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  • 99
    Thermo Fisher tris hcl
    Tris Hcl, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 29239 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore tris hcl
    Tris Hcl, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 61632 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore tris hcl buffer
    Tris Hcl Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 5208 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad tris hcl gels
    Tris Hcl Gels, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 95/100, based on 2301 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad tris hcl gel
    Tris Hcl Gel, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 1782 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher m tris hcl
    M Tris Hcl, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2028 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Bio-Rad tris hcl
    Recombinant N-His 6 -GpsA protein is an active G3P dehydrogenase in vitro. R. prowazekii gpsA was cloned into pET15b, heterologously expressed in E. coli BL21(DE3), and purified with N-His 6 -GpsA. (A) Purified protein was resolved on a 4 to 15% <t>Tris-HCl</t> <t>SDS-polyacrylamide</t> gel and visualized by Imperial protein staining and Western blot analysis using anti-His 6 monoclonal antibody (sizes [in kDa] are indicated on the left). The mass spectrometry analysis coverage map shows matched peptides in bold font (30% coverage). (B) N-His 6 -GpsA-catalyzed conversion of [ 32 P]DHAP to [ 32 P]G3P was verified by paper chromatography. [ 32 P]ATP, [ 32 P]DHAP, [ 32 P]G3P, and [ 32 P]orthophosphate were used as standards. The reaction mixtures were incubated for 60 min at room temperature prior to chromatographic analysis. Lane A, a negative-control reaction mixture containing 6 μM [ 32 P]DHAP and 100 μM NADPH only; lane B, a negative-control reaction mixture containing [ 32 P]DHAP and 2.5 μg ml −1 N-His 6 -GpsA protein only; lane C, the N-His 6 -GpsA-catalyzed conversion of [ 32 P]DHAP to [ 32 P]G3P absolutely required the presence of NADPH. The density of each compound on the chromatograph is expressed as a percentage of the total lane density (summarized in tabular form to the right of each spot on the chromatograph). (C) N-His 6 -GpsA-catalyzed conversion of DHAP to G3P was measured spectrophotometrically at OD 340 by following the oxidation of NADPH or NADH over time. Error bars represent standard deviations. The reaction mixtures contained 1.0 μg ml −1 N-His 6 -GpsA, 500 μM DHAP, and 100 μM NAD(P)H in modified buffer A; and the reactions were carried out at room temperature.
    Tris Hcl, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 93/100, based on 5491 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad tris hcl ready gels
    Recombinant N-His 6 -GpsA protein is an active G3P dehydrogenase in vitro. R. prowazekii gpsA was cloned into pET15b, heterologously expressed in E. coli BL21(DE3), and purified with N-His 6 -GpsA. (A) Purified protein was resolved on a 4 to 15% <t>Tris-HCl</t> <t>SDS-polyacrylamide</t> gel and visualized by Imperial protein staining and Western blot analysis using anti-His 6 monoclonal antibody (sizes [in kDa] are indicated on the left). The mass spectrometry analysis coverage map shows matched peptides in bold font (30% coverage). (B) N-His 6 -GpsA-catalyzed conversion of [ 32 P]DHAP to [ 32 P]G3P was verified by paper chromatography. [ 32 P]ATP, [ 32 P]DHAP, [ 32 P]G3P, and [ 32 P]orthophosphate were used as standards. The reaction mixtures were incubated for 60 min at room temperature prior to chromatographic analysis. Lane A, a negative-control reaction mixture containing 6 μM [ 32 P]DHAP and 100 μM NADPH only; lane B, a negative-control reaction mixture containing [ 32 P]DHAP and 2.5 μg ml −1 N-His 6 -GpsA protein only; lane C, the N-His 6 -GpsA-catalyzed conversion of [ 32 P]DHAP to [ 32 P]G3P absolutely required the presence of NADPH. The density of each compound on the chromatograph is expressed as a percentage of the total lane density (summarized in tabular form to the right of each spot on the chromatograph). (C) N-His 6 -GpsA-catalyzed conversion of DHAP to G3P was measured spectrophotometrically at OD 340 by following the oxidation of NADPH or NADH over time. Error bars represent standard deviations. The reaction mixtures contained 1.0 μg ml −1 N-His 6 -GpsA, 500 μM DHAP, and 100 μM NAD(P)H in modified buffer A; and the reactions were carried out at room temperature.
    Tris Hcl Ready Gels, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 713 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad tris hcl polyacrylamide gels
