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    Name:
    DTT Molecular Grade
    Description:
    Antioxidant for stabilizing enzymes and other proteins containing sulfhydryl groups
    Catalog Number:
    p1171
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    Category:
    Nucleic Acid Extraction Analysis Quantitation Analysis In vitro Transcription
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    Structured Review

    Promega dtt
    RNA binding activities of wild-type (WT) and mutant helicase proteins. The wild-type or mutant protein was incubated with labeled short ssRNA under standard reaction conditions. One-half microgram of the wild-type or mutant helicase protein was incubated with 1.3 pmol of radiolabeled 26-mer ssRNA oligonucleotides in 20 μl of helicase reaction buffer (30 mM <t>Tris-HCl</t> [pH 7.5], 3 mM MgCl 2 , 10 mM <t>DTT,</t> and 1 μl of RNasin [Promega]). As a control, labeled RNA without VP6 was incubated in one reaction mixture. The binding reaction mixture was incubated at 37°C for 15 min; then the reaction was terminated by the addition of 5× RNA loading dye containing 0.5% Nonidet P-40, and the mixture was subsequently electrophoresed on a 4% polyacrylamide-0.5× Tris-borate-EDTA gel buffer. The bound complex was visualized by autoradiography. (A) Autoradiogram of PAGE showing the positions of bound RNA of the wild-type and three mutant VP6 proteins and free unbound RNA. An RNA probe without VP6 was also resolved as a control (indicated as RNA). (B) The radioactivity of labeled proteins in each reaction mixture was estimated by phosphorimager as described in Materials and Methods, and the efficiencies of the RNA binding activities of the three mutant proteins were compared with that of the wild-type VP6. The RNA probe control is indicated as C. The figure shows the mean values of the results of three different experiments.
    Antioxidant for stabilizing enzymes and other proteins containing sulfhydryl groups
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    dtt - by Bioz Stars, 2020-01
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    Images

    1) Product Images from "Defining the Structure-Function Relationships of Bluetongue Virus Helicase Protein VP6"

    Article Title: Defining the Structure-Function Relationships of Bluetongue Virus Helicase Protein VP6

    Journal: Journal of Virology

    doi: 10.1128/JVI.77.21.11347-11356.2003

    RNA binding activities of wild-type (WT) and mutant helicase proteins. The wild-type or mutant protein was incubated with labeled short ssRNA under standard reaction conditions. One-half microgram of the wild-type or mutant helicase protein was incubated with 1.3 pmol of radiolabeled 26-mer ssRNA oligonucleotides in 20 μl of helicase reaction buffer (30 mM Tris-HCl [pH 7.5], 3 mM MgCl 2 , 10 mM DTT, and 1 μl of RNasin [Promega]). As a control, labeled RNA without VP6 was incubated in one reaction mixture. The binding reaction mixture was incubated at 37°C for 15 min; then the reaction was terminated by the addition of 5× RNA loading dye containing 0.5% Nonidet P-40, and the mixture was subsequently electrophoresed on a 4% polyacrylamide-0.5× Tris-borate-EDTA gel buffer. The bound complex was visualized by autoradiography. (A) Autoradiogram of PAGE showing the positions of bound RNA of the wild-type and three mutant VP6 proteins and free unbound RNA. An RNA probe without VP6 was also resolved as a control (indicated as RNA). (B) The radioactivity of labeled proteins in each reaction mixture was estimated by phosphorimager as described in Materials and Methods, and the efficiencies of the RNA binding activities of the three mutant proteins were compared with that of the wild-type VP6. The RNA probe control is indicated as C. The figure shows the mean values of the results of three different experiments.
    Figure Legend Snippet: RNA binding activities of wild-type (WT) and mutant helicase proteins. The wild-type or mutant protein was incubated with labeled short ssRNA under standard reaction conditions. One-half microgram of the wild-type or mutant helicase protein was incubated with 1.3 pmol of radiolabeled 26-mer ssRNA oligonucleotides in 20 μl of helicase reaction buffer (30 mM Tris-HCl [pH 7.5], 3 mM MgCl 2 , 10 mM DTT, and 1 μl of RNasin [Promega]). As a control, labeled RNA without VP6 was incubated in one reaction mixture. The binding reaction mixture was incubated at 37°C for 15 min; then the reaction was terminated by the addition of 5× RNA loading dye containing 0.5% Nonidet P-40, and the mixture was subsequently electrophoresed on a 4% polyacrylamide-0.5× Tris-borate-EDTA gel buffer. The bound complex was visualized by autoradiography. (A) Autoradiogram of PAGE showing the positions of bound RNA of the wild-type and three mutant VP6 proteins and free unbound RNA. An RNA probe without VP6 was also resolved as a control (indicated as RNA). (B) The radioactivity of labeled proteins in each reaction mixture was estimated by phosphorimager as described in Materials and Methods, and the efficiencies of the RNA binding activities of the three mutant proteins were compared with that of the wild-type VP6. The RNA probe control is indicated as C. The figure shows the mean values of the results of three different experiments.

    Techniques Used: RNA Binding Assay, Mutagenesis, Incubation, Labeling, Binding Assay, Autoradiography, Polyacrylamide Gel Electrophoresis, Radioactivity

    Analysis of VP6-RNA complexes by gel filtration chromatography. Purified VP6 was incubated with S7 transcript in 30 mM Tris-HCl (pH 7.5), 10 mM DTT, 3 mM MgCl 2 , and 2.5 U of RNasin. The reaction mixture was incubated at 37°C for 30 min. Products were loaded on to an equilibrated Hi-Load Superdex-200 gel filtration column and eluted with same buffer at a flow rate of 0.3 ml/min as described in Materials and Methods. The column was calibrated with a set of globular protein standards (Amersham Pharmacia Biotech) consisting of ferritin (440 kDa), catalase (232 kDa), aldolase (158 kDa), albumin (43 kDa), and myoglobulin (17 kDa) prior to the VP6 loading. The arrows point to the positions of their elution. Note that the three peaks of VP6 eluted near the positions of catalase, aldolase, and albumin, which are equivalent to the hexamer, tetramer, and monomer of VP6, respectively.
    Figure Legend Snippet: Analysis of VP6-RNA complexes by gel filtration chromatography. Purified VP6 was incubated with S7 transcript in 30 mM Tris-HCl (pH 7.5), 10 mM DTT, 3 mM MgCl 2 , and 2.5 U of RNasin. The reaction mixture was incubated at 37°C for 30 min. Products were loaded on to an equilibrated Hi-Load Superdex-200 gel filtration column and eluted with same buffer at a flow rate of 0.3 ml/min as described in Materials and Methods. The column was calibrated with a set of globular protein standards (Amersham Pharmacia Biotech) consisting of ferritin (440 kDa), catalase (232 kDa), aldolase (158 kDa), albumin (43 kDa), and myoglobulin (17 kDa) prior to the VP6 loading. The arrows point to the positions of their elution. Note that the three peaks of VP6 eluted near the positions of catalase, aldolase, and albumin, which are equivalent to the hexamer, tetramer, and monomer of VP6, respectively.

    Techniques Used: Filtration, Chromatography, Purification, Incubation, Flow Cytometry

    ATPase activities of the wild-type (WT) and mutant VP6 proteins. One-half microgram of wild-type VP6 or each VP6 mutant was incubated at 37°C for 15 min in a reaction mixture (20 μl) containing 30 mM Tris-HCl (pH 7.5), 3 mM MgCl 2 , 1 mM MnCl 2 , 10 mM DTT, 50 mg of bovine serum albumin per ml, 2 μg of poly(U), and 1 μCi of [γ- 32 P]ATP. The reaction product was analyzed by spotting 2-μl samples onto polyethyleneimine-cellulose plates, and the chromatograms were developed in 0.75 M potassium phosphate (pH 3.5). The plates were subsequently dried and autoradiographed. The amount of radioactivity at each position was estimated by phosphorimager, and the efficiencies of the ATPase activities of the three mutants were compared, with that of the wild type arbitrarily assigned a value of 100%. The control (C) represents a reaction mixture without VP6. The figure shows the mean values of the results of three different experiments.
    Figure Legend Snippet: ATPase activities of the wild-type (WT) and mutant VP6 proteins. One-half microgram of wild-type VP6 or each VP6 mutant was incubated at 37°C for 15 min in a reaction mixture (20 μl) containing 30 mM Tris-HCl (pH 7.5), 3 mM MgCl 2 , 1 mM MnCl 2 , 10 mM DTT, 50 mg of bovine serum albumin per ml, 2 μg of poly(U), and 1 μCi of [γ- 32 P]ATP. The reaction product was analyzed by spotting 2-μl samples onto polyethyleneimine-cellulose plates, and the chromatograms were developed in 0.75 M potassium phosphate (pH 3.5). The plates were subsequently dried and autoradiographed. The amount of radioactivity at each position was estimated by phosphorimager, and the efficiencies of the ATPase activities of the three mutants were compared, with that of the wild type arbitrarily assigned a value of 100%. The control (C) represents a reaction mixture without VP6. The figure shows the mean values of the results of three different experiments.

    Techniques Used: Mutagenesis, Incubation, Radioactivity

    ATP binding activities of the wild-type (WT) and mutant VP6 proteins. One-half microgram of protein was mixed with 0.5 μM oxidized [α- 32 P]ATP (400 Ci/mmol) in 50 mM Tris-HCl (pH 7.5), 5 mM MgCl 2 , and 1 mM DTT. The reaction was allowed to proceed overnight on ice in the presence of 7.5 mM NaBH 3 CN. The reaction was stopped by the addition of 5× SDS sample buffer, the mixture was boiled, and protein complexes were loaded onto an SDS-10% PAGE gel. After electrophoresis, the gel was dried and autoradiographed to determine the distribution of labeled proteins. (A) Autoradiogram of the E 157 N and K 110 N mutants and wild-type VP6 bound to [α- 32 P]ATP. (B) The efficiencies of the ATP binding activities of the mutant proteins were compared with that of the wild-type VP6 as described in Materials and Methods. The figure represents the mean values of the results of three different experiments.
    Figure Legend Snippet: ATP binding activities of the wild-type (WT) and mutant VP6 proteins. One-half microgram of protein was mixed with 0.5 μM oxidized [α- 32 P]ATP (400 Ci/mmol) in 50 mM Tris-HCl (pH 7.5), 5 mM MgCl 2 , and 1 mM DTT. The reaction was allowed to proceed overnight on ice in the presence of 7.5 mM NaBH 3 CN. The reaction was stopped by the addition of 5× SDS sample buffer, the mixture was boiled, and protein complexes were loaded onto an SDS-10% PAGE gel. After electrophoresis, the gel was dried and autoradiographed to determine the distribution of labeled proteins. (A) Autoradiogram of the E 157 N and K 110 N mutants and wild-type VP6 bound to [α- 32 P]ATP. (B) The efficiencies of the ATP binding activities of the mutant proteins were compared with that of the wild-type VP6 as described in Materials and Methods. The figure represents the mean values of the results of three different experiments.

