinant human sNASP. ( A ) Integral distribution of the sedimentation coefficient of sNASP at different ionic strengths: 25 mM NaCl ( black triangles ); 150 mM NaCl ( black circles ); 500 mM NaCl, 20 mM Tris-HCl (pH

    Techniques Used: Recombinant, Sedimentation

    Tertiary structure of sNASP. ( A ) SDS-PAGE analysis of the time course of digestion of NASP by trypsin (E:S 1:1000) in 100 mM NaCl, 20 mM Tris-HCl (pH 7.5). ( lane 1 ) Undigested sample; ( lanes 2–5 ) peptides generated after 5, 10, 20, and 40 min
    Figure Legend Snippet: Tertiary structure of sNASP. ( A ) SDS-PAGE analysis of the time course of digestion of NASP by trypsin (E:S 1:1000) in 100 mM NaCl, 20 mM Tris-HCl (pH 7.5). ( lane 1 ) Undigested sample; ( lanes 2–5 ) peptides generated after 5, 10, 20, and 40 min

    Techniques Used: SDS Page, Generated

    Determination of the secondary structure of sNASP. ( A ) CD spectrum. The spectrum was recorded at 20°C in 100 mM NaCl, 40 mM Tris-HCl (pH 8.0) buffer. ( B ) Secondary structure prediction using the hierarchical neural network protein sequence analysis
    Figure Legend Snippet: Determination of the secondary structure of sNASP. ( A ) CD spectrum. The spectrum was recorded at 20°C in 100 mM NaCl, 40 mM Tris-HCl (pH 8.0) buffer. ( B ) Secondary structure prediction using the hierarchical neural network protein sequence analysis

    Techniques Used: Sequencing

    Binding of histones to sNASP. HeLa cell core histones H2A-H2B, H3-H4, and linker histones H1 and their trypsin-resistant core H1c were mixed with sNASP in the presence of 100 mM NaCl, 10 mM Tris-HCl (pH 7.5) at increasing histone:sNASP molar ratios (0
    Figure Legend Snippet: Binding of histones to sNASP. HeLa cell core histones H2A-H2B, H3-H4, and linker histones H1 and their trypsin-resistant core H1c were mixed with sNASP in the presence of 100 mM NaCl, 10 mM Tris-HCl (pH 7.5) at increasing histone:sNASP molar ratios (0

    Techniques Used: Binding Assay

    36) Product Images from "Outer Membrane Vesicles Mediate Transport of Biologically Active Vibrio cholerae Cytolysin (VCC) from V. cholerae Strains"

    Article Title: Outer Membrane Vesicles Mediate Transport of Biologically Active Vibrio cholerae Cytolysin (VCC) from V. cholerae Strains

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0106731

    Hemolytic and cytotoxic effects of OMV-associated VCC. (A) Quantification of hemolytic activity of VCC in OMVs and comparison with activity of purified VCC. Contact hemolytic assay using 5% rabbit red blood cells was performed as described in the materials and methods. (B) Cytotoxic effect on HeLa cells (a) Control cells were treated with Tris buffer; (b) cells were treated with OMVs from wild type NOVC strain V:5/04; (c) cells were treated with OMVs from the Δ vcc mutant. After 6 h the HeLa cells were fixed, permeabilized, and then subjected to staining as described in the materials and methods. Staining, actin filament (green) and nucleus (blue). Magnification, ×1000. Bars = 10 µm.
    Figure Legend Snippet: Hemolytic and cytotoxic effects of OMV-associated VCC. (A) Quantification of hemolytic activity of VCC in OMVs and comparison with activity of purified VCC. Contact hemolytic assay using 5% rabbit red blood cells was performed as described in the materials and methods. (B) Cytotoxic effect on HeLa cells (a) Control cells were treated with Tris buffer; (b) cells were treated with OMVs from wild type NOVC strain V:5/04; (c) cells were treated with OMVs from the Δ vcc mutant. After 6 h the HeLa cells were fixed, permeabilized, and then subjected to staining as described in the materials and methods. Staining, actin filament (green) and nucleus (blue). Magnification, ×1000. Bars = 10 µm.

    Techniques Used: Activity Assay, Purification, Hemolytic Assay, Mutagenesis, Staining

    VCC is tightly associated with NOVC OMVs. (A) Dissociation assays using OMVs from the NOVC strain V:5/04. Samples of OMVs in 20 mM Tris-HCl pH 8.0 were treated for 60 min on ice in the presence of: 20 mM Tris-HCl pH 8.0 (buffer), NaCl (1 M), Na 2 CO 3 (0.1 M), urea (0.8 M), SDS (1%), respectively. Samples were then centrifuged and the resulting OMVs (lanes 1, 3, 5, 7, 9) and Sup (lanes 2, 4 6, 8, 10) were analyzed by immunoblot using anti-VCC antibody. (B) Proteinase K protection assay. An equal amount of vesicles from the wild type NOVC strain V:5/04 was treated with 1.0 µg ml −1 of proteinase K (PK) in the presence or absence of 1% SDS. Samples were subjected to immunoblot analysis using anti-VCC antiserum and the OmpU protein (detected by anti-OmpU antiserum) was used as internal control.
    Figure Legend Snippet: VCC is tightly associated with NOVC OMVs. (A) Dissociation assays using OMVs from the NOVC strain V:5/04. Samples of OMVs in 20 mM Tris-HCl pH 8.0 were treated for 60 min on ice in the presence of: 20 mM Tris-HCl pH 8.0 (buffer), NaCl (1 M), Na 2 CO 3 (0.1 M), urea (0.8 M), SDS (1%), respectively. Samples were then centrifuged and the resulting OMVs (lanes 1, 3, 5, 7, 9) and Sup (lanes 2, 4 6, 8, 10) were analyzed by immunoblot using anti-VCC antibody. (B) Proteinase K protection assay. An equal amount of vesicles from the wild type NOVC strain V:5/04 was treated with 1.0 µg ml −1 of proteinase K (PK) in the presence or absence of 1% SDS. Samples were subjected to immunoblot analysis using anti-VCC antiserum and the OmpU protein (detected by anti-OmpU antiserum) was used as internal control.