    Recombinant N-His 6 -GpsA protein is an active G3P dehydrogenase in vitro. R. prowazekii gpsA was cloned into pET15b, heterologously expressed in E. coli BL21(DE3), and purified with N-His 6 -GpsA. (A) Purified protein was resolved on a 4 to 15% <t>Tris-HCl</t> <t>SDS-polyacrylamide</t> gel and visualized by Imperial protein staining and Western blot analysis using anti-His 6 monoclonal antibody (sizes [in kDa] are indicated on the left). The mass spectrometry analysis coverage map shows matched peptides in bold font (30% coverage). (B) N-His 6 -GpsA-catalyzed conversion of [ 32 P]DHAP to [ 32 P]G3P was verified by paper chromatography. [ 32 P]ATP, [ 32 P]DHAP, [ 32 P]G3P, and [ 32 P]orthophosphate were used as standards. The reaction mixtures were incubated for 60 min at room temperature prior to chromatographic analysis. Lane A, a negative-control reaction mixture containing 6 μM [ 32 P]DHAP and 100 μM NADPH only; lane B, a negative-control reaction mixture containing [ 32 P]DHAP and 2.5 μg ml −1 N-His 6 -GpsA protein only; lane C, the N-His 6 -GpsA-catalyzed conversion of [ 32 P]DHAP to [ 32 P]G3P absolutely required the presence of NADPH. The density of each compound on the chromatograph is expressed as a percentage of the total lane density (summarized in tabular form to the right of each spot on the chromatograph). (C) N-His 6 -GpsA-catalyzed conversion of DHAP to G3P was measured spectrophotometrically at OD 340 by following the oxidation of NADPH or NADH over time. Error bars represent standard deviations. The reaction mixtures contained 1.0 μg ml −1 N-His 6 -GpsA, 500 μM DHAP, and 100 μM NAD(P)H in modified buffer A; and the reactions were carried out at room temperature.
    Tris Hcl Polyacrylamide Gels, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 685 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Fisher Scientific tris hcl
    Activities of recombinant E. coli native and mutant (K192A) YjeF proteins ( A ) Assays (100 µl) contained 25 mM <t>Tris-HCl,</t> pH 8.0, 5 mM KCl, 2 mM MgCl 2 , 0.1 mg mL −1 BSA, 40 µM <t>NADHX</t> (containing approximately equal amounts of S and R forms), and 1 mM ADP. Reactions were started by adding 2 µg of native YjeF and absorbance was monitored at 340 nm at 22°C. ( B ) Assays were performed as above except that reactions were started by adding 2 µg of K192A YjeF (closed circles). In separate assays, 2 µg of Arabidopsis NAD(P)HX epimerase domain protein (E) was added at 4 min (open diamonds). Spontaneous epimerization of NAD(P)HX was undetectable in the conditions and time frame of the assay. Data are means of three replicates; S.E. was
    Tris Hcl, supplied by Fisher Scientific, used in various techniques. Bioz Stars score: 93/100, based on 929 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore m tris hcl buffer
    Activities of recombinant E. coli native and mutant (K192A) YjeF proteins ( A ) Assays (100 µl) contained 25 mM <t>Tris-HCl,</t> pH 8.0, 5 mM KCl, 2 mM MgCl 2 , 0.1 mg mL −1 BSA, 40 µM <t>NADHX</t> (containing approximately equal amounts of S and R forms), and 1 mM ADP. Reactions were started by adding 2 µg of native YjeF and absorbance was monitored at 340 nm at 22°C. ( B ) Assays were performed as above except that reactions were started by adding 2 µg of K192A YjeF (closed circles). In separate assays, 2 µg of Arabidopsis NAD(P)HX epimerase domain protein (E) was added at 4 min (open diamonds). Spontaneous epimerization of NAD(P)HX was undetectable in the conditions and time frame of the assay. Data are means of three replicates; S.E. was
    M Tris Hcl Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 415 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Bio-Rad tris hcl gradient gels
    Activities of recombinant E. coli native and mutant (K192A) YjeF proteins ( A ) Assays (100 µl) contained 25 mM <t>Tris-HCl,</t> pH 8.0, 5 mM KCl, 2 mM MgCl 2 , 0.1 mg mL −1 BSA, 40 µM <t>NADHX</t> (containing approximately equal amounts of S and R forms), and 1 mM ADP. Reactions were started by adding 2 µg of native YjeF and absorbance was monitored at 340 nm at 22°C. ( B ) Assays were performed as above except that reactions were started by adding 2 µg of K192A YjeF (closed circles). In separate assays, 2 µg of Arabidopsis NAD(P)HX epimerase domain protein (E) was added at 4 min (open diamonds). Spontaneous epimerization of NAD(P)HX was undetectable in the conditions and time frame of the assay. Data are means of three replicates; S.E. was
    Tris Hcl Gradient Gels, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 91/100, based on 471 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad criterion tris hcl gels
    Experimental strategy for comparing peptide- vs protein-level enrichments. (A) Experimental design applying on-resin iTRAQ-labeling. (B) Silver-staining image of <t>SDS–PAGE</t> of enriched Cys-peptides. For each lane of the 4–20% <t>Tris–HCl</t> precast gel, 5 of 30 μl of DTT-eluted Cys-peptides was loaded. The higher protein bands in protein-level enrichment samples indicate incomplete on-resin digestion. (C) An MS/MS spectrum of the Cys-peptide GVLECQVSR from obscurin protein and its zoom-in spectrum of reporter-ion region showing the SNO abundance increased in response to GSNO treatment.