    Techniques Used: Binding Assay, Mutagenesis, Polyacrylamide Gel Electrophoresis, Electrophoresis, Labeling

    2) Product Images from "Defining the Structure-Function Relationships of Bluetongue Virus Helicase Protein VP6"

    Article Title: Defining the Structure-Function Relationships of Bluetongue Virus Helicase Protein VP6

    Journal: Journal of Virology

    doi: 10.1128/JVI.77.21.11347-11356.2003

    RNA binding activities of wild-type (WT) and mutant helicase proteins. The wild-type or mutant protein was incubated with labeled short ssRNA under standard reaction conditions. One-half microgram of the wild-type or mutant helicase protein was incubated with 1.3 pmol of radiolabeled 26-mer ssRNA oligonucleotides in 20 μl of helicase reaction buffer (30 mM Tris-HCl [pH 7.5], 3 mM MgCl 2 , 10 mM DTT, and 1 μl of RNasin [Promega]). As a control, labeled RNA without VP6 was incubated in one reaction mixture. The binding reaction mixture was incubated at 37°C for 15 min; then the reaction was terminated by the addition of 5× RNA loading dye containing 0.5% Nonidet P-40, and the mixture was subsequently electrophoresed on a 4% polyacrylamide-0.5× Tris-borate-EDTA gel buffer. The bound complex was visualized by autoradiography. (A) Autoradiogram of PAGE showing the positions of bound RNA of the wild-type and three mutant VP6 proteins and free unbound RNA. An RNA probe without VP6 was also resolved as a control (indicated as RNA). (B) The radioactivity of labeled proteins in each reaction mixture was estimated by phosphorimager as described in Materials and Methods, and the efficiencies of the RNA binding activities of the three mutant proteins were compared with that of the wild-type VP6. The RNA probe control is indicated as C. The figure shows the mean values of the results of three different experiments.
    Figure Legend Snippet: RNA binding activities of wild-type (WT) and mutant helicase proteins. The wild-type or mutant protein was incubated with labeled short ssRNA under standard reaction conditions. One-half microgram of the wild-type or mutant helicase protein was incubated with 1.3 pmol of radiolabeled 26-mer ssRNA oligonucleotides in 20 μl of helicase reaction buffer (30 mM Tris-HCl [pH 7.5], 3 mM MgCl 2 , 10 mM DTT, and 1 μl of RNasin [Promega]). As a control, labeled RNA without VP6 was incubated in one reaction mixture. The binding reaction mixture was incubated at 37°C for 15 min; then the reaction was terminated by the addition of 5× RNA loading dye containing 0.5% Nonidet P-40, and the mixture was subsequently electrophoresed on a 4% polyacrylamide-0.5× Tris-borate-EDTA gel buffer. The bound complex was visualized by autoradiography. (A) Autoradiogram of PAGE showing the positions of bound RNA of the wild-type and three mutant VP6 proteins and free unbound RNA. An RNA probe without VP6 was also resolved as a control (indicated as RNA). (B) The radioactivity of labeled proteins in each reaction mixture was estimated by phosphorimager as described in Materials and Methods, and the efficiencies of the RNA binding activities of the three mutant proteins were compared with that of the wild-type VP6. The RNA probe control is indicated as C. The figure shows the mean values of the results of three different experiments.

    Techniques Used: RNA Binding Assay, Mutagenesis, Incubation, Labeling, Binding Assay, Autoradiography, Polyacrylamide Gel Electrophoresis, Radioactivity

    Analysis of VP6-RNA complexes by gel filtration chromatography. Purified VP6 was incubated with S7 transcript in 30 mM Tris-HCl (pH 7.5), 10 mM DTT, 3 mM MgCl 2 , and 2.5 U of RNasin. The reaction mixture was incubated at 37°C for 30 min. Products were loaded on to an equilibrated Hi-Load Superdex-200 gel filtration column and eluted with same buffer at a flow rate of 0.3 ml/min as described in Materials and Methods. The column was calibrated with a set of globular protein standards (Amersham Pharmacia Biotech) consisting of ferritin (440 kDa), catalase (232 kDa), aldolase (158 kDa), albumin (43 kDa), and myoglobulin (17 kDa) prior to the VP6 loading. The arrows point to the positions of their elution. Note that the three peaks of VP6 eluted near the positions of catalase, aldolase, and albumin, which are equivalent to the hexamer, tetramer, and monomer of VP6, respectively.
    Figure Legend Snippet: Analysis of VP6-RNA complexes by gel filtration chromatography. Purified VP6 was incubated with S7 transcript in 30 mM Tris-HCl (pH 7.5), 10 mM DTT, 3 mM MgCl 2 , and 2.5 U of RNasin. The reaction mixture was incubated at 37°C for 30 min. Products were loaded on to an equilibrated Hi-Load Superdex-200 gel filtration column and eluted with same buffer at a flow rate of 0.3 ml/min as described in Materials and Methods. The column was calibrated with a set of globular protein standards (Amersham Pharmacia Biotech) consisting of ferritin (440 kDa), catalase (232 kDa), aldolase (158 kDa), albumin (43 kDa), and myoglobulin (17 kDa) prior to the VP6 loading. The arrows point to the positions of their elution. Note that the three peaks of VP6 eluted near the positions of catalase, aldolase, and albumin, which are equivalent to the hexamer, tetramer, and monomer of VP6, respectively.

    Techniques Used: Filtration, Chromatography, Purification, Incubation, Flow Cytometry

    ATPase activities of the wild-type (WT) and mutant VP6 proteins. One-half microgram of wild-type VP6 or each VP6 mutant was incubated at 37°C for 15 min in a reaction mixture (20 μl) containing 30 mM Tris-HCl (pH 7.5), 3 mM MgCl 2 , 1 mM MnCl 2 , 10 mM DTT, 50 mg of bovine serum albumin per ml, 2 μg of poly(U), and 1 μCi of [γ- 32 P]ATP. The reaction product was analyzed by spotting 2-μl samples onto polyethyleneimine-cellulose plates, and the chromatograms were developed in 0.75 M potassium phosphate (pH 3.5). The plates were subsequently dried and autoradiographed. The amount of radioactivity at each position was estimated by phosphorimager, and the efficiencies of the ATPase activities of the three mutants were compared, with that of the wild type arbitrarily assigned a value of 100%. The control (C) represents a reaction mixture without VP6. The figure shows the mean values of the results of three different experiments.
    Figure Legend Snippet: ATPase activities of the wild-type (WT) and mutant VP6 proteins. One-half microgram of wild-type VP6 or each VP6 mutant was incubated at 37°C for 15 min in a reaction mixture (20 μl) containing 30 mM Tris-HCl (pH 7.5), 3 mM MgCl 2 , 1 mM MnCl 2 , 10 mM DTT, 50 mg of bovine serum albumin per ml, 2 μg of poly(U), and 1 μCi of [γ- 32 P]ATP. The reaction product was analyzed by spotting 2-μl samples onto polyethyleneimine-cellulose plates, and the chromatograms were developed in 0.75 M potassium phosphate (pH 3.5). The plates were subsequently dried and autoradiographed. The amount of radioactivity at each position was estimated by phosphorimager, and the efficiencies of the ATPase activities of the three mutants were compared, with that of the wild type arbitrarily assigned a value of 100%. The control (C) represents a reaction mixture without VP6. The figure shows the mean values of the results of three different experiments.

    Techniques Used: Mutagenesis, Incubation, Radioactivity

    ATP binding activities of the wild-type (WT) and mutant VP6 proteins. One-half microgram of protein was mixed with 0.5 μM oxidized [α- 32 P]ATP (400 Ci/mmol) in 50 mM Tris-HCl (pH 7.5), 5 mM MgCl 2 , and 1 mM DTT. The reaction was allowed to proceed overnight on ice in the presence of 7.5 mM NaBH 3 CN. The reaction was stopped by the addition of 5× SDS sample buffer, the mixture was boiled, and protein complexes were loaded onto an SDS-10% PAGE gel. After electrophoresis, the gel was dried and autoradiographed to determine the distribution of labeled proteins. (A) Autoradiogram of the E 157 N and K 110 N mutants and wild-type VP6 bound to [α- 32 P]ATP. (B) The efficiencies of the ATP binding activities of the mutant proteins were compared with that of the wild-type VP6 as described in Materials and Methods. The figure represents the mean values of the results of three different experiments.
    Figure Legend Snippet: ATP binding activities of the wild-type (WT) and mutant VP6 proteins. One-half microgram of protein was mixed with 0.5 μM oxidized [α- 32 P]ATP (400 Ci/mmol) in 50 mM Tris-HCl (pH 7.5), 5 mM MgCl 2 , and 1 mM DTT. The reaction was allowed to proceed overnight on ice in the presence of 7.5 mM NaBH 3 CN. The reaction was stopped by the addition of 5× SDS sample buffer, the mixture was boiled, and protein complexes were loaded onto an SDS-10% PAGE gel. After electrophoresis, the gel was dried and autoradiographed to determine the distribution of labeled proteins. (A) Autoradiogram of the E 157 N and K 110 N mutants and wild-type VP6 bound to [α- 32 P]ATP. (B) The efficiencies of the ATP binding activities of the mutant proteins were compared with that of the wild-type VP6 as described in Materials and Methods. The figure represents the mean values of the results of three different experiments.