    Techniques Used:

    37) Product Images from "Plant tRNA ligases are multifunctional enzymes that have diverged in sequence and substrate specificity from RNA ligases of other phylogenetic origins"

    Article Title: Plant tRNA ligases are multifunctional enzymes that have diverged in sequence and substrate specificity from RNA ligases of other phylogenetic origins

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gki174

    Isolation of wheat germ tRNA ligase. ( A ) Purification scheme. RNA ligase was purified from the soluble protein fraction of wheat embryos (S100 extract) by six consecutive steps. ( B ) As substrate for assaying tRNA ligase activity, we have used a natural modified Nicotiana pre-tRNA Tyr (NtY9-T7-M1). The arrows in the two 4 nt bulge loops indicate the 3′ and 5′ splice sites and dots identify the anticodon. ( C ) Fractionation of wheat germ tRNA ligase by Cibacron Blue Trisacryl M chromatography and ligation activity assay. Partially purified tRNA ligase from the Heparin Sepharose column was applied onto a Blue-Trisacryl M column. Elution of tRNA ligase was performed with a gradient of 150–800 mM KCl. Fractions of 5 ml were collected. Splicing endonuclease co-eluted with tRNA ligase at this purification step, generating 3′ and 5′ tRNA halves. Reaction mixtures (20 μl) contained 20 mM Tris–HCl, pH 7.5, 6 mM Mg(OAc) 2 , 80 μM spermine, 1 mM ATP, 0.5 mM GTP, 0.1 mM DTT, 0.5% Triton X-100, 40 fmol (4 × 10 4 c.p.m.) of T7-transcript (NtY9-T7-M1) and 2 μl from eluted fractions. Incubation was for 30 min at 37°C. Products were analysed on a 12.5% polyacrylamide/8 M urea gel. tRNA ligase activity elutes between 280 and 540 mM KCl as revealed by the detection of mature, spliced tRNA.
    Figure Legend Snippet: Isolation of wheat germ tRNA ligase. ( A ) Purification scheme. RNA ligase was purified from the soluble protein fraction of wheat embryos (S100 extract) by six consecutive steps. ( B ) As substrate for assaying tRNA ligase activity, we have used a natural modified Nicotiana pre-tRNA Tyr (NtY9-T7-M1). The arrows in the two 4 nt bulge loops indicate the 3′ and 5′ splice sites and dots identify the anticodon. ( C ) Fractionation of wheat germ tRNA ligase by Cibacron Blue Trisacryl M chromatography and ligation activity assay. Partially purified tRNA ligase from the Heparin Sepharose column was applied onto a Blue-Trisacryl M column. Elution of tRNA ligase was performed with a gradient of 150–800 mM KCl. Fractions of 5 ml were collected. Splicing endonuclease co-eluted with tRNA ligase at this purification step, generating 3′ and 5′ tRNA halves. Reaction mixtures (20 μl) contained 20 mM Tris–HCl, pH 7.5, 6 mM Mg(OAc) 2 , 80 μM spermine, 1 mM ATP, 0.5 mM GTP, 0.1 mM DTT, 0.5% Triton X-100, 40 fmol (4 × 10 4 c.p.m.) of T7-transcript (NtY9-T7-M1) and 2 μl from eluted fractions. Incubation was for 30 min at 37°C. Products were analysed on a 12.5% polyacrylamide/8 M urea gel. tRNA ligase activity elutes between 280 and 540 mM KCl as revealed by the detection of mature, spliced tRNA.

    Techniques Used: Isolation, Purification, Activity Assay, Modification, Fractionation, Chromatography, Ligation, Incubation

    38) Product Images from "Moonlighting adenylyl cyclases in plants – an Arabidopsis thaliana 9-cis-epoxycarotenoid dioxygenase as point in case"

    Article Title: Moonlighting adenylyl cyclases in plants – an Arabidopsis thaliana 9-cis-epoxycarotenoid dioxygenase as point in case

    Journal: bioRxiv

    doi: 10.1101/2021.03.23.436544

    Experimental validation of AC activity in vitro . ( A ) Complementation of E. coli cyaA with AtNCED3 AC fragments AtNCED3 211-440 (left) and AtNCED3 S311P/D313T (right). ( B ) cAMP generated by AtNCED3 211-440 and AtNCED3 S311P/D313T . Reaction mixtures contain 10 μg of AtNCED3 211-440 or AtNCED3 S311P/D313T , 50 mM Tris-HCl pH 8; 2 mM IBMX, 1 mM ATP and 5 mM MnCl 2 . Measurements of three independent experiments are represented as mean ± SE and were subjected to unpaired, one-tailed Student’s t -test (Two-Sample Assuming Unequal Variances) where * = P
    Figure Legend Snippet: Experimental validation of AC activity in vitro . ( A ) Complementation of E. coli cyaA with AtNCED3 AC fragments AtNCED3 211-440 (left) and AtNCED3 S311P/D313T (right). ( B ) cAMP generated by AtNCED3 211-440 and AtNCED3 S311P/D313T . Reaction mixtures contain 10 μg of AtNCED3 211-440 or AtNCED3 S311P/D313T , 50 mM Tris-HCl pH 8; 2 mM IBMX, 1 mM ATP and 5 mM MnCl 2 . Measurements of three independent experiments are represented as mean ± SE and were subjected to unpaired, one-tailed Student’s t -test (Two-Sample Assuming Unequal Variances) where * = P

    Techniques Used: Activity Assay, In Vitro, Generated, One-tailed Test

    Related Articles

    other:

    Article Title: DNA Interaction Studies of a New Platinum(II) Complex Containing Different Aromatic Dinitrogen Ligands
    Article Snippet: Experimental All chemicals such as K2 PtCl4 , 4,7-dimethyl-1,10-phenanthroline, and 4,4-dimethyl-2,2-bypyridine, were purchased from Merck, and Tris-HCl and highly polymerized calf thymus DNA (CT-DNA) were purchased from Sigma Co..