    Criterion Tris Hcl Gels, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 441 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Agilent technologies tris hcl buffer
    Superoxide accelerates the aerobic oxidation of THP. Oxygen consumption was recorded at 25 °C in 2 mL of 50 mM <t>Tris/HCl</t> buffer, pH 7.5, containing 0.3 mM <t>EDTA</t> after the serial additions of 0.5 mM xanthine, 39 nM XO, 5 mM THP, and 80 units/mL of
    Tris Hcl Buffer, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 92/100, based on 370 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad m tris hcl
    Superoxide accelerates the aerobic oxidation of THP. Oxygen consumption was recorded at 25 °C in 2 mL of 50 mM <t>Tris/HCl</t> buffer, pH 7.5, containing 0.3 mM <t>EDTA</t> after the serial additions of 0.5 mM xanthine, 39 nM XO, 5 mM THP, and 80 units/mL of
    M Tris Hcl, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 93/100, based on 483 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Bio-Rad tris hcl gradient gel
    Delocalisation of ERG-associated protein with SET domain (ESET) from promyelocytic leukaemia (PML) nuclear bodies promotes the interaction of small ubiquitin-related modifier (SUMO)ylated ESET and Oct4 . (a) In PML (green)-depleted embryonic stem (ES) cells (white border), ESET (red) punctate foci were delocalised from PML nuclear bodies. Nuclei are labelled in blue. Scale bar, 3 μm. Cultures also contain cells which were not depleted of PML, where ESET exhibit punctate foci (arrow heads) associated with PML bodies. (b) ES cells transfected with Pml short hairpin RNA (shRNA) for 4 days were immunoprecipitated (IP) with anti-ESET antibody (kind gift of HH Ng; see text) under mild conditions in buffer containing digitonin and ethidium bromide, and were subjected to western blotting (WB) with the antibodies indicated. Note that more Oct4 is precipitated by ESET in Pml knockdown ES cells (*lane 4, top panel). Asterisk (lane 3, middle panel) indicates PML being precipitated by ESET in control cells transfected with an empty vector but not in Pml knockdown ES cells (compare with lane 4, middle panel). (c) ES cells transfected with Pml shRNA for 4 days were immunoprecipitated (IP) with anti-Oct4 antibody in buffer containing NP40, N <t>-ethylmaleimide,</t> and ethidium bromide and subjected to WB using 4% to 15% <t>Tris-HCl</t> gradient gel. More SUMOylated ESET was precipitated by Oct4 in two independent experiments (Exp 1 and Exp 2) in Pml knockdown cells as indicated (*; lane 4 and 6, top panel).
    Tris Hcl Gradient Gel, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 91/100, based on 373 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Carl Roth GmbH tris hcl
    Alternating the media 10 mM <t>Tris-HCl</t> and <t>1×</t> PBS buffer for three consecutive runs. ( A ) Shows the minimum shift when exchanging the media. ( B ) Illustrates the changes in amplitude and its phase at a frequency of 251 Hz, when exchanging the media. Comparing the signals, when plotted in the same graph, reveals that the read-out systems detected the media exchange simultaneously, without influencing each other ( C ).
    Tris Hcl, supplied by Carl Roth GmbH, used in various techniques. Bioz Stars score: 93/100, based on 600 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Recombinant N-His 6 -GpsA protein is an active G3P dehydrogenase in vitro. R. prowazekii gpsA was cloned into pET15b, heterologously expressed in E. coli BL21(DE3), and purified with N-His 6 -GpsA. (A) Purified protein was resolved on a 4 to 15% Tris-HCl SDS-polyacrylamide gel and visualized by Imperial protein staining and Western blot analysis using anti-His 6 monoclonal antibody (sizes [in kDa] are indicated on the left). The mass spectrometry analysis coverage map shows matched peptides in bold font (30% coverage). (B) N-His 6 -GpsA-catalyzed conversion of [ 32 P]DHAP to [ 32 P]G3P was verified by paper chromatography. [ 32 P]ATP, [ 32 P]DHAP, [ 32 P]G3P, and [ 32 P]orthophosphate were used as standards. The reaction mixtures were incubated for 60 min at room temperature prior to chromatographic analysis. Lane A, a negative-control reaction mixture containing 6 μM [ 32 P]DHAP and 100 μM NADPH only; lane B, a negative-control reaction mixture containing [ 32 P]DHAP and 2.5 μg ml −1 N-His 6 -GpsA protein only; lane C, the N-His 6 -GpsA-catalyzed conversion of [ 32 P]DHAP to [ 32 P]G3P absolutely required the presence of NADPH. The density of each compound on the chromatograph is expressed as a percentage of the total lane density (summarized in tabular form to the right of each spot on the chromatograph). (C) N-His 6 -GpsA-catalyzed conversion of DHAP to G3P was measured spectrophotometrically at OD 340 by following the oxidation of NADPH or NADH over time. Error bars represent standard deviations. The reaction mixtures contained 1.0 μg ml −1 N-His 6 -GpsA, 500 μM DHAP, and 100 μM NAD(P)H in modified buffer A; and the reactions were carried out at room temperature.