    Techniques Used: Binding Assay, Mutagenesis, Polyacrylamide Gel Electrophoresis, Electrophoresis, Labeling

    3) Product Images from "Activation of OASIS family, ER stress transducers, is dependent on its stabilization"

    Article Title: Activation of OASIS family, ER stress transducers, is dependent on its stabilization

    Journal: Cell Death and Differentiation

    doi: 10.1038/cdd.2012.77

    Stabilization of BBF2H7 and OASIS under ER stress conditions enhances transcription of their target genes for chondrogenesis and osteogenesis. ( a ) Reporter assay with overexpression of BBF2H7 and OASIS. (Left) ATDC5 cells were transiently transfected with a luciferase reporter driven by a 1.0-kb Sec23a promoter (pGL3-Sec23a-luc), a Renilla vector and an empty vector (control) or a vector expressing BBF2H7. (Right) MC3T3-E1 cells were transiently transfected with a luciferase reporter driven by a 2.3-kb Col1a1 promoter (pGL3-Col1a1-luc), a Renilla vector and an empty vector (control) or a vector expressing OASIS. The relative luciferase activities were determined with or without (NT) treatment with 5 μ M lactacystin (Lact), 1 μ M thapsigargin (Tg), 3 μ g/ml tunicamycin (Tm) or 1 mM DTT and/or 5 μ M MG132 for 12 h. ( b ) Reporter assay with the knockdown of BBF2H7 and OASIS. (Left) ATDC5 cells were transiently transfected with BBF2H7 siRNA, and then transfected with a luciferase reporter driven by the Sec23a promoter (pGL3-Sec23a-luc) and a Renilla vector. (Right) MC3T3-E1 cells were transiently transfected with OASIS siRNA, and then transfected with a luciferase reporter driven by the Col1a1 promoter (pGL3-Col1a1-luc) and a Renilla vector. The relative luciferase activities in the cells were determined with or without treatment with 5 μ M MG132 or 1 μ M Tg for 12 h (mean±S.D.; n =3; * P
    Figure Legend Snippet: Stabilization of BBF2H7 and OASIS under ER stress conditions enhances transcription of their target genes for chondrogenesis and osteogenesis. ( a ) Reporter assay with overexpression of BBF2H7 and OASIS. (Left) ATDC5 cells were transiently transfected with a luciferase reporter driven by a 1.0-kb Sec23a promoter (pGL3-Sec23a-luc), a Renilla vector and an empty vector (control) or a vector expressing BBF2H7. (Right) MC3T3-E1 cells were transiently transfected with a luciferase reporter driven by a 2.3-kb Col1a1 promoter (pGL3-Col1a1-luc), a Renilla vector and an empty vector (control) or a vector expressing OASIS. The relative luciferase activities were determined with or without (NT) treatment with 5 μ M lactacystin (Lact), 1 μ M thapsigargin (Tg), 3 μ g/ml tunicamycin (Tm) or 1 mM DTT and/or 5 μ M MG132 for 12 h. ( b ) Reporter assay with the knockdown of BBF2H7 and OASIS. (Left) ATDC5 cells were transiently transfected with BBF2H7 siRNA, and then transfected with a luciferase reporter driven by the Sec23a promoter (pGL3-Sec23a-luc) and a Renilla vector. (Right) MC3T3-E1 cells were transiently transfected with OASIS siRNA, and then transfected with a luciferase reporter driven by the Col1a1 promoter (pGL3-Col1a1-luc) and a Renilla vector. The relative luciferase activities in the cells were determined with or without treatment with 5 μ M MG132 or 1 μ M Tg for 12 h (mean±S.D.; n =3; * P

    Techniques Used: Reporter Assay, Over Expression, Transfection, Luciferase, Plasmid Preparation, Expressing

    Full-length BBF2H7 and OASIS are easily degraded under normal conditions and are elevated under ER stress conditions. ( a ) Predicted peptide features of mouse BBF2H7, OASIS and ATF6 α . ( b ) Endogenous BiP interacts with endogenous ATF6 α but not with OASIS or BBF2H7. Co-immunoprecipitation (IP) of BiP and ATF6 α , OASIS or BBF2H7 in MC3T3-E1 (left) or HeLa (right) cells. The cell lysates were immunoprecipitated with the anti-KDEL antibody, which recognizes BiP, and then immunoblotted (IB) with anti-ATF6 α , anti-OASIS (left) or anti-BBF2H7 (right). ( c and d ) Expression of exogenous OASIS ( c ) and BBF2H7 ( d ). MEF tet-off-FLAG-OASIS or -BBF2H7 cells were cultured in medium containing 1 μ g/ml DOX (lane 1). Flag-tagged full-length (F) or activated N-terminal (N) OASIS or BBF2H7 expression was induced by culturing the cells in DOX-free medium for 12 h in the absence or presence of the ER stressor thapsigargin (Tg; 1 μ M), and/or the proteasomal inhibitors MG132 (3 μ M) or lactacystin (lactacys; 5 μ M). Xbp1 and β-actin mRNA levels were measured by RT-PCR. The spliced form of Xbp1 mRNA ( Xbp1-s ) appeared upon treatment with the ER stressor but not with the proteasomal inhibitor. Xbp1-u represents the unspliced form of Xbp1. Equal amounts of total protein or RNA were analyzed in each lane. ( e ) Expression of endogenous BBF2H7 and ATF6 α in HeLa cells. Cells were incubated with 3 μ M MG132, 3 μ M lactacystin, 1 mM DTT (an ER stressor), or DTT with MG132 for 4 h, and lysates were immunoblotted with anti-ATF6 α or anti-BBF2H7. Xbp1 and β-actin mRNA levels were measured by RT-PCR. ( f ) Expression of endogenous BBF2H7 and OASIS in C6 glioma cells. Cells were incubated with 3 μ M MG132 or 1 mM DTT for the indicated times. (Upper) The lysates were western blotted with anti-BBF2H7 or anti-OASIS. (Lower) The levels of Xbp1 , Bbf2h7 , Oasis and β-actin mRNAs were measured by RT-PCR. ( g ) C6 glioma cells were incubated in the presence or absence of 3 μ M MG132 for 8 h, then co-stained with anti-BBF2H7 or anti-OASIS and anti-KDEL. MG132 treatment increased the immunoreactivity of BBF2H7 and OASIS both in the ER and in the nucleus
    Figure Legend Snippet: Full-length BBF2H7 and OASIS are easily degraded under normal conditions and are elevated under ER stress conditions. ( a ) Predicted peptide features of mouse BBF2H7, OASIS and ATF6 α . ( b ) Endogenous BiP interacts with endogenous ATF6 α but not with OASIS or BBF2H7. Co-immunoprecipitation (IP) of BiP and ATF6 α , OASIS or BBF2H7 in MC3T3-E1 (left) or HeLa (right) cells. The cell lysates were immunoprecipitated with the anti-KDEL antibody, which recognizes BiP, and then immunoblotted (IB) with anti-ATF6 α , anti-OASIS (left) or anti-BBF2H7 (right). ( c and d ) Expression of exogenous OASIS ( c ) and BBF2H7 ( d ). MEF tet-off-FLAG-OASIS or -BBF2H7 cells were cultured in medium containing 1 μ g/ml DOX (lane 1). Flag-tagged full-length (F) or activated N-terminal (N) OASIS or BBF2H7 expression was induced by culturing the cells in DOX-free medium for 12 h in the absence or presence of the ER stressor thapsigargin (Tg; 1 μ M), and/or the proteasomal inhibitors MG132 (3 μ M) or lactacystin (lactacys; 5 μ M). Xbp1 and β-actin mRNA levels were measured by RT-PCR. The spliced form of Xbp1 mRNA ( Xbp1-s ) appeared upon treatment with the ER stressor but not with the proteasomal inhibitor. Xbp1-u represents the unspliced form of Xbp1. Equal amounts of total protein or RNA were analyzed in each lane. ( e ) Expression of endogenous BBF2H7 and ATF6 α in HeLa cells. Cells were incubated with 3 μ M MG132, 3 μ M lactacystin, 1 mM DTT (an ER stressor), or DTT with MG132 for 4 h, and lysates were immunoblotted with anti-ATF6 α or anti-BBF2H7. Xbp1 and β-actin mRNA levels were measured by RT-PCR. ( f ) Expression of endogenous BBF2H7 and OASIS in C6 glioma cells. Cells were incubated with 3 μ M MG132 or 1 mM DTT for the indicated times. (Upper) The lysates were western blotted with anti-BBF2H7 or anti-OASIS. (Lower) The levels of Xbp1 , Bbf2h7 , Oasis and β-actin mRNAs were measured by RT-PCR. ( g ) C6 glioma cells were incubated in the presence or absence of 3 μ M MG132 for 8 h, then co-stained with anti-BBF2H7 or anti-OASIS and anti-KDEL. MG132 treatment increased the immunoreactivity of BBF2H7 and OASIS both in the ER and in the nucleus

    Techniques Used: Immunoprecipitation, Expressing, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Incubation, Western Blot, Staining

    4) Product Images from "The VCP-UBXN1 Complex Mediates Triage of Ubiquitylated Cytosolic Proteins Bound to the BAG6 Complex"

    Article Title: The VCP-UBXN1 Complex Mediates Triage of Ubiquitylated Cytosolic Proteins Bound to the BAG6 Complex