    Article Title: Synthesis Characterization and DNA Interaction Studies of a New Zn(II) Complex Containing Different Dinitrogen Aromatic Ligands
    Article Snippet: Experimental All chemicals such as Zn(NO3 )2 ·6H2 O, 4,7-diphenyl-1,10-phenanthroline (DIP), and 4,7-dimethyl-1,10-phenanthroline (DMP) were purchased from Merck, and Tris-HCl highly polymerized calf thymus DNA (CT-DNA) were purchased from Sigma Co.

    Centrifugation:

    Article Title: CFTR mutations altering CFTR fragmentation
    Article Snippet: Cells were in growth phase at ~80% confluence and cell lysis was performed using the following protocol unless specified in the Figure legend. .. Cells were washed with ice-cold PBS, scraped from the plates, pelleted by centrifugation (800 g for 5 min at 22°C) and lysed by the addition of ice-cold buffer consisting of 1% (v/v) Nonidet P40, 50 mM Tris/HCl (pH 7.5) and 150 mM NaCl with fresh protease inhibitor cocktail (Calbiochem). ..

    Protease Inhibitor:

    Article Title: CFTR mutations altering CFTR fragmentation
    Article Snippet: Cells were in growth phase at ~80% confluence and cell lysis was performed using the following protocol unless specified in the Figure legend. .. Cells were washed with ice-cold PBS, scraped from the plates, pelleted by centrifugation (800 g for 5 min at 22°C) and lysed by the addition of ice-cold buffer consisting of 1% (v/v) Nonidet P40, 50 mM Tris/HCl (pH 7.5) and 150 mM NaCl with fresh protease inhibitor cocktail (Calbiochem). ..

    Article Title: Structural analysis of the complex between influenza B nucleoprotein and human importin-α
    Article Snippet: Escherichia coli BL21 (DE3) cells transformed with the corresponding plasmids were induced 12 hours by adding 0.3 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 18 °C and collected by centrifugation. .. For B/NP constructs, pellets were resuspended and sonicated in lysis buffer composed of 50 mM Tris-HCl pH 7.5, 300 mM NaCl, 1 M NDSB201 (Sigma), 5 mM β-mercaptoethanol (β-ME) and cOmplete EDTA-free protease inhibitor cocktail (Roche). .. For importin-α7, pellets were resuspended and sonicated in lysis buffer composed of 50 mM Tris-HCl pH 8, 500 mM NaCl, 1 mM β-ME and cOmplete EDTA-free protease inhibitor cocktail.

    Filtration:

    Article Title: USP7S-dependent inactivation of Mule regulates DNA damage signalling and repair
    Article Snippet: In vitro ubiquitylation and deubiquitylation assays Mule (25 pmol) was self-ubiquitylated in the presence of 7 pmol E1, 65 pmol UbcH7 and 6 pmol ubiquitin in buffer containing 25 mM Tris–HCl, pH 8.0, 4 mM adenosine triphosphate, 5 mM MgCl2 , 200 µM CaCl2, 1 mM DTT and 10 µM MG-132 for 1 h at 30°C with shaking. .. Ubiquitin and E2 were removed from the reaction mixture by filtration and buffer exchange into deubiquitylation buffer containing 50 mM Tris–HCl, pH 8.0, 150 mM KCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 5% glycerol and 2 mM DTT using Amicon Ultra 30K units (Millipore). .. For the deubiquitylation assay, ubiquitylated Mule (0.6 pmol) was incubated with equal amounts of wild-type USP7S or inactive C223S USP7S mutant (1.2 and 2.4 pmol) in deubiquitylation buffer for 30 min at 30°C with shaking.

    Buffer Exchange:

    Article Title: USP7S-dependent inactivation of Mule regulates DNA damage signalling and repair
    Article Snippet: In vitro ubiquitylation and deubiquitylation assays Mule (25 pmol) was self-ubiquitylated in the presence of 7 pmol E1, 65 pmol UbcH7 and 6 pmol ubiquitin in buffer containing 25 mM Tris–HCl, pH 8.0, 4 mM adenosine triphosphate, 5 mM MgCl2 , 200 µM CaCl2, 1 mM DTT and 10 µM MG-132 for 1 h at 30°C with shaking. .. Ubiquitin and E2 were removed from the reaction mixture by filtration and buffer exchange into deubiquitylation buffer containing 50 mM Tris–HCl, pH 8.0, 150 mM KCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 5% glycerol and 2 mM DTT using Amicon Ultra 30K units (Millipore). .. For the deubiquitylation assay, ubiquitylated Mule (0.6 pmol) was incubated with equal amounts of wild-type USP7S or inactive C223S USP7S mutant (1.2 and 2.4 pmol) in deubiquitylation buffer for 30 min at 30°C with shaking.

    Activity Assay:

    Article Title: Efficiency of Incorporation and Chain Termination Determines the Inhibition Potency of 2′-Modified Nucleotide Analogs against Hepatitis C Virus Polymerase
    Article Snippet: .. The HCV polymerase activity was measured as the incorporation of radiolabeled nucleotide monophosphates into acid-insoluble RNA products using HCV NS5B and complementary internal ribosome entry site (IRES)-derived RNA templates as described previously , with the following modifications: HCV polymerase reaction mixtures contained 50 nM 5′-untranslated region (UTR) RNA template, 1 μM tritiated CTP (18.8 Ci/mmol), 1 μM ATP, 1 μM GTP, 0.5 μM UTP, 40 mM Tris-HCl (pH 8.0), 20 mM NaCl, 3 mM DTT, 4 mM MgCl2 , serial diluted inhibitor, and 100 nM NS5B enzyme in 96-well MultiScreen plates (EMD Millipore, Billerica, MA). .. Reaction mixtures were incubated for 2 h at 30°C and stopped by the addition of equal volumes of 20% (vol/vol) trichloroacetic acid.