    Journal: Journal of Bacteriology

    Article Title: Rickettsia prowazekii Uses an sn-Glycerol-3-Phosphate Dehydrogenase and a Novel Dihydroxyacetone Phosphate Transport System To Supply Triose Phosphate for Phospholipid Biosynthesis ▿

    doi: 10.1128/JB.00443-10

    Figure Lengend Snippet: Recombinant N-His 6 -GpsA protein is an active G3P dehydrogenase in vitro. R. prowazekii gpsA was cloned into pET15b, heterologously expressed in E. coli BL21(DE3), and purified with N-His 6 -GpsA. (A) Purified protein was resolved on a 4 to 15% Tris-HCl SDS-polyacrylamide gel and visualized by Imperial protein staining and Western blot analysis using anti-His 6 monoclonal antibody (sizes [in kDa] are indicated on the left). The mass spectrometry analysis coverage map shows matched peptides in bold font (30% coverage). (B) N-His 6 -GpsA-catalyzed conversion of [ 32 P]DHAP to [ 32 P]G3P was verified by paper chromatography. [ 32 P]ATP, [ 32 P]DHAP, [ 32 P]G3P, and [ 32 P]orthophosphate were used as standards. The reaction mixtures were incubated for 60 min at room temperature prior to chromatographic analysis. Lane A, a negative-control reaction mixture containing 6 μM [ 32 P]DHAP and 100 μM NADPH only; lane B, a negative-control reaction mixture containing [ 32 P]DHAP and 2.5 μg ml −1 N-His 6 -GpsA protein only; lane C, the N-His 6 -GpsA-catalyzed conversion of [ 32 P]DHAP to [ 32 P]G3P absolutely required the presence of NADPH. The density of each compound on the chromatograph is expressed as a percentage of the total lane density (summarized in tabular form to the right of each spot on the chromatograph). (C) N-His 6 -GpsA-catalyzed conversion of DHAP to G3P was measured spectrophotometrically at OD 340 by following the oxidation of NADPH or NADH over time. Error bars represent standard deviations. The reaction mixtures contained 1.0 μg ml −1 N-His 6 -GpsA, 500 μM DHAP, and 100 μM NAD(P)H in modified buffer A; and the reactions were carried out at room temperature.