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00154-18

    UBXN1 recognizes ubiquitylated substrates in a BAG6-dependent manner. (A) Wild-type and UBXN1 KO cells were transfected with OPG-TASK and HA-ubiquitin (Ub). Cells were denatured in 2% SDS lysis buffer, and HA-ubiquitin was affinity purified and probed for ubiquitylated OPG-TASK (*, heavy and light chains). (B) HEK-293T cells were transfected with BAG6 siRNA, OPG-TASK, and Myc-UBXN1. Myc-UBXN1 immunoprecipitates were probed for OPG-TASK. The fraction of OPG-TASK bound to UBXN1 was calculated by densitometry and normalized to the levels of OPG-TASK in the cell lysate. (C) HA-PrP mut was transiently expressed in HEK-293T cells that were treated with 1 μM bortezomib for 2 h. Stabilized HA-PrP mut was immunoprecipitated via the HA tag and probed for UBXN1. GFP was used as a transfection control. (D) Domains within UBXN1. UBA, ubiquitin associated; cc, coiled coil; UBX, ubiquitin X. HEK-293T cells were transfected with HA-PrP mut and wild-type Myc-UBXN1 or the UBA M13A F15A mutant (UBA*). Cells were treated with bortezomib for 2 h, and Myc-UBXN1 was immunopurified. (E) HeLa Flp-in T-REX cells were transfected with HA-PrP mut , Myc-UBXN1, and His-ubiquitin and treated with 1 μM bortezomib, as indicated. Consecutive nondenaturing (for Myc-UBXN1) and denaturing (for HA-PrP mut ) immunoprecipitations were performed, and immunoprecipitates were probed for His-ubiquitin to demonstrate that PrP bound to UBXN1 is ubiquitylated. (F) HEK-293T cells were transfected with Myc-UBXN1 and treated with 1 μM bortezomib or 1.5 μM DTT for 6 h. Myc-UBXN1 was purified from cell lysates and probed for BAG6. The levels of BAG6 bound to UBXN1 were determined by densitometry and normalized to BAG6 levels in the whole-cell lysate ( n = 3).
    Figure Legend Snippet: UBXN1 recognizes ubiquitylated substrates in a BAG6-dependent manner. (A) Wild-type and UBXN1 KO cells were transfected with OPG-TASK and HA-ubiquitin (Ub). Cells were denatured in 2% SDS lysis buffer, and HA-ubiquitin was affinity purified and probed for ubiquitylated OPG-TASK (*, heavy and light chains). (B) HEK-293T cells were transfected with BAG6 siRNA, OPG-TASK, and Myc-UBXN1. Myc-UBXN1 immunoprecipitates were probed for OPG-TASK. The fraction of OPG-TASK bound to UBXN1 was calculated by densitometry and normalized to the levels of OPG-TASK in the cell lysate. (C) HA-PrP mut was transiently expressed in HEK-293T cells that were treated with 1 μM bortezomib for 2 h. Stabilized HA-PrP mut was immunoprecipitated via the HA tag and probed for UBXN1. GFP was used as a transfection control. (D) Domains within UBXN1. UBA, ubiquitin associated; cc, coiled coil; UBX, ubiquitin X. HEK-293T cells were transfected with HA-PrP mut and wild-type Myc-UBXN1 or the UBA M13A F15A mutant (UBA*). Cells were treated with bortezomib for 2 h, and Myc-UBXN1 was immunopurified. (E) HeLa Flp-in T-REX cells were transfected with HA-PrP mut , Myc-UBXN1, and His-ubiquitin and treated with 1 μM bortezomib, as indicated. Consecutive nondenaturing (for Myc-UBXN1) and denaturing (for HA-PrP mut ) immunoprecipitations were performed, and immunoprecipitates were probed for His-ubiquitin to demonstrate that PrP bound to UBXN1 is ubiquitylated. (F) HEK-293T cells were transfected with Myc-UBXN1 and treated with 1 μM bortezomib or 1.5 μM DTT for 6 h. Myc-UBXN1 was purified from cell lysates and probed for BAG6. The levels of BAG6 bound to UBXN1 were determined by densitometry and normalized to BAG6 levels in the whole-cell lysate ( n = 3).

    Techniques Used: Transfection, Lysis, Affinity Purification, Immunoprecipitation, Mutagenesis, Purification

    Loss of UBXN1 sensitizes cells to proteotoxic stress. (A) HeLa Flp-in T-REX cells were transfected with VCAM1-GFP and treated with 100 nM CT08 for 24 h. A total of 1 μM bortezomib was added during the final 8 h of CT08 treatment. Cells were fixed, stained for calnexin, and imaged. In CT08-treated cells, VCAM-GFP no longer localized to the ER. Cotreatment with bortezomib and CT08 was cytotoxic, and few cells remained to be quantified; however, in surviving cells, VCAM-GFP was localized to an aggresome-like structure next to the nucleus. Data for 50 cells were quantified in each of 2 independent experiments. (B) Quantification of data in panel A. (C) To determine the migration of mature and unglycosylated wild-type PrP, cell lysates expressing HA-PrP were treated with EndoH and resolved on SDS-PAGE gels. (D) Wild-type HA-PrP-expressing HeLa Flp-in T-REX cells were treated with 1 μM CT08 or 1 μM bortezomib for 6 h, as indicated. Cells were fractionated into soluble and pellet fractions. Mature and unglycosylated PrPs are indicated. (E) Wild-type and UBXN1 KO cells were plated in triplicate into 96-well plates and treated with the indicated concentrations of bortezomib, CT08, DTT, or tunicamycin (Tu) for 24 h. Cell viability was measured and normalized to the value for DMSO-treated controls for each cell line. **, P ≤ 0.01 (as determined by one-way ANOVA) ( n = 2).
    Figure Legend Snippet: Loss of UBXN1 sensitizes cells to proteotoxic stress. (A) HeLa Flp-in T-REX cells were transfected with VCAM1-GFP and treated with 100 nM CT08 for 24 h. A total of 1 μM bortezomib was added during the final 8 h of CT08 treatment. Cells were fixed, stained for calnexin, and imaged. In CT08-treated cells, VCAM-GFP no longer localized to the ER. Cotreatment with bortezomib and CT08 was cytotoxic, and few cells remained to be quantified; however, in surviving cells, VCAM-GFP was localized to an aggresome-like structure next to the nucleus. Data for 50 cells were quantified in each of 2 independent experiments. (B) Quantification of data in panel A. (C) To determine the migration of mature and unglycosylated wild-type PrP, cell lysates expressing HA-PrP were treated with EndoH and resolved on SDS-PAGE gels. (D) Wild-type HA-PrP-expressing HeLa Flp-in T-REX cells were treated with 1 μM CT08 or 1 μM bortezomib for 6 h, as indicated. Cells were fractionated into soluble and pellet fractions. Mature and unglycosylated PrPs are indicated. (E) Wild-type and UBXN1 KO cells were plated in triplicate into 96-well plates and treated with the indicated concentrations of bortezomib, CT08, DTT, or tunicamycin (Tu) for 24 h. Cell viability was measured and normalized to the value for DMSO-treated controls for each cell line. **, P ≤ 0.01 (as determined by one-way ANOVA) ( n = 2).

    Techniques Used: Transfection, Staining, Migration, Expressing, SDS Page

    5) Product Images from "Scalable Purification and Characterization of the Anticancer Lunasin Peptide from Soybean"

    Article Title: Scalable Purification and Characterization of the Anticancer Lunasin Peptide from Soybean

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0035409

    Mass spectrometry of the purified lunasin-containing complex. (A, top panel) Deconvoluted spectrum of purified lunasin complex. The most abundant isotopic mass in the spectrum is at 14109.3 Da. The mass signal adjacent to lunasin complex (14207.3 Da) is the adduct of lunasin complex with phosphoric acid (plus 98 Da). (A, middle panel) Deconvoluted spectrum of reduced lunasin complex. The most abundant isotopic masses shown in the spectrum are lunasin (5141.3 Da) and soybean albumin long chain (8975.1 Da). (A, bottom panel) Deconvoluted spectrum of lunasin complex treated with DTT and IAA. The most abundant masses shown in the spectrum are lunasin (5256.3 Da) and soybean albumin long chain (9317.2 Da). The monoisotopic masses are 5139.28 Da and 5253.33 Da for lunasin and lunasin treated with DTT and IAA respectively. The monoisotopic masses of lunasin complex and soybean albumin long chain were too low to be detected. (B) Sequence of 2S albumin preproprotein. Sequence in red is corresponds to our purified lunasin and its monoisotopic and average molecular weights are 5139.27 and 5142.43 Da, respectively. Sequence in blue corresponds to soybean albumin long chain and its monoisotopic and average molecular weights are 8969.05 and 8975.17 Da, respectively.
    Figure Legend Snippet: Mass spectrometry of the purified lunasin-containing complex. (A, top panel) Deconvoluted spectrum of purified lunasin complex. The most abundant isotopic mass in the spectrum is at 14109.3 Da. The mass signal adjacent to lunasin complex (14207.3 Da) is the adduct of lunasin complex with phosphoric acid (plus 98 Da). (A, middle panel) Deconvoluted spectrum of reduced lunasin complex. The most abundant isotopic masses shown in the spectrum are lunasin (5141.3 Da) and soybean albumin long chain (8975.1 Da). (A, bottom panel) Deconvoluted spectrum of lunasin complex treated with DTT and IAA. The most abundant masses shown in the spectrum are lunasin (5256.3 Da) and soybean albumin long chain (9317.2 Da). The monoisotopic masses are 5139.28 Da and 5253.33 Da for lunasin and lunasin treated with DTT and IAA respectively. The monoisotopic masses of lunasin complex and soybean albumin long chain were too low to be detected. (B) Sequence of 2S albumin preproprotein. Sequence in red is corresponds to our purified lunasin and its monoisotopic and average molecular weights are 5139.27 and 5142.43 Da, respectively. Sequence in blue corresponds to soybean albumin long chain and its monoisotopic and average molecular weights are 8969.05 and 8975.17 Da, respectively.

    Techniques Used: Mass Spectrometry, Purification, Sequencing

    Mass spectrometry of the purified lunasin. (A, top panel) Deconvoluted MS Spectra of purified lunasin. The monoisotopic mass of the purified lunasin was found to be 5139.25 Da, which is 114.02 Da higher than the expected monoisotopic mass (5025.23 Da) for the 43 amino-acid form of lunasin described in the literature. The mass difference suggests that the predominant form of our purified lunasin contains 44 amino acids and that it contains an additional asparagine residue. (A, middle panel) Deconvoluted spectrum of lunasin reduced with DTT. Reduction with DTT did not cause a mass shift, indicating there is no disulfide bond present in the purified lunasin. (A, bottom panel) Deconvoluted spectrum of lunasin complex treated with DTT and IAA. The monoisotopic mass of lunasin shifted to 5253.29 Da after alkylation with IAA, which is 114.04 Da higher than unalkylated lunasin. This mass shift confirmed that lunasin has two free cysteine residues as expected. (B) MS/MS spectrum of C-terminal peptide of lunasin. Calculated b and Y ions for the peptide GDDDDDDDDDN are shown in the table inset. The matched b (red) and Y (blue) ions detected match very well the expected fragment ion values for this peptide. Signals corresponding to the loss of one (green) or more H 2 O molecules, which are expected in MS/MS spectra of peptides with multiple acidic residues, are also evident in the spectrum. These [b – H 2 O] signals are consistent with the presence of the GDDDDDDDDDN peptide. This analysis confirmed that the residue at the C-terminus of lunasin purified from soybean is asparagine rather than aspartic acid.
    Figure Legend Snippet: Mass spectrometry of the purified lunasin. (A, top panel) Deconvoluted MS Spectra of purified lunasin. The monoisotopic mass of the purified lunasin was found to be 5139.25 Da, which is 114.02 Da higher than the expected monoisotopic mass (5025.23 Da) for the 43 amino-acid form of lunasin described in the literature. The mass difference suggests that the predominant form of our purified lunasin contains 44 amino acids and that it contains an additional asparagine residue. (A, middle panel) Deconvoluted spectrum of lunasin reduced with DTT. Reduction with DTT did not cause a mass shift, indicating there is no disulfide bond present in the purified lunasin. (A, bottom panel) Deconvoluted spectrum of lunasin complex treated with DTT and IAA. The monoisotopic mass of lunasin shifted to 5253.29 Da after alkylation with IAA, which is 114.04 Da higher than unalkylated lunasin. This mass shift confirmed that lunasin has two free cysteine residues as expected. (B) MS/MS spectrum of C-terminal peptide of lunasin. Calculated b and Y ions for the peptide GDDDDDDDDDN are shown in the table inset. The matched b (red) and Y (blue) ions detected match very well the expected fragment ion values for this peptide. Signals corresponding to the loss of one (green) or more H 2 O molecules, which are expected in MS/MS spectra of peptides with multiple acidic residues, are also evident in the spectrum. These [b – H 2 O] signals are consistent with the presence of the GDDDDDDDDDN peptide. This analysis confirmed that the residue at the C-terminus of lunasin purified from soybean is asparagine rather than aspartic acid.