    Translocation Assay:

    Article Title: Structure of mycobacterial 3′-to-5′ RNA:DNA helicase Lhr bound to a ssDNA tracking strand highlights distinctive features of a novel family of bacterial helicases
    Article Snippet: The labeled DNA was purified by electrophoresis through a 18% native polyacrylamide gel and eluted from an excised gel slice by overnight incubation at 4°C in 400 μl of 10 mM Tris–HCl, pH 7.5, 1 mM EDTA and 50 mM NaCl. .. Translocation reaction mixtures (10 μl) containing 20 mM Tris–HCl, pH 7.0, 50 mM NaCl, 1 mM DTT, 1 mM CaCl2 , 1 mM ATP, 100 nM (1 pmol) biotinylated 32 P-labeled DNA and 4 μM streptavidin (Sigma) were pre-incubated at room temperature for 10 min to form streptavidin–DNA (SA–DNA) complexes. .. The mixtures were supplemented with 40 μM free Biotin (Fisher) and the translocation reactions were initiated by adding Lhr-(1–856).

    Construct:

    Article Title: Structural analysis of the complex between influenza B nucleoprotein and human importin-α
    Article Snippet: Escherichia coli BL21 (DE3) cells transformed with the corresponding plasmids were induced 12 hours by adding 0.3 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 18 °C and collected by centrifugation. .. For B/NP constructs, pellets were resuspended and sonicated in lysis buffer composed of 50 mM Tris-HCl pH 7.5, 300 mM NaCl, 1 M NDSB201 (Sigma), 5 mM β-mercaptoethanol (β-ME) and cOmplete EDTA-free protease inhibitor cocktail (Roche). .. For importin-α7, pellets were resuspended and sonicated in lysis buffer composed of 50 mM Tris-HCl pH 8, 500 mM NaCl, 1 mM β-ME and cOmplete EDTA-free protease inhibitor cocktail.

    Sonication:

    Article Title: Structural analysis of the complex between influenza B nucleoprotein and human importin-α
    Article Snippet: Escherichia coli BL21 (DE3) cells transformed with the corresponding plasmids were induced 12 hours by adding 0.3 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 18 °C and collected by centrifugation. .. For B/NP constructs, pellets were resuspended and sonicated in lysis buffer composed of 50 mM Tris-HCl pH 7.5, 300 mM NaCl, 1 M NDSB201 (Sigma), 5 mM β-mercaptoethanol (β-ME) and cOmplete EDTA-free protease inhibitor cocktail (Roche). .. For importin-α7, pellets were resuspended and sonicated in lysis buffer composed of 50 mM Tris-HCl pH 8, 500 mM NaCl, 1 mM β-ME and cOmplete EDTA-free protease inhibitor cocktail.

    Lysis:

    Article Title: Structural analysis of the complex between influenza B nucleoprotein and human importin-α
    Article Snippet: Escherichia coli BL21 (DE3) cells transformed with the corresponding plasmids were induced 12 hours by adding 0.3 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 18 °C and collected by centrifugation. .. For B/NP constructs, pellets were resuspended and sonicated in lysis buffer composed of 50 mM Tris-HCl pH 7.5, 300 mM NaCl, 1 M NDSB201 (Sigma), 5 mM β-mercaptoethanol (β-ME) and cOmplete EDTA-free protease inhibitor cocktail (Roche). .. For importin-α7, pellets were resuspended and sonicated in lysis buffer composed of 50 mM Tris-HCl pH 8, 500 mM NaCl, 1 mM β-ME and cOmplete EDTA-free protease inhibitor cocktail.