    Article Snippet: Protein purification was verified by electrophoresis using a 4 to 15% Tris-HCl, SDS-polyacrylamide gel (Bio-Rad) and visualized by staining with Imperial protein stain (Bio-Rad).

    Techniques: Recombinant, In Vitro, Clone Assay, Purification, Staining, Western Blot, Mass Spectrometry, Paper Chromatography, Incubation, Negative Control, Modification

    Activities of recombinant E. coli native and mutant (K192A) YjeF proteins ( A ) Assays (100 µl) contained 25 mM Tris-HCl, pH 8.0, 5 mM KCl, 2 mM MgCl 2 , 0.1 mg mL −1 BSA, 40 µM NADHX (containing approximately equal amounts of S and R forms), and 1 mM ADP. Reactions were started by adding 2 µg of native YjeF and absorbance was monitored at 340 nm at 22°C. ( B ) Assays were performed as above except that reactions were started by adding 2 µg of K192A YjeF (closed circles). In separate assays, 2 µg of Arabidopsis NAD(P)HX epimerase domain protein (E) was added at 4 min (open diamonds). Spontaneous epimerization of NAD(P)HX was undetectable in the conditions and time frame of the assay. Data are means of three replicates; S.E. was

    Journal: Bioscience Reports

    Article Title: Evidence that the metabolite repair enzyme NAD(P)HX epimerase has a moonlighting function

    doi: 10.1042/BSR20180223

    Figure Lengend Snippet: Activities of recombinant E. coli native and mutant (K192A) YjeF proteins ( A ) Assays (100 µl) contained 25 mM Tris-HCl, pH 8.0, 5 mM KCl, 2 mM MgCl 2 , 0.1 mg mL −1 BSA, 40 µM NADHX (containing approximately equal amounts of S and R forms), and 1 mM ADP. Reactions were started by adding 2 µg of native YjeF and absorbance was monitored at 340 nm at 22°C. ( B ) Assays were performed as above except that reactions were started by adding 2 µg of K192A YjeF (closed circles). In separate assays, 2 µg of Arabidopsis NAD(P)HX epimerase domain protein (E) was added at 4 min (open diamonds). Spontaneous epimerization of NAD(P)HX was undetectable in the conditions and time frame of the assay. Data are means of three replicates; S.E. was

    Article Snippet: For assay of NADHX activity, cell pellets were resuspended in 1 ml of 25 mM Tris-HCl, pH 8.0, 300 mM KCl, 1 mM 2-mercaptoethanol on ice and sonicated (Fisher Scientific Ultrasonic Dismembrator, model 150E; 5 × 3 s pulses at 70% power, cooling on ice for 30–60 s between pulses).

    Techniques: Recombinant, Mutagenesis

    Experimental strategy for comparing peptide- vs protein-level enrichments. (A) Experimental design applying on-resin iTRAQ-labeling. (B) Silver-staining image of SDS–PAGE of enriched Cys-peptides. For each lane of the 4–20% Tris–HCl precast gel, 5 of 30 μl of DTT-eluted Cys-peptides was loaded. The higher protein bands in protein-level enrichment samples indicate incomplete on-resin digestion. (C) An MS/MS spectrum of the Cys-peptide GVLECQVSR from obscurin protein and its zoom-in spectrum of reporter-ion region showing the SNO abundance increased in response to GSNO treatment.