    Techniques Used: Mass Spectrometry, Purification

    6) Product Images from "Murine osteoclasts secrete serine protease HtrA1 capable of degrading osteoprotegerin in the bone microenvironment"

    Article Title: Murine osteoclasts secrete serine protease HtrA1 capable of degrading osteoprotegerin in the bone microenvironment

    Journal: Communications Biology

    doi: 10.1038/s42003-019-0334-5

    HtrA1 recognizes the three-dimensional structure and cleaves osteoprotegerin (OPG). a OPG 22–196 (2 μg) was incubated with HtrA1 (0.5 μg) at 37 °C for the indicated times. The reaction mixture was treated with dithiothreitol (DTT, 10 mM) and iodoacetamide (IAA). Conventional treatment after incubation (−) was compared with reducing OPG 22–196 by pre-treatment with DTT and IAA before incubation (+). OPG fragment sequences were identified using sequence analysis software (Protein Pilot). Amino-acid residues contained in the detected peptides were counted. b OPG 22–196 (2 μg) was incubated with HtrA1 (0.5 μg) at 37 °C for 5 min. The reaction mixture was treated with DTT and IAA, and subjected to MALDI-TOF MS. The measurement of native OPG 22–196 was shown at 0 min. The measurement of reduced carbamidomethylated OPG fragments were shown after the incubation with HtrA1 for 5 min. m/z indicates the mass-to-charge ratio. Intact OPG 22–196 and reduced carbamidomethylated OPG 22–196 were detected before and after the incubation with HtrA1 ( m / z 19778 and 20750, respectively). Before the incubation, a doubly charged ion of OPG ( m / z 9909) was also detected (0 min). Two characteristic OPG fragment peaks (OPG 22–90 , OPG 91–196 , m / z 8673 and 12205) derived from OPG 22–196 were detected 5 min after the reaction. c After the incubation of OPG 22–196 with HtrA1, the reaction mixture was treated with DTT and IAA. The sequences of OPG fragments were identified using Protein Pilot. Detections of the C- and N-terminal residues in OPG fragments were counted. Leucine 90 showed the highest peak at 5 min (arrow).
    Figure Legend Snippet: HtrA1 recognizes the three-dimensional structure and cleaves osteoprotegerin (OPG). a OPG 22–196 (2 μg) was incubated with HtrA1 (0.5 μg) at 37 °C for the indicated times. The reaction mixture was treated with dithiothreitol (DTT, 10 mM) and iodoacetamide (IAA). Conventional treatment after incubation (−) was compared with reducing OPG 22–196 by pre-treatment with DTT and IAA before incubation (+). OPG fragment sequences were identified using sequence analysis software (Protein Pilot). Amino-acid residues contained in the detected peptides were counted. b OPG 22–196 (2 μg) was incubated with HtrA1 (0.5 μg) at 37 °C for 5 min. The reaction mixture was treated with DTT and IAA, and subjected to MALDI-TOF MS. The measurement of native OPG 22–196 was shown at 0 min. The measurement of reduced carbamidomethylated OPG fragments were shown after the incubation with HtrA1 for 5 min. m/z indicates the mass-to-charge ratio. Intact OPG 22–196 and reduced carbamidomethylated OPG 22–196 were detected before and after the incubation with HtrA1 ( m / z 19778 and 20750, respectively). Before the incubation, a doubly charged ion of OPG ( m / z 9909) was also detected (0 min). Two characteristic OPG fragment peaks (OPG 22–90 , OPG 91–196 , m / z 8673 and 12205) derived from OPG 22–196 were detected 5 min after the reaction. c After the incubation of OPG 22–196 with HtrA1, the reaction mixture was treated with DTT and IAA. The sequences of OPG fragments were identified using Protein Pilot. Detections of the C- and N-terminal residues in OPG fragments were counted. Leucine 90 showed the highest peak at 5 min (arrow).

    Techniques Used: Incubation, Sequencing, Software, Mass Spectrometry, Derivative Assay

    HtrA1 degrades osteoprotegerin (OPG), whereas MMP9 does not. a Full-length OPG (20 ng) was incubated with HtrA1, mutated HtrA1 (S328A), and MMP9 at 37 °C for 30, 60, and 120 min, and the remaining OPG was detected by western blotting. Amounts of proteases used were also shown. HtrA1 degraded OPG within 30 min, whereas neither mutated HtrA1 (S328A) nor MMP9. b Full-length OPG (2 μg) was incubated with HtrA1, HtrA1 (S328A), and MMP9 (0.5 μg each) at 37 °C for the indicated times. The reaction mixture was treated with dithiothreitol (DTT) and iodoacetamide (IAA), and subjected to mass spectrometry. The sequence of the OPG fragment was identified using sequence analysis software (Protein Pilot, AB SCIEX), and the number of OPG fragments was counted. Degradation of OPG by HtrA1 increased with longer reaction times. c Amino-acid residues contained in peptides detected by mass spectrometry were counted at each reaction time. The positions of the N terminus and C terminus of OPG were 22 and 401, respectively
    Figure Legend Snippet: HtrA1 degrades osteoprotegerin (OPG), whereas MMP9 does not. a Full-length OPG (20 ng) was incubated with HtrA1, mutated HtrA1 (S328A), and MMP9 at 37 °C for 30, 60, and 120 min, and the remaining OPG was detected by western blotting. Amounts of proteases used were also shown. HtrA1 degraded OPG within 30 min, whereas neither mutated HtrA1 (S328A) nor MMP9. b Full-length OPG (2 μg) was incubated with HtrA1, HtrA1 (S328A), and MMP9 (0.5 μg each) at 37 °C for the indicated times. The reaction mixture was treated with dithiothreitol (DTT) and iodoacetamide (IAA), and subjected to mass spectrometry. The sequence of the OPG fragment was identified using sequence analysis software (Protein Pilot, AB SCIEX), and the number of OPG fragments was counted. Degradation of OPG by HtrA1 increased with longer reaction times. c Amino-acid residues contained in peptides detected by mass spectrometry were counted at each reaction time. The positions of the N terminus and C terminus of OPG were 22 and 401, respectively

    Techniques Used: Incubation, Western Blot, Mass Spectrometry, Sequencing, Software

    7) Product Images from "Canonical nucleosides can be utilized by T4 DNA ligase as universal template bases at ligation junctions"

    Article Title: Canonical nucleosides can be utilized by T4 DNA ligase as universal template bases at ligation junctions

    Journal: Nucleic Acids Research

    doi:

    Time-dependent ligation reactions. The optimum reaction conditions were used, which are 30 mM Tris–HCl (pH 7.5), 10 mM DTT, 3 mM MgCl 2 , 10 µM ATP, 20% DMSO, 2.5 pmol acceptor molecule, 70 fmol donor molecule, 3.125 pmol C4 template and 3 U T4 DNA ligase at 22°C. The graph shows the plots and fitted curves of each of the four ligation reactions analyzed (see inset for oligonucleotide sequences). The observed rate constants ( k obs ) for each reaction are given on the graph. The data points are the average of two independent assays.
    Figure Legend Snippet: Time-dependent ligation reactions. The optimum reaction conditions were used, which are 30 mM Tris–HCl (pH 7.5), 10 mM DTT, 3 mM MgCl 2 , 10 µM ATP, 20% DMSO, 2.5 pmol acceptor molecule, 70 fmol donor molecule, 3.125 pmol C4 template and 3 U T4 DNA ligase at 22°C. The graph shows the plots and fitted curves of each of the four ligation reactions analyzed (see inset for oligonucleotide sequences). The observed rate constants ( k obs ) for each reaction are given on the graph. The data points are the average of two independent assays.

    Techniques Used: Ligation

    Optimization of ligation reactions. ( A and B ) ATP and MgCl 2 concentration-dependent ligations using (c + p c)/T2. The MgCl 2 concentration was first optimized under standard reaction conditions of 30 mM Tris–HCl (pH 7.5), 10 mM DTT, 1 mM ATP, 2.5 pmol acceptor molecule, 70 fmol donor molecule, 3.125 pmol template and 3 U T4 DNA ligase at 30°C for 18 h. An optimum concentration of 3 mM MgCl 2 was found, which was then used in the subsequent ATP-dependent assay. ( C ) DMSO-dependent ligation reaction using (c + p c)/C2 in 3 mM MgCl 2 and 10 µM ATP. ( D ) Time-dependent ligation reaction using (c + p c)/T2 in 3 mM MgCl 2 , 10 µM ATP and 20% DMSO. In each case, product is represented by squares and intermediate by circles.
    Figure Legend Snippet: Optimization of ligation reactions. ( A and B ) ATP and MgCl 2 concentration-dependent ligations using (c + p c)/T2. The MgCl 2 concentration was first optimized under standard reaction conditions of 30 mM Tris–HCl (pH 7.5), 10 mM DTT, 1 mM ATP, 2.5 pmol acceptor molecule, 70 fmol donor molecule, 3.125 pmol template and 3 U T4 DNA ligase at 30°C for 18 h. An optimum concentration of 3 mM MgCl 2 was found, which was then used in the subsequent ATP-dependent assay. ( C ) DMSO-dependent ligation reaction using (c + p c)/C2 in 3 mM MgCl 2 and 10 µM ATP. ( D ) Time-dependent ligation reaction using (c + p c)/T2 in 3 mM MgCl 2 , 10 µM ATP and 20% DMSO. In each case, product is represented by squares and intermediate by circles.