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  • 99
    Millipore m tris hcl
    Immunochemistry of whole-cell extracts with anti-(1→5)-α-arabinan probe, LM6. A, Immunoblotting on nitrocellulose with LM6 of material separated by SDS-PAGE. Material (10 μg of protein per lane) from WT MG fruit (lanes 1–3) and Cnr MG fruit (lanes 4–6) were prepared in sample buffer immediately (lanes 1 and 4) or after incubation in <t>Tris-buffer</t> (lanes 2 and 5) or incubation in Tris-buffer plus <t>Pronase</t> E (lanes 3 and 6). Significant amounts of LM6-reactive, Pronase E-sensitive material entered the gel from Cnr , but not WT material. R indicates top of resolving gel. M shows M r markers. B, Immunodot assay on nitrocellulose of material (0.4 μg of protein per dot) analyzed in A showing abundance of LM6 epitope in all samples.
    M Tris Hcl, supplied by Millipore, 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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    98
    Millipore tris hcl sds page gels
    ET treatment does not affect protein synthesis. Primary hepatocytes treated were treated with 25 μg/ml ET and 250 ng/ml TM as indicated in media with 10 percent the normal amount of methionine and cysteine, and EasyTag ExpreSS 35 S (Perkin Elmer NEG772002MC) was added to a final concentration of 50 mCi/ml. After the indicated times, cell lysates were collected in 1% <t>SDS</t> 100 mM <t>Tris,</t> pH 8.8, denatured by heating, spotted onto gridded 3MM filter paper, and air dried. Filter was then precipitated by serial incubations in ice-cold 10% trichloroacetic acid (TCA) for 60 min., 5% TCA at room temperature for 5 min., 5% TCA preheated to 75° for 5 min., 5% TCA at room temperature for 5 min., and finally 2:1 ethanol:ether preheated to 37° for 5 min. Filter was then air dried, sliced into individual samples, and immersed in scintillation cocktail, and counted. n = 6 samples per condition. The modest effect of this dose of TM on protein synthesis is consistent with previous findings ( Rutkowski et al., 2006 )
    Tris Hcl Sds Page Gels, supplied by Millipore, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Millipore tris hcl
    Qualitative analysis of ssDNA and <t>dsDNA</t> hydrolysis activities of LHK-Exo. ( A ) dsDNA exonuclease activities. Agarose gel showing aliquots taken (0–15 min) from an incubation of LHK-Exo (30 µg, 0.41 nmol of trimers) and BamHI-linearized pET28a (1.8 µg, 0.54 pmol) in <t>Tris–HCl</t> (pH 8.0, 50 mM), 50 mM NaCl, 7.5 mM MgCl 2 at 37°C. ( B ) Polarity of dsDNA digestion. A total of 6 µg of LHK-Exo (82 pmol of trimers, lanes 2–5) or λ-exonuclease (74 pmol of trimers, lanes 6–9) protein was incubated with 0.1 µg (0.23 pmol) of a 712-bp linear 5′-phosphorylated dsDNA substrate (‘unmodified’; lanes 2, 3, 6 and 7), or an analogous 5′-phosphorylated linear dsDNA substrate containing three consecutive ‘nuclease-resistant’ phosphorothioate linkages at its 5′-termini (‘PT-modified’; lanes 4, 5, 8, 9). Assays were quenched immediately (0 min) or incubated at 37°C for 20 min, before analysis of digestion products on 1% agarose gels. ( C ) Digestion of 5′-phosphorylated ssDNA. Reaction mixtures (80 µl) containing LHK-Exo (4.5 µg, 61.4 pmol of trimers) and 5′-PO 4 -(dT) 50 (0.4 nmol) in 25 mM Tris–HCl (pH 8.0), 7.5 mM MgCl 2 , 1 mM DTT were incubated at 37°C. Aliquots (20 µl) were withdrawn after 0, 0.5, 5 and 20 min, and immediately quenched. Reaction products were analyzed by denaturing gel electrophoresis. ( D ) Digestion of non-phosphorylated ssDNA. Analogous sets of assays were performed using non-phosphorylated 50-mers of oligothymidine [5′-OH-(dT) 50 ]. Fluorescent gel images were scanned after SYBR Gold staining. A ssDNA ladder [Oligo Length Standards 20/100 Ladder (IDT)] is included in lane 1.
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    Image Search Results


    Immunochemistry of whole-cell extracts with anti-(1→5)-α-arabinan probe, LM6. A, Immunoblotting on nitrocellulose with LM6 of material separated by SDS-PAGE. Material (10 μg of protein per lane) from WT MG fruit (lanes 1–3) and Cnr MG fruit (lanes 4–6) were prepared in sample buffer immediately (lanes 1 and 4) or after incubation in Tris-buffer (lanes 2 and 5) or incubation in Tris-buffer plus Pronase E (lanes 3 and 6). Significant amounts of LM6-reactive, Pronase E-sensitive material entered the gel from Cnr , but not WT material. R indicates top of resolving gel. M shows M r markers. B, Immunodot assay on nitrocellulose of material (0.4 μg of protein per dot) analyzed in A showing abundance of LM6 epitope in all samples.

    Journal: Plant Physiology

    Article Title: Altered Middle Lamella Homogalacturonan and Disrupted Deposition of (1- > 5)-?-l-Arabinan in the Pericarp of Cnr, a Ripening Mutant of Tomato 1

    doi:

    Figure Lengend Snippet: Immunochemistry of whole-cell extracts with anti-(1→5)-α-arabinan probe, LM6. A, Immunoblotting on nitrocellulose with LM6 of material separated by SDS-PAGE. Material (10 μg of protein per lane) from WT MG fruit (lanes 1–3) and Cnr MG fruit (lanes 4–6) were prepared in sample buffer immediately (lanes 1 and 4) or after incubation in Tris-buffer (lanes 2 and 5) or incubation in Tris-buffer plus Pronase E (lanes 3 and 6). Significant amounts of LM6-reactive, Pronase E-sensitive material entered the gel from Cnr , but not WT material. R indicates top of resolving gel. M shows M r markers. B, Immunodot assay on nitrocellulose of material (0.4 μg of protein per dot) analyzed in A showing abundance of LM6 epitope in all samples.

    Article Snippet: The powder (2 mL) was suspended in 50 m m Tris-HCl, pH 6.5, or 50 m m Tris-HCl, pH 6.5, containing Pronase E (Sigma) at 1 mg mL−1 .

    Techniques: SDS Page, Incubation

    ET treatment does not affect protein synthesis. Primary hepatocytes treated were treated with 25 μg/ml ET and 250 ng/ml TM as indicated in media with 10 percent the normal amount of methionine and cysteine, and EasyTag ExpreSS 35 S (Perkin Elmer NEG772002MC) was added to a final concentration of 50 mCi/ml. After the indicated times, cell lysates were collected in 1% SDS 100 mM Tris, pH 8.8, denatured by heating, spotted onto gridded 3MM filter paper, and air dried. Filter was then precipitated by serial incubations in ice-cold 10% trichloroacetic acid (TCA) for 60 min., 5% TCA at room temperature for 5 min., 5% TCA preheated to 75° for 5 min., 5% TCA at room temperature for 5 min., and finally 2:1 ethanol:ether preheated to 37° for 5 min. Filter was then air dried, sliced into individual samples, and immersed in scintillation cocktail, and counted. n = 6 samples per condition. The modest effect of this dose of TM on protein synthesis is consistent with previous findings ( Rutkowski et al., 2006 )

    Journal: bioRxiv

    Article Title: NADPH and glutathione redox link TCA cycle activity to endoplasmic reticulum stress

    doi: 10.1101/822775

    Figure Lengend Snippet: ET treatment does not affect protein synthesis. Primary hepatocytes treated were treated with 25 μg/ml ET and 250 ng/ml TM as indicated in media with 10 percent the normal amount of methionine and cysteine, and EasyTag ExpreSS 35 S (Perkin Elmer NEG772002MC) was added to a final concentration of 50 mCi/ml. After the indicated times, cell lysates were collected in 1% SDS 100 mM Tris, pH 8.8, denatured by heating, spotted onto gridded 3MM filter paper, and air dried. Filter was then precipitated by serial incubations in ice-cold 10% trichloroacetic acid (TCA) for 60 min., 5% TCA at room temperature for 5 min., 5% TCA preheated to 75° for 5 min., 5% TCA at room temperature for 5 min., and finally 2:1 ethanol:ether preheated to 37° for 5 min. Filter was then air dried, sliced into individual samples, and immersed in scintillation cocktail, and counted. n = 6 samples per condition. The modest effect of this dose of TM on protein synthesis is consistent with previous findings ( Rutkowski et al., 2006 )

    Article Snippet: Samples were run on Tris-tricine or Tris-HCl SDS-PAGE gels and transferred to 0.45 µm Immobilon-P Polyvinylidene Fluoride (PVDF) (Millipore) for Western blotting using ECL Prime substrate (GE Healthcare). qRT-PCR, including primer validation by standard curve and melt curve analysis, was as described ( ).

    Techniques: Concentration Assay

    Inhibiting Glutathione Reductase attenuates ER stress. (A) Primary hepatocytes were treated with 25 µg/mL ET for 4 h followed by addition of 10 mM DTT for 0, 30, or 60 min. Protein lysates were treated with 4 µM mm(PEG) 24 prior to SDS-PAGE and immunoblotting to detect endogenous albumin. The percentage of oxidized albumin is given below each group, with statistical comparison between ET-treated and non-treated cells. (B) Primary hepatocytes were treated with 25 μg/ml ET or 25 µM 2-AAPA for 8 h. Levels of total (GSx tot ), reduced (GSH), and oxidized (GSSG) glutathione, and the ratio of GSSG to GSH were measured fluorometrically and expressed relative to untreated cells. (C) Splicing of Xbp1 mRNA was measured by conventional RT-PCR, and (D) mRNA expression of UPR markers was measured by qRT-PCR in cells treated for 8 h with TM and/or 2-AAPA.

    Journal: bioRxiv

    Article Title: NADPH and glutathione redox link TCA cycle activity to endoplasmic reticulum stress

    doi: 10.1101/822775

    Figure Lengend Snippet: Inhibiting Glutathione Reductase attenuates ER stress. (A) Primary hepatocytes were treated with 25 µg/mL ET for 4 h followed by addition of 10 mM DTT for 0, 30, or 60 min. Protein lysates were treated with 4 µM mm(PEG) 24 prior to SDS-PAGE and immunoblotting to detect endogenous albumin. The percentage of oxidized albumin is given below each group, with statistical comparison between ET-treated and non-treated cells. (B) Primary hepatocytes were treated with 25 μg/ml ET or 25 µM 2-AAPA for 8 h. Levels of total (GSx tot ), reduced (GSH), and oxidized (GSSG) glutathione, and the ratio of GSSG to GSH were measured fluorometrically and expressed relative to untreated cells. (C) Splicing of Xbp1 mRNA was measured by conventional RT-PCR, and (D) mRNA expression of UPR markers was measured by qRT-PCR in cells treated for 8 h with TM and/or 2-AAPA.

    Article Snippet: Samples were run on Tris-tricine or Tris-HCl SDS-PAGE gels and transferred to 0.45 µm Immobilon-P Polyvinylidene Fluoride (PVDF) (Millipore) for Western blotting using ECL Prime substrate (GE Healthcare). qRT-PCR, including primer validation by standard curve and melt curve analysis, was as described ( ).

    Techniques: SDS Page, Reverse Transcription Polymerase Chain Reaction, Expressing, Quantitative RT-PCR

    Qualitative analysis of ssDNA and dsDNA hydrolysis activities of LHK-Exo. ( A ) dsDNA exonuclease activities. Agarose gel showing aliquots taken (0–15 min) from an incubation of LHK-Exo (30 µg, 0.41 nmol of trimers) and BamHI-linearized pET28a (1.8 µg, 0.54 pmol) in Tris–HCl (pH 8.0, 50 mM), 50 mM NaCl, 7.5 mM MgCl 2 at 37°C. ( B ) Polarity of dsDNA digestion. A total of 6 µg of LHK-Exo (82 pmol of trimers, lanes 2–5) or λ-exonuclease (74 pmol of trimers, lanes 6–9) protein was incubated with 0.1 µg (0.23 pmol) of a 712-bp linear 5′-phosphorylated dsDNA substrate (‘unmodified’; lanes 2, 3, 6 and 7), or an analogous 5′-phosphorylated linear dsDNA substrate containing three consecutive ‘nuclease-resistant’ phosphorothioate linkages at its 5′-termini (‘PT-modified’; lanes 4, 5, 8, 9). Assays were quenched immediately (0 min) or incubated at 37°C for 20 min, before analysis of digestion products on 1% agarose gels. ( C ) Digestion of 5′-phosphorylated ssDNA. Reaction mixtures (80 µl) containing LHK-Exo (4.5 µg, 61.4 pmol of trimers) and 5′-PO 4 -(dT) 50 (0.4 nmol) in 25 mM Tris–HCl (pH 8.0), 7.5 mM MgCl 2 , 1 mM DTT were incubated at 37°C. Aliquots (20 µl) were withdrawn after 0, 0.5, 5 and 20 min, and immediately quenched. Reaction products were analyzed by denaturing gel electrophoresis. ( D ) Digestion of non-phosphorylated ssDNA. Analogous sets of assays were performed using non-phosphorylated 50-mers of oligothymidine [5′-OH-(dT) 50 ]. Fluorescent gel images were scanned after SYBR Gold staining. A ssDNA ladder [Oligo Length Standards 20/100 Ladder (IDT)] is included in lane 1.