    Journal: Free radical biology & medicine

    Article Title: Quantitative site-specific reactivity profiling of S-nitrosylation in mouse skeletal muscle using cysteinyl peptide enrichment coupled with mass spectrometry

    doi: 10.1016/j.freeradbiomed.2012.12.010

    Figure Lengend Snippet: Experimental strategy for comparing peptide- vs protein-level enrichments. (A) Experimental design applying on-resin iTRAQ-labeling. (B) Silver-staining image of SDS–PAGE of enriched Cys-peptides. For each lane of the 4–20% Tris–HCl precast gel, 5 of 30 μl of DTT-eluted Cys-peptides was loaded. The higher protein bands in protein-level enrichment samples indicate incomplete on-resin digestion. (C) An MS/MS spectrum of the Cys-peptide GVLECQVSR from obscurin protein and its zoom-in spectrum of reporter-ion region showing the SNO abundance increased in response to GSNO treatment.

    Article Snippet: Tris/glycine/sodium dodecyl sulfate (SDS) buffer, Laemmli sample loading buffer, and Tris–HCl precast gel with 4–20% linear gradient were all from Bio-Rad Laboratories (Hercules, CA, USA).

    Techniques: Labeling, Silver Staining, SDS Page, Mass Spectrometry

    Superoxide accelerates the aerobic oxidation of THP. Oxygen consumption was recorded at 25 °C in 2 mL of 50 mM Tris/HCl buffer, pH 7.5, containing 0.3 mM EDTA after the serial additions of 0.5 mM xanthine, 39 nM XO, 5 mM THP, and 80 units/mL of

    Journal: Biochemistry

    Article Title: Flavin-linked Erv-family sulfhydryl oxidases release superoxide anion during catalytic turnover

    doi: 10.1021/bi201672h

    Figure Lengend Snippet: Superoxide accelerates the aerobic oxidation of THP. Oxygen consumption was recorded at 25 °C in 2 mL of 50 mM Tris/HCl buffer, pH 7.5, containing 0.3 mM EDTA after the serial additions of 0.5 mM xanthine, 39 nM XO, 5 mM THP, and 80 units/mL of

    Article Snippet: Here, 39 nM of XO was added to 1 mL of 50 mM Tris/HCl buffer, pH 7.5, containing 0.3 mM EDTA, 0.5 mM xanthine and 50 μM cytochrome c and the reaction was followed at 550 nm in an Agilent 8453 spectrophotometer.

    Techniques:

    THP as a substrate of Erv2p in the oxygen electrode. The apparent turnover numbers were calculated using 100 nM Erv2p adding the indicated concentration of THP to 2 mL of 50 mM Tris/HCl buffer (pH 7.5, 25 °C) containing 0.3 mM EDTA in the absence

    Journal: Biochemistry

    Article Title: Flavin-linked Erv-family sulfhydryl oxidases release superoxide anion during catalytic turnover

    doi: 10.1021/bi201672h

    Figure Lengend Snippet: THP as a substrate of Erv2p in the oxygen electrode. The apparent turnover numbers were calculated using 100 nM Erv2p adding the indicated concentration of THP to 2 mL of 50 mM Tris/HCl buffer (pH 7.5, 25 °C) containing 0.3 mM EDTA in the absence

    Article Snippet: Here, 39 nM of XO was added to 1 mL of 50 mM Tris/HCl buffer, pH 7.5, containing 0.3 mM EDTA, 0.5 mM xanthine and 50 μM cytochrome c and the reaction was followed at 550 nm in an Agilent 8453 spectrophotometer.

    Techniques: Concentration Assay

    Delocalisation of ERG-associated protein with SET domain (ESET) from promyelocytic leukaemia (PML) nuclear bodies promotes the interaction of small ubiquitin-related modifier (SUMO)ylated ESET and Oct4 . (a) In PML (green)-depleted embryonic stem (ES) cells (white border), ESET (red) punctate foci were delocalised from PML nuclear bodies. Nuclei are labelled in blue. Scale bar, 3 μm. Cultures also contain cells which were not depleted of PML, where ESET exhibit punctate foci (arrow heads) associated with PML bodies. (b) ES cells transfected with Pml short hairpin RNA (shRNA) for 4 days were immunoprecipitated (IP) with anti-ESET antibody (kind gift of HH Ng; see text) under mild conditions in buffer containing digitonin and ethidium bromide, and were subjected to western blotting (WB) with the antibodies indicated. Note that more Oct4 is precipitated by ESET in Pml knockdown ES cells (*lane 4, top panel). Asterisk (lane 3, middle panel) indicates PML being precipitated by ESET in control cells transfected with an empty vector but not in Pml knockdown ES cells (compare with lane 4, middle panel). (c) ES cells transfected with Pml shRNA for 4 days were immunoprecipitated (IP) with anti-Oct4 antibody in buffer containing NP40, N -ethylmaleimide, and ethidium bromide and subjected to WB using 4% to 15% Tris-HCl gradient gel. More SUMOylated ESET was precipitated by Oct4 in two independent experiments (Exp 1 and Exp 2) in Pml knockdown cells as indicated (*; lane 4 and 6, top panel).