    Techniques Used: Ligation, Concentration Assay

    8) Product Images from "Exploring the Conserved Role of MANF in the Unfolded Protein Response in Drosophila melanogaster"

    Article Title: Exploring the Conserved Role of MANF in the Unfolded Protein Response in Drosophila melanogaster

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0151550

    Drug-induced ER stress upregulates DmManf expression. A–B) In Schneider 2 (S2) cells, ER stress was induced by thapsigargin (TG), tunicamycin (TM) and dithiothreitol (DTT). DMSO was used as a control treatment. A) The mRNA levels of DmManf and Hsc3 were analysed by qPCR, values were normalised to control treatment (DMSO). B) RT-PCR and agarose gel electrophoresis analysis revealed two transcripts of Xbp1 , unspliced ( Xbp1 u ) and spliced ( Xbp1 s ). RpL32 was used as a loading control. C–D) qPCR analysis of Hsc3 and Xbp1 expression in DmManf mutant (C) and DmManf overexpressing (D) larvae. Expression of Hsc3 was not altered but Xbp1s mRNA level was increased in response to overexpression of DmManf . The overexpression of DmManf resulted in 165-fold increase in DmManf mRNA level (±23, P
    Figure Legend Snippet: Drug-induced ER stress upregulates DmManf expression. A–B) In Schneider 2 (S2) cells, ER stress was induced by thapsigargin (TG), tunicamycin (TM) and dithiothreitol (DTT). DMSO was used as a control treatment. A) The mRNA levels of DmManf and Hsc3 were analysed by qPCR, values were normalised to control treatment (DMSO). B) RT-PCR and agarose gel electrophoresis analysis revealed two transcripts of Xbp1 , unspliced ( Xbp1 u ) and spliced ( Xbp1 s ). RpL32 was used as a loading control. C–D) qPCR analysis of Hsc3 and Xbp1 expression in DmManf mutant (C) and DmManf overexpressing (D) larvae. Expression of Hsc3 was not altered but Xbp1s mRNA level was increased in response to overexpression of DmManf . The overexpression of DmManf resulted in 165-fold increase in DmManf mRNA level (±23, P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Mutagenesis, Over Expression

    9) Product Images from "Dynamic flexibility of the ATPase p97 is important for its interprotomer motion transmission"

    Article Title: Dynamic flexibility of the ATPase p97 is important for its interprotomer motion transmission

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

    doi: 10.1073/pnas.1205853109

    Introduction of a disulfide bond to limit flexibility of the D1-D2 linker. ( A ) Rationale of selection of G610 and R465 for introducing a disulfide bond lock between the D1-D2 linker and its neighboring protomer. Residues G610 and R465 are shown in blue and green spheres, respectively. All cysteine residues present in wild-type p97 are located far from residues 610 and 465, shown in purple spheres. ( Lower Right ) A close-up view of the corresponding region of the overview figure. ( B ) Mass spectrum of trypsin-digested wild-type p97 and R465C/G610C double-cysteine mutant under nonreducing or reducing conditions. A peak at m/z of 5325.3 was found in R465C/G610C double-cysteine mutant but not in wild-type protein. This peak, which was eliminated by DTT treatment, corresponds to the potential digested protein fragment containing C465-C610 (theoretical value is 5,322.6). These results confirmed formation of disulfide bond between C465 and G610.
    Figure Legend Snippet: Introduction of a disulfide bond to limit flexibility of the D1-D2 linker. ( A ) Rationale of selection of G610 and R465 for introducing a disulfide bond lock between the D1-D2 linker and its neighboring protomer. Residues G610 and R465 are shown in blue and green spheres, respectively. All cysteine residues present in wild-type p97 are located far from residues 610 and 465, shown in purple spheres. ( Lower Right ) A close-up view of the corresponding region of the overview figure. ( B ) Mass spectrum of trypsin-digested wild-type p97 and R465C/G610C double-cysteine mutant under nonreducing or reducing conditions. A peak at m/z of 5325.3 was found in R465C/G610C double-cysteine mutant but not in wild-type protein. This peak, which was eliminated by DTT treatment, corresponds to the potential digested protein fragment containing C465-C610 (theoretical value is 5,322.6). These results confirmed formation of disulfide bond between C465 and G610.

    Techniques Used: Selection, Mutagenesis

    10) Product Images from "RNA structure analysis assisted by capillary electrophoresis"

    Article Title: RNA structure analysis assisted by capillary electrophoresis

    Journal: Nucleic Acids Research

    doi:

    Analysis of structurally heterogeneous BRCA1 Ex1a and transcript truncation to obtain a homogeneous structure. ( A ) Non-denaturing 10% polyacrylamide gel electrophoresis of 5′-end radiolabeled Ex1a transcript treated as follows: lane 1, dissolved in water and incubated at 20°C for 30 min; lane 2, heated to 75°C for 1 min (denaturation) and cooled slowly to 20°C (renaturation); lane 3, dissolved in the structure-probing buffer (10 mM Tris–HCl pH 7.2, 10 mM magnesium ions, 40 mM NaCl) and incubated at 20°C for 30 min; lane 4, dissolved as described for lane 3 and subjected to the denaturation/renaturation procedure; lane 5, dissolved as described for lane 3, carrier RNA added to a final concentration of 8 µM, and incubated at 20°C for 30 min; lane 6, carrier RNA added and subjected to denaturation/renaturation. ( B ) CE in non-denaturing conditions of Ex1a transcript fluorescently labeled at its 3′ end with TdT and: [R110]dUTP, [RG6]dUTP, [TAMRA]dUTP (shadowed peaks); TAMRA-500 internal standard (gray line). ( C ) CE in non-denaturing polymer at temperatures: 30, 45 and 60°C of Ex1a transcript end labeled with [R110]dUTP and Klenow fragment. ( D ) Non-denaturing 10% polyacrylamide gel electrophoresis of 5′-end radiolabeled Ex1a transcript (0.5 µM) (lane 1), and the same transcript hybridized with 18 nt of Rex1a oligodeoxynucleotide complementary to its 3′ end (lane 2). Hybridization of transcript (1 µM) with oligodeoxynucleotide (1 µM) was performed in 15 mM Tris–HCl (pH 7.2), 10 mM MgCl 2 , 1.5 mM DTT by heating the sample at 90°C for 1 min and fast cooling. Arrowhead indicates the position of hybrid migration. ( E ) CE in non-denaturing conditions of Ex1a102nt transcript labeled with TdT and [RG6]dUTP (gray line indicates ROX-500 internal standard).
    Figure Legend Snippet: Analysis of structurally heterogeneous BRCA1 Ex1a and transcript truncation to obtain a homogeneous structure. ( A ) Non-denaturing 10% polyacrylamide gel electrophoresis of 5′-end radiolabeled Ex1a transcript treated as follows: lane 1, dissolved in water and incubated at 20°C for 30 min; lane 2, heated to 75°C for 1 min (denaturation) and cooled slowly to 20°C (renaturation); lane 3, dissolved in the structure-probing buffer (10 mM Tris–HCl pH 7.2, 10 mM magnesium ions, 40 mM NaCl) and incubated at 20°C for 30 min; lane 4, dissolved as described for lane 3 and subjected to the denaturation/renaturation procedure; lane 5, dissolved as described for lane 3, carrier RNA added to a final concentration of 8 µM, and incubated at 20°C for 30 min; lane 6, carrier RNA added and subjected to denaturation/renaturation. ( B ) CE in non-denaturing conditions of Ex1a transcript fluorescently labeled at its 3′ end with TdT and: [R110]dUTP, [RG6]dUTP, [TAMRA]dUTP (shadowed peaks); TAMRA-500 internal standard (gray line). ( C ) CE in non-denaturing polymer at temperatures: 30, 45 and 60°C of Ex1a transcript end labeled with [R110]dUTP and Klenow fragment. ( D ) Non-denaturing 10% polyacrylamide gel electrophoresis of 5′-end radiolabeled Ex1a transcript (0.5 µM) (lane 1), and the same transcript hybridized with 18 nt of Rex1a oligodeoxynucleotide complementary to its 3′ end (lane 2). Hybridization of transcript (1 µM) with oligodeoxynucleotide (1 µM) was performed in 15 mM Tris–HCl (pH 7.2), 10 mM MgCl 2 , 1.5 mM DTT by heating the sample at 90°C for 1 min and fast cooling. Arrowhead indicates the position of hybrid migration. ( E ) CE in non-denaturing conditions of Ex1a102nt transcript labeled with TdT and [RG6]dUTP (gray line indicates ROX-500 internal standard).

    Techniques Used: Polyacrylamide Gel Electrophoresis, Incubation, Concentration Assay, Labeling, Hybridization, Migration

    11) Product Images from "Regulation of the catalytic function of topoisomerase II alpha through association with RNA"

    Article Title: Regulation of the catalytic function of topoisomerase II alpha through association with RNA

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkn614

    Characterization of the RNA-binding activity associated with topoisomerase IIα. ( A ) Either ssRNA or a partial dsRNA (2.5 nM) was incubated with an increasing amount of topoisomerase IIα (7.35, 14.7 or 29.4 nM) in a reaction mixture (10 μl) consisting of 20 mM HEPES-NaOH, pH 7.4, 2 mM DTT, 0.05% NP-40 and 7 mM MgCl 2 . ( B ) ATP (2 mM) or ( C ) increasing concentration of salt (0–300 mM) was included in a standard reaction mixture (see Materials and Methods section), containing topoisomerase IIα (7.35 nM) and 60-bp flush-ended dsRNA. ( D ) Supershift assay was performed by sequentially incubating topoisomerase IIα (7.35 nM) (lanes 2–4 and 6–7) with 60-bp flush-ended dsRNA and monoclonal antibody (0.125 or 0.25 μg) specific for the C-terminal domain of topoisomerase IIα (aa 1245–1361) (lanes 3–5). All RNA–topoisomerase IIα complexes were detected as described in Figure 1 .
    Figure Legend Snippet: Characterization of the RNA-binding activity associated with topoisomerase IIα. ( A ) Either ssRNA or a partial dsRNA (2.5 nM) was incubated with an increasing amount of topoisomerase IIα (7.35, 14.7 or 29.4 nM) in a reaction mixture (10 μl) consisting of 20 mM HEPES-NaOH, pH 7.4, 2 mM DTT, 0.05% NP-40 and 7 mM MgCl 2 . ( B ) ATP (2 mM) or ( C ) increasing concentration of salt (0–300 mM) was included in a standard reaction mixture (see Materials and Methods section), containing topoisomerase IIα (7.35 nM) and 60-bp flush-ended dsRNA. ( D ) Supershift assay was performed by sequentially incubating topoisomerase IIα (7.35 nM) (lanes 2–4 and 6–7) with 60-bp flush-ended dsRNA and monoclonal antibody (0.125 or 0.25 μg) specific for the C-terminal domain of topoisomerase IIα (aa 1245–1361) (lanes 3–5). All RNA–topoisomerase IIα complexes were detected as described in Figure 1 .

    Techniques Used: RNA Binding Assay, Activity Assay, Incubation, Concentration Assay

    12) Product Images from "PhTX-II a Basic Myotoxic Phospholipase A2 from Porthidium hyoprora Snake Venom, Pharmacological Characterization and Amino Acid Sequence by Mass Spectrometry"

    Article Title: PhTX-II a Basic Myotoxic Phospholipase A2 from Porthidium hyoprora Snake Venom, Pharmacological Characterization and Amino Acid Sequence by Mass Spectrometry

    Journal: Toxins

    doi: 10.3390/toxins6113077

    Chromatographic and electrophoretic profile of Porthidium hyoprora venom fractioning on a µ-Bondapack C18 column, monitoring elution profile at 280 nm. Emphasized in black is fraction 11 ( * ) characterized as PhTX-II PLA 2 ; Insert: Electrophoretic profile in Tricine SDS-PAGE (1) Molecular mass markers; (2) PhTX-II not reduced; (3) PhTX-II reduced with DTT (1 M).
    Figure Legend Snippet: Chromatographic and electrophoretic profile of Porthidium hyoprora venom fractioning on a µ-Bondapack C18 column, monitoring elution profile at 280 nm. Emphasized in black is fraction 11 ( * ) characterized as PhTX-II PLA 2 ; Insert: Electrophoretic profile in Tricine SDS-PAGE (1) Molecular mass markers; (2) PhTX-II not reduced; (3) PhTX-II reduced with DTT (1 M).

    Techniques Used: Proximity Ligation Assay, SDS Page

    13) Product Images from "Association of Dnmt3a and thymine DNA glycosylase links DNA methylation with base-excision repair"

    Article Title: Association of Dnmt3a and thymine DNA glycosylase links DNA methylation with base-excision repair

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl1052

    Dnmt3a stimulates the glycosylase activity of TDG. ( A ) Purified recombinant 6xHis-tagged Dnmt3a, APE and TDG proteins (Coomassie blue staining). The purity of Dnmt3a, APE and TDG was about 85, 90 and 80%, respectively, as determined by densitometry. Lower panel, the G/T mismatch-containing substrate (27 bp) used for the glycosylase activity assay. The upper T-containing strand was labeled with 32 P at the 5′ end. ( B ) Glycosylase activity assay for TDG in the absence and presence of Dnmt3a or APE. The reactions in lanes 1–9 contained 25 mM HEPES (pH 7.8), 0.5 mM EDTA, 0.5 mM DTT, 0.5 mg/ml BSA and 10 nM substrate DNA. The reactions in lanes 10–12 contained 50 mM NaCl and 2 mM MgCl 2 in addition to the above and show no difference, ruling out any possibility that the stimulation is only due to the addition of salt from the Dnmt3a prep. All reactions were incubated at 30°C for 1 h. The excision of unpaired thymine resulted in breakage of the upper strand upon subsequent hot alkaline treatment. The generation of the 13mer breakage product was examined by separation on a denaturing gel followed by quantification with phosphoimaging. The lower panel is a bar graph representation of TDG enzymatic assays. The activity of TDG alone was set to 1. The relative activity (given on the y -axis) for each reaction is the average of three experiments ±SD. ( C ) The kinetics of the base-excision reaction of TDG with and without Dnmt3a. DNA substrate (10 nM) was incubated with TDG (4 nM) alone or with Dnmt3a (20 nM) in the reaction buffer without NaCl for different time periods indicated on the top. The lower panel is a line graph representation in which the activity for each reaction at different time points is the average of three experiments ±SD. ( D ) Specific stimulation of TDG glycosylase activity by Dnmt3a. The glycosylase reaction was carried out for 30 min at 30°C. The upper panel shows the lack of stimulation of two other glycosylases UDG and SMUG1 by Dnmt3a. The DNA substrate used here had the same sequence as shown in (A) except that a G/U mismatch was incorporated in the place of G/T. The lower panel shows the absence of stimulation by M.SssI, M.HhaI and Dnmt2 on the glycosylase activity of TDG.
    Figure Legend Snippet: Dnmt3a stimulates the glycosylase activity of TDG. ( A ) Purified recombinant 6xHis-tagged Dnmt3a, APE and TDG proteins (Coomassie blue staining). The purity of Dnmt3a, APE and TDG was about 85, 90 and 80%, respectively, as determined by densitometry. Lower panel, the G/T mismatch-containing substrate (27 bp) used for the glycosylase activity assay. The upper T-containing strand was labeled with 32 P at the 5′ end. ( B ) Glycosylase activity assay for TDG in the absence and presence of Dnmt3a or APE. The reactions in lanes 1–9 contained 25 mM HEPES (pH 7.8), 0.5 mM EDTA, 0.5 mM DTT, 0.5 mg/ml BSA and 10 nM substrate DNA. The reactions in lanes 10–12 contained 50 mM NaCl and 2 mM MgCl 2 in addition to the above and show no difference, ruling out any possibility that the stimulation is only due to the addition of salt from the Dnmt3a prep. All reactions were incubated at 30°C for 1 h. The excision of unpaired thymine resulted in breakage of the upper strand upon subsequent hot alkaline treatment. The generation of the 13mer breakage product was examined by separation on a denaturing gel followed by quantification with phosphoimaging. The lower panel is a bar graph representation of TDG enzymatic assays. The activity of TDG alone was set to 1. The relative activity (given on the y -axis) for each reaction is the average of three experiments ±SD. ( C ) The kinetics of the base-excision reaction of TDG with and without Dnmt3a. DNA substrate (10 nM) was incubated with TDG (4 nM) alone or with Dnmt3a (20 nM) in the reaction buffer without NaCl for different time periods indicated on the top. The lower panel is a line graph representation in which the activity for each reaction at different time points is the average of three experiments ±SD. ( D ) Specific stimulation of TDG glycosylase activity by Dnmt3a. The glycosylase reaction was carried out for 30 min at 30°C. The upper panel shows the lack of stimulation of two other glycosylases UDG and SMUG1 by Dnmt3a. The DNA substrate used here had the same sequence as shown in (A) except that a G/U mismatch was incorporated in the place of G/T. The lower panel shows the absence of stimulation by M.SssI, M.HhaI and Dnmt2 on the glycosylase activity of TDG.

    Techniques Used: Activity Assay, Purification, Recombinant, Staining, Labeling, Incubation, Sequencing

    14) Product Images from "Terminal deoxynucleotidyl transferase catalyzes the reaction of DNA phosphorylation"

    Article Title: Terminal deoxynucleotidyl transferase catalyzes the reaction of DNA phosphorylation

    Journal: Nucleic Acids Research

    doi:

    Primer extension by compounds VI catalyzed by TDT P+ (Series A) and TDT P– (Series B). ( A ) Lane 1, primer+ enzyme (control); lane 2, as in lane 1 + 0.5 µM ddTTP; lane 3, as in lane 1 + 5 µM dTTP; lanes 4–6, as in lane 1 + 5, 50 and 500 µM VIa , respectively; lane 7, synthetic 3′-methylphosphonylated oligodeoxynucleotide VIII . ( B ) Lane 1, primer + enzyme (control); lane 2, as in lane 1 + 0.1 µM ddTTP; lane 3, as in lane 1 + 1 µM ddTTP and 10 µM dTTP; lanes 4–6, as in lane 1 + 100, 300 and 600 µM VIb , respectively; lanes 7 and 8, as in lane 6, primer extension reaction followed by addition of 0.05 and 0.1 M DTT, respectively; lanes 9–11, as in lane 1 + 100, 300 and 600 µM VIc , respectively; lanes 12 and 13, as in lane 11, primer extension reaction followed by addition of 0.05 and 0.1 M DTT, respectively; lanes 14–17, as in lane 1 + 100, 300, 600 and 1000 µM VId , respectively.
    Figure Legend Snippet: Primer extension by compounds VI catalyzed by TDT P+ (Series A) and TDT P– (Series B). ( A ) Lane 1, primer+ enzyme (control); lane 2, as in lane 1 + 0.5 µM ddTTP; lane 3, as in lane 1 + 5 µM dTTP; lanes 4–6, as in lane 1 + 5, 50 and 500 µM VIa , respectively; lane 7, synthetic 3′-methylphosphonylated oligodeoxynucleotide VIII . ( B ) Lane 1, primer + enzyme (control); lane 2, as in lane 1 + 0.1 µM ddTTP; lane 3, as in lane 1 + 1 µM ddTTP and 10 µM dTTP; lanes 4–6, as in lane 1 + 100, 300 and 600 µM VIb , respectively; lanes 7 and 8, as in lane 6, primer extension reaction followed by addition of 0.05 and 0.1 M DTT, respectively; lanes 9–11, as in lane 1 + 100, 300 and 600 µM VIc , respectively; lanes 12 and 13, as in lane 11, primer extension reaction followed by addition of 0.05 and 0.1 M DTT, respectively; lanes 14–17, as in lane 1 + 100, 300, 600 and 1000 µM VId , respectively.

    Techniques Used:

    15) Product Images from "Terminal deoxynucleotidyl transferase catalyzes the reaction of DNA phosphorylation"

    Article Title: Terminal deoxynucleotidyl transferase catalyzes the reaction of DNA phosphorylation

    Journal: Nucleic Acids Research

    doi:

    Primer extension by compounds VI catalyzed by TDT P+ (Series A) and TDT P– (Series B). ( A ) Lane 1, primer+ enzyme (control); lane 2, as in lane 1 + 0.5 µM ddTTP; lane 3, as in lane 1 + 5 µM dTTP; lanes 4–6, as in lane 1 + 5, 50 and 500 µM VIa , respectively; lane 7, synthetic 3′-methylphosphonylated oligodeoxynucleotide VIII . ( B ) Lane 1, primer + enzyme (control); lane 2, as in lane 1 + 0.1 µM ddTTP; lane 3, as in lane 1 + 1 µM ddTTP and 10 µM dTTP; lanes 4–6, as in lane 1 + 100, 300 and 600 µM VIb , respectively; lanes 7 and 8, as in lane 6, primer extension reaction followed by addition of 0.05 and 0.1 M DTT, respectively; lanes 9–11, as in lane 1 + 100, 300 and 600 µM VIc , respectively; lanes 12 and 13, as in lane 11, primer extension reaction followed by addition of 0.05 and 0.1 M DTT, respectively; lanes 14–17, as in lane 1 + 100, 300, 600 and 1000 µM VId , respectively.
    Figure Legend Snippet: Primer extension by compounds VI catalyzed by TDT P+ (Series A) and TDT P– (Series B). ( A ) Lane 1, primer+ enzyme (control); lane 2, as in lane 1 + 0.5 µM ddTTP; lane 3, as in lane 1 + 5 µM dTTP; lanes 4–6, as in lane 1 + 5, 50 and 500 µM VIa , respectively; lane 7, synthetic 3′-methylphosphonylated oligodeoxynucleotide VIII . ( B ) Lane 1, primer + enzyme (control); lane 2, as in lane 1 + 0.1 µM ddTTP; lane 3, as in lane 1 + 1 µM ddTTP and 10 µM dTTP; lanes 4–6, as in lane 1 + 100, 300 and 600 µM VIb , respectively; lanes 7 and 8, as in lane 6, primer extension reaction followed by addition of 0.05 and 0.1 M DTT, respectively; lanes 9–11, as in lane 1 + 100, 300 and 600 µM VIc , respectively; lanes 12 and 13, as in lane 11, primer extension reaction followed by addition of 0.05 and 0.1 M DTT, respectively; lanes 14–17, as in lane 1 + 100, 300, 600 and 1000 µM VId , respectively.

    Techniques Used:

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    Electrophoresis:

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    Random Hexamer Labeling:

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    Mass Spectrometry:

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    Modification:

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    High Performance Liquid Chromatography:

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    Flow Cytometry:

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    Chromatography:

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    Reverse Transcription Polymerase Chain Reaction:

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    Tandem Mass Spectroscopy:

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    Polymerase Chain Reaction:

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    Pull Down Assay:

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    Size-exclusion Chromatography:

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    Peptide Fractionation:

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    Sequencing:

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    Protein Extraction:

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    Reversed-phase Chromatography:

    Article Title: Remodeling Membrane Binding by Mono-Ubiquitylation
    Article Snippet: Briefly, the proteins in each gel slice were reduced (10 mM dithiotreitol), modified with 40 mM iodoacetamide (at 25 °C), and trypsinized (modified trypsin (Promega)) at a 1:100 enzyme-to-substrate ratio for 18 h at 37 °C. .. The resulting tryptic peptides from each gel slice were resolved by reverse-phase chromatography on 0.075 × 200 mm fused silica capillaries (Aligent Technologies J & W, Santa Clara, CA, USA) packed with Reprosil reversed-phase material (Dr Maisch GmbH, Entringe, Germany).

    Sample Prep:

    Article Title: Full dynamic range proteome analysis of S. cerevisiae by targeted proteomics
    Article Snippet: Paragraph title: Sample preparation ... Pelleted cells were disrupted by glass bead beating, proteins were precipitated by cold acetone, reduced with 12 mM dithiotreitol and alkylated with 40 mM iodoacetamide and digested with sequencing grade trypsin (Promega).

    Liquid Chromatography:

    Article Title: Divergent regulation of functionally distinct γ-tubulin complexes during differentiation
    Article Snippet: The sample was reduced with 10 mM dithiolthreitol for 20 min at 80°C and alkylated with 20 mM iodoacetamide for 45 min at room temperature before digesting with sequencing-grade modified trypsin (Promega) at 37°C overnight. .. Liquid chromatography (LC) tandem mass spectrometry (MS/MS) was performed on 5 µl of the sample using a nanoAcquity UPLC system (Waters) coupled to a Thermo QExactive Plus high-resolution accurate mass tandem mass spectrometer (Thermo Fisher Scientific) via a nanoelectrospray ionization source.

    Agarose Gel Electrophoresis:

    Article Title: PTTG expression in different experimental and human prolactinomas in relation to dopaminergic control of lactotropes
    Article Snippet: Semiquantitative RT-PCR for mouse samples Total RNA (150 ng) was reverse transcribed in a reaction mixture containing 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2 , 10 mM dithiotreitol, 1 mM deoxynucleotide triphosphates (dNTPs), 8 U RNAse inhibitor (Promega, Madison, WI), 1 μg of random hexamers (Biodynamics SRL, Buenos Aires, Argentina) and 200 U Moloney murine leukemia virus transcriptase (Invitrogen Life Technologies, Buenos Aires, Argentina) in a final volume of 20 μl. .. Preliminary experiments using various RNA concentrations and cycle numbers confirmed that these PCR reactions were performed within the linear phase of the PCR amplification reaction and the amplified product was analyzed by 1.8 % agarose gel electrophoresis.

    Incubation:

    Article Title: Viral pathogens hitchhike with insect sperm for paternal transmission
    Article Snippet: .. After extensive wash of the column, bound proteins were eluted, and then the eluted proteins were treated with dithiotreitol (1 mm ) for 30 min followed by alkylation with iodoacetamide (50 mm ) for 30 min The mixture was incubated with trypsin (1 μg/50 μg protein) (Promega) overnight, centrifuged at 14,000 × g for 2 min and the pellets were collected. ..

    Article Title: Gynura procumbens Improved Fertility of Diabetic Rats: Preliminary Study of Sperm Proteomic
    Article Snippet: .. A total of 100 μ g of protein lysate was incubated with dithiotreitol and iodoacetamide for reduction and alkylation steps, followed by overnight trypsinisation by adding 6 ng/ μ L trypsin (PROMEGA Gold, USA). .. The peptide samples were then submitted into an LC system (Dionex 3000 Ultimate RSLCnano) coupled to an LTQ Orbitrap Fusion mass spectrometer (Thermo Fisher, Bremen, Germany).

    Article Title: The activity of bacterial peptidylarginine deiminase is important during formation of dual-species biofilm by periodontal pathogen Porphyromonas gingivalis and opportunistic fungus Candida albicans
    Article Snippet: .. After harvesting the cells by centrifugation (3000 × g, 5 min) and washing them three times with 1 mL of 25 mM ammonium bicarbonate buffer (NH4 HCO3 ), 100 μL of the same buffer with 5 mM dithiotreitol and 1 μg of sequencing-grade trypsin (Promega, Madison, WI, USA) were added, followed by the incubation for 10 min at 37°C. ..

    Concentration Assay:

    Article Title: The activity of bacterial peptidylarginine deiminase is important during formation of dual-species biofilm by periodontal pathogen Porphyromonas gingivalis and opportunistic fungus Candida albicans
    Article Snippet: After harvesting the cells by centrifugation (3000 × g, 5 min) and washing them three times with 1 mL of 25 mM ammonium bicarbonate buffer (NH4 HCO3 ), 100 μL of the same buffer with 5 mM dithiotreitol and 1 μg of sequencing-grade trypsin (Promega, Madison, WI, USA) were added, followed by the incubation for 10 min at 37°C. .. The undigested proteins were removed from the samples by addition of trifluoroacetic acid to the final concentration of 0.1% (v/v) and centrifugation (12 000 × g, 12 min) of any precipitates.

    Cell Counting:

    Article Title: Full dynamic range proteome analysis of S. cerevisiae by targeted proteomics
    Article Snippet: Before lysis aliquots of the cell suspension were subjected to cell counting in a Neubauer chamber and averaged results were expressed as cells/ml. .. Pelleted cells were disrupted by glass bead beating, proteins were precipitated by cold acetone, reduced with 12 mM dithiotreitol and alkylated with 40 mM iodoacetamide and digested with sequencing grade trypsin (Promega).

    Lysis:

    Article Title: Gynura procumbens Improved Fertility of Diabetic Rats: Preliminary Study of Sperm Proteomic
    Article Snippet: These sperm samples were centrifuged and lysed with lysis buffer. .. A total of 100 μ g of protein lysate was incubated with dithiotreitol and iodoacetamide for reduction and alkylation steps, followed by overnight trypsinisation by adding 6 ng/ μ L trypsin (PROMEGA Gold, USA).

    Article Title: Full dynamic range proteome analysis of S. cerevisiae by targeted proteomics
    Article Snippet: Before lysis aliquots of the cell suspension were subjected to cell counting in a Neubauer chamber and averaged results were expressed as cells/ml. .. Pelleted cells were disrupted by glass bead beating, proteins were precipitated by cold acetone, reduced with 12 mM dithiotreitol and alkylated with 40 mM iodoacetamide and digested with sequencing grade trypsin (Promega).

    Electrofocusing:

    Article Title: Full dynamic range proteome analysis of S. cerevisiae by targeted proteomics
    Article Snippet: Pelleted cells were disrupted by glass bead beating, proteins were precipitated by cold acetone, reduced with 12 mM dithiotreitol and alkylated with 40 mM iodoacetamide and digested with sequencing grade trypsin (Promega). .. The peptide mixtures were either directly destined to MS analysis or firstly separated by off-gel electrofocusing (OGE) using a pH 3–10 IPG strip (Amersham Biosciences, Otelfingen, Switzerland), and a 3100 OFFGEL Fractionator (Agilent Technologies) with collection in 24 wells.

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    Promega dtt molecular grade
    Dtt Molecular Grade, supplied by Promega, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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