    Journal: Nucleic Acids Research

    Article Title: Structural and functional insight into the mechanism of an alkaline exonuclease from Laribacter hongkongensis

    doi: 10.1093/nar/gkr660

    Figure Lengend Snippet: Qualitative analysis of ssDNA and dsDNA hydrolysis activities of LHK-Exo. ( A ) dsDNA exonuclease activities. Agarose gel showing aliquots taken (0–15 min) from an incubation of LHK-Exo (30 µg, 0.41 nmol of trimers) and BamHI-linearized pET28a (1.8 µg, 0.54 pmol) in Tris–HCl (pH 8.0, 50 mM), 50 mM NaCl, 7.5 mM MgCl 2 at 37°C. ( B ) Polarity of dsDNA digestion. A total of 6 µg of LHK-Exo (82 pmol of trimers, lanes 2–5) or λ-exonuclease (74 pmol of trimers, lanes 6–9) protein was incubated with 0.1 µg (0.23 pmol) of a 712-bp linear 5′-phosphorylated dsDNA substrate (‘unmodified’; lanes 2, 3, 6 and 7), or an analogous 5′-phosphorylated linear dsDNA substrate containing three consecutive ‘nuclease-resistant’ phosphorothioate linkages at its 5′-termini (‘PT-modified’; lanes 4, 5, 8, 9). Assays were quenched immediately (0 min) or incubated at 37°C for 20 min, before analysis of digestion products on 1% agarose gels. ( C ) Digestion of 5′-phosphorylated ssDNA. Reaction mixtures (80 µl) containing LHK-Exo (4.5 µg, 61.4 pmol of trimers) and 5′-PO 4 -(dT) 50 (0.4 nmol) in 25 mM Tris–HCl (pH 8.0), 7.5 mM MgCl 2 , 1 mM DTT were incubated at 37°C. Aliquots (20 µl) were withdrawn after 0, 0.5, 5 and 20 min, and immediately quenched. Reaction products were analyzed by denaturing gel electrophoresis. ( D ) Digestion of non-phosphorylated ssDNA. Analogous sets of assays were performed using non-phosphorylated 50-mers of oligothymidine [5′-OH-(dT) 50 ]. Fluorescent gel images were scanned after SYBR Gold staining. A ssDNA ladder [Oligo Length Standards 20/100 Ladder (IDT)] is included in lane 1.

    Article Snippet: Determination of digestion processivity LHK-Exo (6 µg, 82 pmol of trimers) protein and 60 ng (0.015 pmol) of linear dsDNA substrate (5′-PO4 -dsDNA, EcoRV-linearized pMal-c2; or 5′-OH-dsDNA, 5′-dephosphorylated EcoRV-linearized pMal-c2) were equilibrated in 180 µl of Tris–HCl (25 mM, pH 8.0), 1 mM DTT, at 25°C for 5 min. A total of 90 µl of Tris–HCl (25 mM, pH 8.0), 1 mM DTT 15 mM MgCl2 was added to initiate the reaction; then exactly 15 s later, excess heparin (Sigma; 360 µg, 20 nmol) in 90 µl of Tris–HCl (25 mM, pH 8.0), 1 mM DTT, 7.5 mM MgCl2 was added to sequester unbound LHK-Exo (total reaction volume 360 µl).

    Techniques: Agarose Gel Electrophoresis, Incubation, Modification, Nucleic Acid Electrophoresis, Staining

    Determination of optimum conditions for LHK-Exo dsDNA digestion activities. PicoGreen fluorescence assays were performed to quantify the amounts of a representative 5′-phosphorylatd dsDNA substrate (PstI-linearized pUC18 that were digested by LHK-Exo under various conditions (reported as a percentage of the initial quantities of DNA). ( A and B ) Optimal concentrations of Mg 2+ and Mn 2+ ions. Amounts of PstI-linearized pUC18 (30 ng, 0.018 pmol) digested by LHK-Exo (8 ng, 0.11 pmol of trimers) in Tris–HCl (25 mM, pH 8.0) containing varying concentrations of Mg 2+ ions (1–15 mM, A) or Mn 2+ ions (0.1–1.7 mM, B), respectively, upon incubation at 25°C for 20 min. ( C ) Optimal pH. Amounts of PstI-linearized pUC18 (30 ng, 0.018 pmol) digested by LHK-Exo (8 ng, 0.11 pmol of trimers) in 25 mM Tris–HCl, 7.5 mM MgCl 2 ; adjusted to the appropriate pH value (pH 7.0–9.0); upon incubation at 25°C for 20 min. ( D ) Optimal temperature. Amounts of PstI-linearized pUC18 (30 ng, 0.018 pmol) digested by LHK-Exo (40 ng, 0.55 pmol of trimers) in Tris–HCl (25 mM, pH 8.0), 7.5 mM MgCl 2 , after incubation for 1 min at the indicated temperature (34–54°C). Four to six independent replicates of each experimental condition were performed, and data are reported as the mean ± standard deviation.

    Journal: Nucleic Acids Research

    Article Title: Structural and functional insight into the mechanism of an alkaline exonuclease from Laribacter hongkongensis

    doi: 10.1093/nar/gkr660

    Figure Lengend Snippet: Determination of optimum conditions for LHK-Exo dsDNA digestion activities. PicoGreen fluorescence assays were performed to quantify the amounts of a representative 5′-phosphorylatd dsDNA substrate (PstI-linearized pUC18 that were digested by LHK-Exo under various conditions (reported as a percentage of the initial quantities of DNA). ( A and B ) Optimal concentrations of Mg 2+ and Mn 2+ ions. Amounts of PstI-linearized pUC18 (30 ng, 0.018 pmol) digested by LHK-Exo (8 ng, 0.11 pmol of trimers) in Tris–HCl (25 mM, pH 8.0) containing varying concentrations of Mg 2+ ions (1–15 mM, A) or Mn 2+ ions (0.1–1.7 mM, B), respectively, upon incubation at 25°C for 20 min. ( C ) Optimal pH. Amounts of PstI-linearized pUC18 (30 ng, 0.018 pmol) digested by LHK-Exo (8 ng, 0.11 pmol of trimers) in 25 mM Tris–HCl, 7.5 mM MgCl 2 ; adjusted to the appropriate pH value (pH 7.0–9.0); upon incubation at 25°C for 20 min. ( D ) Optimal temperature. Amounts of PstI-linearized pUC18 (30 ng, 0.018 pmol) digested by LHK-Exo (40 ng, 0.55 pmol of trimers) in Tris–HCl (25 mM, pH 8.0), 7.5 mM MgCl 2 , after incubation for 1 min at the indicated temperature (34–54°C). Four to six independent replicates of each experimental condition were performed, and data are reported as the mean ± standard deviation.

    Article Snippet: Determination of digestion processivity LHK-Exo (6 µg, 82 pmol of trimers) protein and 60 ng (0.015 pmol) of linear dsDNA substrate (5′-PO4 -dsDNA, EcoRV-linearized pMal-c2; or 5′-OH-dsDNA, 5′-dephosphorylated EcoRV-linearized pMal-c2) were equilibrated in 180 µl of Tris–HCl (25 mM, pH 8.0), 1 mM DTT, at 25°C for 5 min. A total of 90 µl of Tris–HCl (25 mM, pH 8.0), 1 mM DTT 15 mM MgCl2 was added to initiate the reaction; then exactly 15 s later, excess heparin (Sigma; 360 µg, 20 nmol) in 90 µl of Tris–HCl (25 mM, pH 8.0), 1 mM DTT, 7.5 mM MgCl2 was added to sequester unbound LHK-Exo (total reaction volume 360 µl).

    Techniques: Fluorescence, Incubation, Standard Deviation

    Processivity of double strand DNA digestion by wild-type LHK-Exo and Arg12Ala mutant. Time course analysis of the digestion of 5′-phosphorylated double strand DNA (5′-PO 4 -dsDNA: EcoRV-linearized pMal-c2) and 5′-dephosphorylated double strand DNA (5′-OH-dsDNA: 5′dephosphorylated EcoRV-linearized pMal-c2) substrates by wild-type LHK-Exo and the Arg12Ala mutant form using a ‘heparin trap’ approach. A total of 6 µg (82 pmol of trimers) of LHK-Exo or Arg12Ala mutant protein was incubated at 25°C with 60 ng (0.015 pmol) of 5′-PO 4 -dsDNA or 5′-OH-dsDNA in Tris–HCl (25 mM, pH 8.0), 1 mM DTT, 7.5 mM MgCl 2 . After 15 s, excess heparin was added to sequester all unbound protein, and to prevent disassociated protein from re-binding. Aliquots were removed at various time points (0–20 min), and dsDNA levels were determined using fluorescent PicoGreen assays, to enable the extent of DNA digestion to be calculated. In one set of assays, heparin was added to LHK-Exo prior to the addition of dsDNA substrate, to confirm the efficacy of the heparin trap method (filled black squares, green line). Graphs show the mean number of nucleotides digested from each terminus (±SD; y -axis) plotted against the time of analysis (in minutes; x -axis).

    Journal: Nucleic Acids Research

    Article Title: Structural and functional insight into the mechanism of an alkaline exonuclease from Laribacter hongkongensis

    doi: 10.1093/nar/gkr660

    Figure Lengend Snippet: Processivity of double strand DNA digestion by wild-type LHK-Exo and Arg12Ala mutant. Time course analysis of the digestion of 5′-phosphorylated double strand DNA (5′-PO 4 -dsDNA: EcoRV-linearized pMal-c2) and 5′-dephosphorylated double strand DNA (5′-OH-dsDNA: 5′dephosphorylated EcoRV-linearized pMal-c2) substrates by wild-type LHK-Exo and the Arg12Ala mutant form using a ‘heparin trap’ approach. A total of 6 µg (82 pmol of trimers) of LHK-Exo or Arg12Ala mutant protein was incubated at 25°C with 60 ng (0.015 pmol) of 5′-PO 4 -dsDNA or 5′-OH-dsDNA in Tris–HCl (25 mM, pH 8.0), 1 mM DTT, 7.5 mM MgCl 2 . After 15 s, excess heparin was added to sequester all unbound protein, and to prevent disassociated protein from re-binding. Aliquots were removed at various time points (0–20 min), and dsDNA levels were determined using fluorescent PicoGreen assays, to enable the extent of DNA digestion to be calculated. In one set of assays, heparin was added to LHK-Exo prior to the addition of dsDNA substrate, to confirm the efficacy of the heparin trap method (filled black squares, green line). Graphs show the mean number of nucleotides digested from each terminus (±SD; y -axis) plotted against the time of analysis (in minutes; x -axis).

    Article Snippet: Determination of digestion processivity LHK-Exo (6 µg, 82 pmol of trimers) protein and 60 ng (0.015 pmol) of linear dsDNA substrate (5′-PO4 -dsDNA, EcoRV-linearized pMal-c2; or 5′-OH-dsDNA, 5′-dephosphorylated EcoRV-linearized pMal-c2) were equilibrated in 180 µl of Tris–HCl (25 mM, pH 8.0), 1 mM DTT, at 25°C for 5 min. A total of 90 µl of Tris–HCl (25 mM, pH 8.0), 1 mM DTT 15 mM MgCl2 was added to initiate the reaction; then exactly 15 s later, excess heparin (Sigma; 360 µg, 20 nmol) in 90 µl of Tris–HCl (25 mM, pH 8.0), 1 mM DTT, 7.5 mM MgCl2 was added to sequester unbound LHK-Exo (total reaction volume 360 µl).

    Techniques: Mutagenesis, Incubation, Binding Assay