    Journal: Epigenetics & Chromatin

    Article Title: ERG-associated protein with SET domain (ESET)-Oct4 interaction regulates pluripotency and represses the trophectoderm lineage

    doi: 10.1186/1756-8935-2-12

    Figure Lengend Snippet: Delocalisation of ERG-associated protein with SET domain (ESET) from promyelocytic leukaemia (PML) nuclear bodies promotes the interaction of small ubiquitin-related modifier (SUMO)ylated ESET and Oct4 . (a) In PML (green)-depleted embryonic stem (ES) cells (white border), ESET (red) punctate foci were delocalised from PML nuclear bodies. Nuclei are labelled in blue. Scale bar, 3 μm. Cultures also contain cells which were not depleted of PML, where ESET exhibit punctate foci (arrow heads) associated with PML bodies. (b) ES cells transfected with Pml short hairpin RNA (shRNA) for 4 days were immunoprecipitated (IP) with anti-ESET antibody (kind gift of HH Ng; see text) under mild conditions in buffer containing digitonin and ethidium bromide, and were subjected to western blotting (WB) with the antibodies indicated. Note that more Oct4 is precipitated by ESET in Pml knockdown ES cells (*lane 4, top panel). Asterisk (lane 3, middle panel) indicates PML being precipitated by ESET in control cells transfected with an empty vector but not in Pml knockdown ES cells (compare with lane 4, middle panel). (c) ES cells transfected with Pml shRNA for 4 days were immunoprecipitated (IP) with anti-Oct4 antibody in buffer containing NP40, N -ethylmaleimide, and ethidium bromide and subjected to WB using 4% to 15% Tris-HCl gradient gel. More SUMOylated ESET was precipitated by Oct4 in two independent experiments (Exp 1 and Exp 2) in Pml knockdown cells as indicated (*; lane 4 and 6, top panel).

    Article Snippet: Immunoprecipitant (IP) and supernatant were subjected to western blot (WB) with anti-Flag (PML, top panel) and anti-haemagglutinin (HA) (ESET, bottom panel) antibodies. (b) Embryonic stem (ES) cell lysates were immunoprecipitated (IP) with the indicated antibodies in buffer containing NP40 in the presence of N -ethylmaleimide (NEM) and subjected to WB using 4% to 15% Tris-HCl gradient gel.

    Techniques: Transfection, shRNA, Immunoprecipitation, Western Blot, Plasmid Preparation

    ERG-associated protein with SET domain (ESET) localises to promyelocytic leukaemia (PML) nuclear bodies and is post-translationally modified by small ubiquitin-related modifier (SUMO) . (a) ESET (red) localises to distinct, punctate foci in euchromatin regions, as indicated by the absence of DAPI (blue) staining in embryonic stem (ES) cells. Scale bar, 3 μm. (b) ESET (red) localises to distinct, punctate foci that overlaps with PML nuclear bodies (green) in ES cells. Nuclei are labelled in blue. Scale bar, 5 μm. (c) ES cell lysates were immunoprecipitated (IP) with anti-ESET antibody (kind gift of HH Ng; see text) under mild conditions in digitonin-containing buffer and subjected to western blotting (WB) with the antibodies indicated. Rabbit IgG was used as a negative control. (d) Western blot analysis of coimmunoprecipitation experiment in 293T cells transfected with haemagglutinin (HA)-ESET and/or Flag-SUMO-1. S-ESET represents SUMOylated ESET. (e) ES cell lysates were immunoprecipitated (IP) with the indicated antibodies in NP40-containing buffer either in the presence or absence of N -ethylmaleimide (NEM) and subjected to western blotting (WB) using 4% to 15% Tris-HCl gradient gel. A rabbit anti-HA antibody was used as negative control. (f) Quantitative polymerase chain reaction (Q-PCR) analysis of the enrichment of SUMO-1 on different positions along Cdx2 promoter relative to the least enriched region (C5) after normalising against their respective input and IgG controls. Error bars, standard deviation (SD) of three technical replicates.

    Journal: Epigenetics & Chromatin

    Article Title: ERG-associated protein with SET domain (ESET)-Oct4 interaction regulates pluripotency and represses the trophectoderm lineage

    doi: 10.1186/1756-8935-2-12

    Figure Lengend Snippet: ERG-associated protein with SET domain (ESET) localises to promyelocytic leukaemia (PML) nuclear bodies and is post-translationally modified by small ubiquitin-related modifier (SUMO) . (a) ESET (red) localises to distinct, punctate foci in euchromatin regions, as indicated by the absence of DAPI (blue) staining in embryonic stem (ES) cells. Scale bar, 3 μm. (b) ESET (red) localises to distinct, punctate foci that overlaps with PML nuclear bodies (green) in ES cells. Nuclei are labelled in blue. Scale bar, 5 μm. (c) ES cell lysates were immunoprecipitated (IP) with anti-ESET antibody (kind gift of HH Ng; see text) under mild conditions in digitonin-containing buffer and subjected to western blotting (WB) with the antibodies indicated. Rabbit IgG was used as a negative control. (d) Western blot analysis of coimmunoprecipitation experiment in 293T cells transfected with haemagglutinin (HA)-ESET and/or Flag-SUMO-1. S-ESET represents SUMOylated ESET. (e) ES cell lysates were immunoprecipitated (IP) with the indicated antibodies in NP40-containing buffer either in the presence or absence of N -ethylmaleimide (NEM) and subjected to western blotting (WB) using 4% to 15% Tris-HCl gradient gel. A rabbit anti-HA antibody was used as negative control. (f) Quantitative polymerase chain reaction (Q-PCR) analysis of the enrichment of SUMO-1 on different positions along Cdx2 promoter relative to the least enriched region (C5) after normalising against their respective input and IgG controls. Error bars, standard deviation (SD) of three technical replicates.

    Article Snippet: Immunoprecipitant (IP) and supernatant were subjected to western blot (WB) with anti-Flag (PML, top panel) and anti-haemagglutinin (HA) (ESET, bottom panel) antibodies. (b) Embryonic stem (ES) cell lysates were immunoprecipitated (IP) with the indicated antibodies in buffer containing NP40 in the presence of N -ethylmaleimide (NEM) and subjected to WB using 4% to 15% Tris-HCl gradient gel.

    Techniques: Modification, Staining, Immunoprecipitation, Western Blot, Negative Control, Transfection, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Standard Deviation

    Alternating the media 10 mM Tris-HCl and 1× PBS buffer for three consecutive runs. ( A ) Shows the minimum shift when exchanging the media. ( B ) Illustrates the changes in amplitude and its phase at a frequency of 251 Hz, when exchanging the media. Comparing the signals, when plotted in the same graph, reveals that the read-out systems detected the media exchange simultaneously, without influencing each other ( C ).

    Journal: Sensors (Basel, Switzerland)

    Article Title: Combining Electrochemical Impedance Spectroscopy and Surface Plasmon Resonance into one Simultaneous Read-Out System for the Detection of Surface Interactions

    doi: 10.3390/s131114650

    Figure Lengend Snippet: Alternating the media 10 mM Tris-HCl and 1× PBS buffer for three consecutive runs. ( A ) Shows the minimum shift when exchanging the media. ( B ) Illustrates the changes in amplitude and its phase at a frequency of 251 Hz, when exchanging the media. Comparing the signals, when plotted in the same graph, reveals that the read-out systems detected the media exchange simultaneously, without influencing each other ( C ).

    Article Snippet: The results have shown that these media changes could be detected both by EIS and SPR and when changing the medium from 10 mM Tris-HCl to 1× PBS and then back to 10 mM Tris-HCl, no shift in minimum or impedance occurs.

    Techniques: