protein persulfide Search Results


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  • 99
    Thermo Fisher dylight 488 streptavidin
    Visualization of persulfides in normal and cystinotic fibroblasts using the CN-biotin tag switch method. (A) Representative photomicrographs of normal and cystinotic human lung fibroblasts labeled with CN biotin tag-switch assay. The nuclei were stained with DAPI. (B) Intracellular persulfide levels in normal and cystinotic human lung fibroblast cell lysates, labeled using the CN-biotin tag switch reagent. Proteins were visualized using <t>Streptavidin</t> Dylight 488. Artificial color intensity was used and the gradient scale is shown on the right. GAPDH was used as a loading control (bottom). Scale bar: 20 µm.
    Dylight 488 Streptavidin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore biotinylated proteins
    Visualization of persulfides in normal and cystinotic fibroblasts using the CN-biotin tag switch method. (A) Representative photomicrographs of normal and cystinotic human lung fibroblasts labeled with CN biotin tag-switch assay. The nuclei were stained with DAPI. (B) Intracellular persulfide levels in normal and cystinotic human lung fibroblast cell lysates, labeled using the CN-biotin tag switch reagent. Proteins were visualized using <t>Streptavidin</t> Dylight 488. Artificial color intensity was used and the gradient scale is shown on the right. GAPDH was used as a loading control (bottom). Scale bar: 20 µm.
    Biotinylated Proteins, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1392 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Schiff Nutrition International persulfide
    Visualization of persulfides in normal and cystinotic fibroblasts using the CN-biotin tag switch method. (A) Representative photomicrographs of normal and cystinotic human lung fibroblasts labeled with CN biotin tag-switch assay. The nuclei were stained with DAPI. (B) Intracellular persulfide levels in normal and cystinotic human lung fibroblast cell lysates, labeled using the CN-biotin tag switch reagent. Proteins were visualized using <t>Streptavidin</t> Dylight 488. Artificial color intensity was used and the gradient scale is shown on the right. GAPDH was used as a loading control (bottom). Scale bar: 20 µm.
    Persulfide, supplied by Schiff Nutrition International, used in various techniques. Bioz Stars score: 86/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Sino Biological anti biotin antibody
    Visualization of persulfides in normal and cystinotic fibroblasts using the CN-biotin tag switch method. (A) Representative photomicrographs of normal and cystinotic human lung fibroblasts labeled with CN biotin tag-switch assay. The nuclei were stained with DAPI. (B) Intracellular persulfide levels in normal and cystinotic human lung fibroblast cell lysates, labeled using the CN-biotin tag switch reagent. Proteins were visualized using <t>Streptavidin</t> Dylight 488. Artificial color intensity was used and the gradient scale is shown on the right. GAPDH was used as a loading control (bottom). Scale bar: 20 µm.
    Anti Biotin Antibody, supplied by Sino Biological, used in various techniques. Bioz Stars score: 93/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Bio-Rad micro biospinp6columns
    Visualization of persulfides in normal and cystinotic fibroblasts using the CN-biotin tag switch method. (A) Representative photomicrographs of normal and cystinotic human lung fibroblasts labeled with CN biotin tag-switch assay. The nuclei were stained with DAPI. (B) Intracellular persulfide levels in normal and cystinotic human lung fibroblast cell lysates, labeled using the CN-biotin tag switch reagent. Proteins were visualized using <t>Streptavidin</t> Dylight 488. Artificial color intensity was used and the gradient scale is shown on the right. GAPDH was used as a loading control (bottom). Scale bar: 20 µm.
    Micro Biospinp6columns, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 93/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Bio-Rad hek293 cells
    TrxR1 and TRP14 counteract intracellular protein persulfide accumulation. S3 refers to sampling according to Fig. 1 . ( A ) Western blot showing the knockdown of TRP14 and TrxR1 in stably transfected <t>HEK293</t> cells, compared to control cells. GAPDH is applied as a loading control. ( B ) Representative silver-stained gel (of n = 4 experiments) shows that more protein persulfides are detected in TrxR1 and TRP14 knockdown HEK293 cells than in the control (control cells have been transfected with a plasmid that transcribes a scramble shRNA) under normal growth conditions. ( C ) Increases in protein persulfide levels in TrxR1 and TRP14 knockdown HEK293 cells compared to control reached statistical significance (* P
    Hek293 Cells, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 92/100, based on 506 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Bio-Rad silver stained gels
    TrxR1 and TRP14 counteract intracellular protein persulfide accumulation. S3 refers to sampling according to Fig. 1 . ( A ) Western blot showing the knockdown of TRP14 and TrxR1 in stably transfected <t>HEK293</t> cells, compared to control cells. GAPDH is applied as a loading control. ( B ) Representative silver-stained gel (of n = 4 experiments) shows that more protein persulfides are detected in TrxR1 and TRP14 knockdown HEK293 cells than in the control (control cells have been transfected with a plasmid that transcribes a scramble shRNA) under normal growth conditions. ( C ) Increases in protein persulfide levels in TrxR1 and TRP14 knockdown HEK293 cells compared to control reached statistical significance (* P
    Silver Stained Gels, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 91/100, based on 308 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Greiner Bio h2 sn
    TrxR1 and TRP14 counteract intracellular protein persulfide accumulation. S3 refers to sampling according to Fig. 1 . ( A ) Western blot showing the knockdown of TRP14 and TrxR1 in stably transfected <t>HEK293</t> cells, compared to control cells. GAPDH is applied as a loading control. ( B ) Representative silver-stained gel (of n = 4 experiments) shows that more protein persulfides are detected in TrxR1 and TRP14 knockdown HEK293 cells than in the control (control cells have been transfected with a plasmid that transcribes a scramble shRNA) under normal growth conditions. ( C ) Increases in protein persulfide levels in TrxR1 and TRP14 knockdown HEK293 cells compared to control reached statistical significance (* P
    H2 Sn, supplied by Greiner Bio, used in various techniques. Bioz Stars score: 94/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Dojindo Labs ssp4
    Visualization of persulfides and polysulfides in normal fibroblasts in response to exogenous cystine treatment. (A) Photomicrographs of human lung fibroblasts cultured ±200 µM cystine for 1 h and labeled by the CN biotin tag-switch method. Nuclei were stained with DAPI. (B) Western blot analysis of proteins from (A) shows a similar labeling intensity in cells grown ± cystine supplementation. GAPDH was used as a loading control (bottom). (C) Representative photomicrographs show a clear increase in fluorescence in cells treated with cystine when probed with the <t>SSP4</t> reagent. Nuclei were stained with Hoechst. Scale bar: 20 µm.
    Ssp4, supplied by Dojindo Labs, used in various techniques. Bioz Stars score: 91/100, based on 55 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    dtt  (Bio-Rad)
    99
    Bio-Rad dtt
    LC-MS/MS analysis of the tryptic peptide containing Cys60 of LeACO1 and LeACO2 recombinant protein (A) . The table inside the spectrum contains the predicted ion types for the modified peptide, and the ions detected in the spectrum are highlighted in red. A 32 D mass shift in Cys60 were included in the analysis of data by Maxquant software. Immunoblot analysis of recombinant LeACO1 mutation proteins (B) . <t>NaHS-induced</t> persulfidation of LeACO1 mutation was detected using a MBST. Recombinant proteins were pretreated with NaHS (200 μM) and NaHS plus <t>DTT</t> (10 mM) for 20 min, and then the immunoblot analysis carried out. Persulfidated proteins were labeled with biotin and analyzed using an anti-biotin antibody. Input is anti-his signal, which indicate the total protein amount. Analysis of recombinant LeACO1 C60S mutation protein activity (C) . Recombinant proteins were pretreated with NaHS and NaHS plus 10 mM DTT for 20 min, and then enzyme activity was analyzed. Mean values ± SE were calculated from three replicates. Within each set of experiments, bars with different letters are significant different (Duncan’s multiple range tests, P
    Dtt, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 5935 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Bio-Rad imagej
    LC-MS/MS analysis of the tryptic peptide containing Cys60 of LeACO1 and LeACO2 recombinant protein (A) . The table inside the spectrum contains the predicted ion types for the modified peptide, and the ions detected in the spectrum are highlighted in red. A 32 D mass shift in Cys60 were included in the analysis of data by Maxquant software. Immunoblot analysis of recombinant LeACO1 mutation proteins (B) . <t>NaHS-induced</t> persulfidation of LeACO1 mutation was detected using a MBST. Recombinant proteins were pretreated with NaHS (200 μM) and NaHS plus <t>DTT</t> (10 mM) for 20 min, and then the immunoblot analysis carried out. Persulfidated proteins were labeled with biotin and analyzed using an anti-biotin antibody. Input is anti-his signal, which indicate the total protein amount. Analysis of recombinant LeACO1 C60S mutation protein activity (C) . Recombinant proteins were pretreated with NaHS and NaHS plus 10 mM DTT for 20 min, and then enzyme activity was analyzed. Mean values ± SE were calculated from three replicates. Within each set of experiments, bars with different letters are significant different (Duncan’s multiple range tests, P
    Imagej, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 93/100, based on 387 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Agilent technologies poroshell 120
    LC-MS/MS analysis of the tryptic peptide containing Cys60 of LeACO1 and LeACO2 recombinant protein (A) . The table inside the spectrum contains the predicted ion types for the modified peptide, and the ions detected in the spectrum are highlighted in red. A 32 D mass shift in Cys60 were included in the analysis of data by Maxquant software. Immunoblot analysis of recombinant LeACO1 mutation proteins (B) . <t>NaHS-induced</t> persulfidation of LeACO1 mutation was detected using a MBST. Recombinant proteins were pretreated with NaHS (200 μM) and NaHS plus <t>DTT</t> (10 mM) for 20 min, and then the immunoblot analysis carried out. Persulfidated proteins were labeled with biotin and analyzed using an anti-biotin antibody. Input is anti-his signal, which indicate the total protein amount. Analysis of recombinant LeACO1 C60S mutation protein activity (C) . Recombinant proteins were pretreated with NaHS and NaHS plus 10 mM DTT for 20 min, and then enzyme activity was analyzed. Mean values ± SE were calculated from three replicates. Within each set of experiments, bars with different letters are significant different (Duncan’s multiple range tests, P
    Poroshell 120, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 88/100, based on 78 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Schiff Nutrition International site plp
    LC-MS/MS analysis of the tryptic peptide containing Cys60 of LeACO1 and LeACO2 recombinant protein (A) . The table inside the spectrum contains the predicted ion types for the modified peptide, and the ions detected in the spectrum are highlighted in red. A 32 D mass shift in Cys60 were included in the analysis of data by Maxquant software. Immunoblot analysis of recombinant LeACO1 mutation proteins (B) . <t>NaHS-induced</t> persulfidation of LeACO1 mutation was detected using a MBST. Recombinant proteins were pretreated with NaHS (200 μM) and NaHS plus <t>DTT</t> (10 mM) for 20 min, and then the immunoblot analysis carried out. Persulfidated proteins were labeled with biotin and analyzed using an anti-biotin antibody. Input is anti-his signal, which indicate the total protein amount. Analysis of recombinant LeACO1 C60S mutation protein activity (C) . Recombinant proteins were pretreated with NaHS and NaHS plus 10 mM DTT for 20 min, and then enzyme activity was analyzed. Mean values ± SE were calculated from three replicates. Within each set of experiments, bars with different letters are significant different (Duncan’s multiple range tests, P
    Site Plp, supplied by Schiff Nutrition International, used in various techniques. Bioz Stars score: 86/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Journal of Biological Chemistry h2 s
    LC-MS/MS analysis of the tryptic peptide containing Cys60 of LeACO1 and LeACO2 recombinant protein (A) . The table inside the spectrum contains the predicted ion types for the modified peptide, and the ions detected in the spectrum are highlighted in red. A 32 D mass shift in Cys60 were included in the analysis of data by Maxquant software. Immunoblot analysis of recombinant LeACO1 mutation proteins (B) . <t>NaHS-induced</t> persulfidation of LeACO1 mutation was detected using a MBST. Recombinant proteins were pretreated with NaHS (200 μM) and NaHS plus <t>DTT</t> (10 mM) for 20 min, and then the immunoblot analysis carried out. Persulfidated proteins were labeled with biotin and analyzed using an anti-biotin antibody. Input is anti-his signal, which indicate the total protein amount. Analysis of recombinant LeACO1 C60S mutation protein activity (C) . Recombinant proteins were pretreated with NaHS and NaHS plus 10 mM DTT for 20 min, and then enzyme activity was analyzed. Mean values ± SE were calculated from three replicates. Within each set of experiments, bars with different letters are significant different (Duncan’s multiple range tests, P
    H2 S, supplied by Journal of Biological Chemistry, used in various techniques. Bioz Stars score: 92/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore streptavidin coated magnetic beads
    Potential caveats of the ProPerDP method. ( A ) In proteins with more than one free Cys that exhibit different persulfidation properties, because of a nonpersulfidated Cys residue, the protein can stay immobilized on the <t>streptavidin</t> beads despite containing a persulfide, leading to false-negative signals. ( B ) Intermolecular protein disulfide bonds with nonpersulfidated extra Cys residues on one of the polypeptide chains might appear as false-positive signals in the persulfide proteome. Upon reduction, P 2 proteins are cleaved off the beads and could erroneously be present in the persulfide proteome fractions (Sample 3 in Fig. 1 ). ( C ) A potential way to overcome the abovementioned caveats is to digest the alkylated proteins before the pulldown step because it is highly unlikely that both the free and persulfidated Cys [for (A)] or the disulfide and the free Cys moieties [for (B)] will end up in the same peptide using this method. With this approach, the alkylated peptide persulfides could be detected by mass spectrometry after they are cleaved off the beads by the reducing agent. This method improvement is under development in our laboratory.
    Streptavidin Coated Magnetic Beads, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 80 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher phosphate buffered saline pbs
    Potential caveats of the ProPerDP method. ( A ) In proteins with more than one free Cys that exhibit different persulfidation properties, because of a nonpersulfidated Cys residue, the protein can stay immobilized on the <t>streptavidin</t> beads despite containing a persulfide, leading to false-negative signals. ( B ) Intermolecular protein disulfide bonds with nonpersulfidated extra Cys residues on one of the polypeptide chains might appear as false-positive signals in the persulfide proteome. Upon reduction, P 2 proteins are cleaved off the beads and could erroneously be present in the persulfide proteome fractions (Sample 3 in Fig. 1 ). ( C ) A potential way to overcome the abovementioned caveats is to digest the alkylated proteins before the pulldown step because it is highly unlikely that both the free and persulfidated Cys [for (A)] or the disulfide and the free Cys moieties [for (B)] will end up in the same peptide using this method. With this approach, the alkylated peptide persulfides could be detected by mass spectrometry after they are cleaved off the beads by the reducing agent. This method improvement is under development in our laboratory.
    Phosphate Buffered Saline Pbs, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 34431 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Valiant zymolyase 100t
    Potential caveats of the ProPerDP method. ( A ) In proteins with more than one free Cys that exhibit different persulfidation properties, because of a nonpersulfidated Cys residue, the protein can stay immobilized on the <t>streptavidin</t> beads despite containing a persulfide, leading to false-negative signals. ( B ) Intermolecular protein disulfide bonds with nonpersulfidated extra Cys residues on one of the polypeptide chains might appear as false-positive signals in the persulfide proteome. Upon reduction, P 2 proteins are cleaved off the beads and could erroneously be present in the persulfide proteome fractions (Sample 3 in Fig. 1 ). ( C ) A potential way to overcome the abovementioned caveats is to digest the alkylated proteins before the pulldown step because it is highly unlikely that both the free and persulfidated Cys [for (A)] or the disulfide and the free Cys moieties [for (B)] will end up in the same peptide using this method. With this approach, the alkylated peptide persulfides could be detected by mass spectrometry after they are cleaved off the beads by the reducing agent. This method improvement is under development in our laboratory.
    Zymolyase 100t, supplied by Valiant, used in various techniques. Bioz Stars score: 93/100, based on 140 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Xenobiotics h2 s
    Potential caveats of the ProPerDP method. ( A ) In proteins with more than one free Cys that exhibit different persulfidation properties, because of a nonpersulfidated Cys residue, the protein can stay immobilized on the <t>streptavidin</t> beads despite containing a persulfide, leading to false-negative signals. ( B ) Intermolecular protein disulfide bonds with nonpersulfidated extra Cys residues on one of the polypeptide chains might appear as false-positive signals in the persulfide proteome. Upon reduction, P 2 proteins are cleaved off the beads and could erroneously be present in the persulfide proteome fractions (Sample 3 in Fig. 1 ). ( C ) A potential way to overcome the abovementioned caveats is to digest the alkylated proteins before the pulldown step because it is highly unlikely that both the free and persulfidated Cys [for (A)] or the disulfide and the free Cys moieties [for (B)] will end up in the same peptide using this method. With this approach, the alkylated peptide persulfides could be detected by mass spectrometry after they are cleaved off the beads by the reducing agent. This method improvement is under development in our laboratory.
    H2 S, supplied by Xenobiotics, used in various techniques. Bioz Stars score: 93/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore n hydroxysuccinimide nhs
    Potential caveats of the ProPerDP method. ( A ) In proteins with more than one free Cys that exhibit different persulfidation properties, because of a nonpersulfidated Cys residue, the protein can stay immobilized on the <t>streptavidin</t> beads despite containing a persulfide, leading to false-negative signals. ( B ) Intermolecular protein disulfide bonds with nonpersulfidated extra Cys residues on one of the polypeptide chains might appear as false-positive signals in the persulfide proteome. Upon reduction, P 2 proteins are cleaved off the beads and could erroneously be present in the persulfide proteome fractions (Sample 3 in Fig. 1 ). ( C ) A potential way to overcome the abovementioned caveats is to digest the alkylated proteins before the pulldown step because it is highly unlikely that both the free and persulfidated Cys [for (A)] or the disulfide and the free Cys moieties [for (B)] will end up in the same peptide using this method. With this approach, the alkylated peptide persulfides could be detected by mass spectrometry after they are cleaved off the beads by the reducing agent. This method improvement is under development in our laboratory.
    N Hydroxysuccinimide Nhs, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2694 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Agilent technologies electrospray lc ms
    Potential caveats of the ProPerDP method. ( A ) In proteins with more than one free Cys that exhibit different persulfidation properties, because of a nonpersulfidated Cys residue, the protein can stay immobilized on the <t>streptavidin</t> beads despite containing a persulfide, leading to false-negative signals. ( B ) Intermolecular protein disulfide bonds with nonpersulfidated extra Cys residues on one of the polypeptide chains might appear as false-positive signals in the persulfide proteome. Upon reduction, P 2 proteins are cleaved off the beads and could erroneously be present in the persulfide proteome fractions (Sample 3 in Fig. 1 ). ( C ) A potential way to overcome the abovementioned caveats is to digest the alkylated proteins before the pulldown step because it is highly unlikely that both the free and persulfidated Cys [for (A)] or the disulfide and the free Cys moieties [for (B)] will end up in the same peptide using this method. With this approach, the alkylated peptide persulfides could be detected by mass spectrometry after they are cleaved off the beads by the reducing agent. This method improvement is under development in our laboratory.
    Electrospray Lc Ms, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 88/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Thermo Fisher iab
    Potential caveats of the ProPerDP method. ( A ) In proteins with more than one free Cys that exhibit different persulfidation properties, because of a nonpersulfidated Cys residue, the protein can stay immobilized on the <t>streptavidin</t> beads despite containing a persulfide, leading to false-negative signals. ( B ) Intermolecular protein disulfide bonds with nonpersulfidated extra Cys residues on one of the polypeptide chains might appear as false-positive signals in the persulfide proteome. Upon reduction, P 2 proteins are cleaved off the beads and could erroneously be present in the persulfide proteome fractions (Sample 3 in Fig. 1 ). ( C ) A potential way to overcome the abovementioned caveats is to digest the alkylated proteins before the pulldown step because it is highly unlikely that both the free and persulfidated Cys [for (A)] or the disulfide and the free Cys moieties [for (B)] will end up in the same peptide using this method. With this approach, the alkylated peptide persulfides could be detected by mass spectrometry after they are cleaved off the beads by the reducing agent. This method improvement is under development in our laboratory.
    Iab, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 35 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Lonza hek293 cells
    TrxR1 and TRP14 counteract intracellular protein persulfide accumulation. S3 refers to sampling according to Fig. 1 . ( A ) Western blot showing the knockdown of TRP14 and TrxR1 in stably transfected <t>HEK293</t> cells, compared to control cells. GAPDH is applied as a loading control. ( B ) Representative silver-stained gel (of n = 4 experiments) shows that more protein persulfides are detected in TrxR1 and TRP14 knockdown HEK293 cells than in the control (control cells have been transfected with a plasmid that transcribes a scramble shRNA) under normal growth conditions. ( C ) Increases in protein persulfide levels in TrxR1 and TRP14 knockdown HEK293 cells compared to control reached statistical significance (* P
    Hek293 Cells, supplied by Lonza, used in various techniques. Bioz Stars score: 93/100, based on 495 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Lonza balanced salt solution
    TrxR1 and TRP14 counteract intracellular protein persulfide accumulation. S3 refers to sampling according to Fig. 1 . ( A ) Western blot showing the knockdown of TRP14 and TrxR1 in stably transfected <t>HEK293</t> cells, compared to control cells. GAPDH is applied as a loading control. ( B ) Representative silver-stained gel (of n = 4 experiments) shows that more protein persulfides are detected in TrxR1 and TRP14 knockdown HEK293 cells than in the control (control cells have been transfected with a plasmid that transcribes a scramble shRNA) under normal growth conditions. ( C ) Increases in protein persulfide levels in TrxR1 and TRP14 knockdown HEK293 cells compared to control reached statistical significance (* P
    Balanced Salt Solution, supplied by Lonza, used in various techniques. Bioz Stars score: 92/100, based on 172 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TrxR1 and TRP14 counteract intracellular protein persulfide accumulation. S3 refers to sampling according to Fig. 1 . ( A ) Western blot showing the knockdown of TRP14 and TrxR1 in stably transfected <t>HEK293</t> cells, compared to control cells. GAPDH is applied as a loading control. ( B ) Representative silver-stained gel (of n = 4 experiments) shows that more protein persulfides are detected in TrxR1 and TRP14 knockdown HEK293 cells than in the control (control cells have been transfected with a plasmid that transcribes a scramble shRNA) under normal growth conditions. ( C ) Increases in protein persulfide levels in TrxR1 and TRP14 knockdown HEK293 cells compared to control reached statistical significance (* P
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    TrxR1 and TRP14 counteract intracellular protein persulfide accumulation. S3 refers to sampling according to Fig. 1 . ( A ) Western blot showing the knockdown of TRP14 and TrxR1 in stably transfected <t>HEK293</t> cells, compared to control cells. GAPDH is applied as a loading control. ( B ) Representative silver-stained gel (of n = 4 experiments) shows that more protein persulfides are detected in TrxR1 and TRP14 knockdown HEK293 cells than in the control (control cells have been transfected with a plasmid that transcribes a scramble shRNA) under normal growth conditions. ( C ) Increases in protein persulfide levels in TrxR1 and TRP14 knockdown HEK293 cells compared to control reached statistical significance (* P
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    Image Search Results


    Visualization of persulfides in normal and cystinotic fibroblasts using the CN-biotin tag switch method. (A) Representative photomicrographs of normal and cystinotic human lung fibroblasts labeled with CN biotin tag-switch assay. The nuclei were stained with DAPI. (B) Intracellular persulfide levels in normal and cystinotic human lung fibroblast cell lysates, labeled using the CN-biotin tag switch reagent. Proteins were visualized using Streptavidin Dylight 488. Artificial color intensity was used and the gradient scale is shown on the right. GAPDH was used as a loading control (bottom). Scale bar: 20 µm.

    Journal: Journal of the American Chemical Society

    Article Title: Biosynthesis and Reactivity of Cysteine Persulfides in Signaling

    doi: 10.1021/jacs.5b10494

    Figure Lengend Snippet: Visualization of persulfides in normal and cystinotic fibroblasts using the CN-biotin tag switch method. (A) Representative photomicrographs of normal and cystinotic human lung fibroblasts labeled with CN biotin tag-switch assay. The nuclei were stained with DAPI. (B) Intracellular persulfide levels in normal and cystinotic human lung fibroblast cell lysates, labeled using the CN-biotin tag switch reagent. Proteins were visualized using Streptavidin Dylight 488. Artificial color intensity was used and the gradient scale is shown on the right. GAPDH was used as a loading control (bottom). Scale bar: 20 µm.

    Article Snippet: For visualization of persulfide levels by fluorescence microscopy, cells were plated in ibidi dishes (ibidi, Martinsried, Germany) until they reached confluency, fixed using methanol and permeabilized with acetone and then labeled for persulfide detection using the CN-biotin tag-switch assay method, as previously described., Briefly, fixed cells were first incubated with 10 mM 2-(methylsulfonyl)-1,3-benzothiazole overnight, washed 5 times with PBS and then incubated for 1 h at 37 °C with 2 mM CN-biotin, prepared as described., DyLight 488 Streptavidin (Pierce, ThermoFisher Scientific) was used to visualize biotinylated proteins.

    Techniques: Labeling, Staining

    TrxR1 and TRP14 counteract intracellular protein persulfide accumulation. S3 refers to sampling according to Fig. 1 . ( A ) Western blot showing the knockdown of TRP14 and TrxR1 in stably transfected HEK293 cells, compared to control cells. GAPDH is applied as a loading control. ( B ) Representative silver-stained gel (of n = 4 experiments) shows that more protein persulfides are detected in TrxR1 and TRP14 knockdown HEK293 cells than in the control (control cells have been transfected with a plasmid that transcribes a scramble shRNA) under normal growth conditions. ( C ) Increases in protein persulfide levels in TrxR1 and TRP14 knockdown HEK293 cells compared to control reached statistical significance (* P

    Journal: Science Advances

    Article Title: A novel persulfide detection method reveals protein persulfide- and polysulfide-reducing functions of thioredoxin and glutathione systems

    doi: 10.1126/sciadv.1500968

    Figure Lengend Snippet: TrxR1 and TRP14 counteract intracellular protein persulfide accumulation. S3 refers to sampling according to Fig. 1 . ( A ) Western blot showing the knockdown of TRP14 and TrxR1 in stably transfected HEK293 cells, compared to control cells. GAPDH is applied as a loading control. ( B ) Representative silver-stained gel (of n = 4 experiments) shows that more protein persulfides are detected in TrxR1 and TRP14 knockdown HEK293 cells than in the control (control cells have been transfected with a plasmid that transcribes a scramble shRNA) under normal growth conditions. ( C ) Increases in protein persulfide levels in TrxR1 and TRP14 knockdown HEK293 cells compared to control reached statistical significance (* P

    Article Snippet: Estimation of endogenous protein persulfide levels Protein persulfide levels in intact HEK293 cells and mouse liver tissue samples were estimated by densitometric analysis of the corresponding silver-stained gels using the ImageJ [National Institutes of Health (NIH)] or Quantity One (Bio-Rad) software.

    Techniques: Sampling, Western Blot, Stable Transfection, Transfection, Staining, Plasmid Preparation, shRNA

    Visualization of persulfides and polysulfides in normal fibroblasts in response to exogenous cystine treatment. (A) Photomicrographs of human lung fibroblasts cultured ±200 µM cystine for 1 h and labeled by the CN biotin tag-switch method. Nuclei were stained with DAPI. (B) Western blot analysis of proteins from (A) shows a similar labeling intensity in cells grown ± cystine supplementation. GAPDH was used as a loading control (bottom). (C) Representative photomicrographs show a clear increase in fluorescence in cells treated with cystine when probed with the SSP4 reagent. Nuclei were stained with Hoechst. Scale bar: 20 µm.

    Journal: Journal of the American Chemical Society

    Article Title: Biosynthesis and Reactivity of Cysteine Persulfides in Signaling

    doi: 10.1021/jacs.5b10494

    Figure Lengend Snippet: Visualization of persulfides and polysulfides in normal fibroblasts in response to exogenous cystine treatment. (A) Photomicrographs of human lung fibroblasts cultured ±200 µM cystine for 1 h and labeled by the CN biotin tag-switch method. Nuclei were stained with DAPI. (B) Western blot analysis of proteins from (A) shows a similar labeling intensity in cells grown ± cystine supplementation. GAPDH was used as a loading control (bottom). (C) Representative photomicrographs show a clear increase in fluorescence in cells treated with cystine when probed with the SSP4 reagent. Nuclei were stained with Hoechst. Scale bar: 20 µm.

    Article Snippet: In addition, enzymes like mercaptopyruvate sulfurtransferase and rhodanese, which stabilize Cys−SSH intermediates in their active sites, use stereoelectronic control to transfer the outer sulfur to thiophilic acceptors., Hence, SSP4 can detect hydropolysulfides, polysulfides and protein bound persulfides at cysteines with low p K a values but is unlikely to detect LMW persulfides in solution.

    Techniques: Cell Culture, Labeling, Staining, Western Blot, Fluorescence

    LC-MS/MS analysis of the tryptic peptide containing Cys60 of LeACO1 and LeACO2 recombinant protein (A) . The table inside the spectrum contains the predicted ion types for the modified peptide, and the ions detected in the spectrum are highlighted in red. A 32 D mass shift in Cys60 were included in the analysis of data by Maxquant software. Immunoblot analysis of recombinant LeACO1 mutation proteins (B) . NaHS-induced persulfidation of LeACO1 mutation was detected using a MBST. Recombinant proteins were pretreated with NaHS (200 μM) and NaHS plus DTT (10 mM) for 20 min, and then the immunoblot analysis carried out. Persulfidated proteins were labeled with biotin and analyzed using an anti-biotin antibody. Input is anti-his signal, which indicate the total protein amount. Analysis of recombinant LeACO1 C60S mutation protein activity (C) . Recombinant proteins were pretreated with NaHS and NaHS plus 10 mM DTT for 20 min, and then enzyme activity was analyzed. Mean values ± SE were calculated from three replicates. Within each set of experiments, bars with different letters are significant different (Duncan’s multiple range tests, P

    Journal: Frontiers in Plant Science

    Article Title: Ethylene-Induced Hydrogen Sulfide Negatively Regulates Ethylene Biosynthesis by Persulfidation of ACO in Tomato Under Osmotic Stress

    doi: 10.3389/fpls.2018.01517

    Figure Lengend Snippet: LC-MS/MS analysis of the tryptic peptide containing Cys60 of LeACO1 and LeACO2 recombinant protein (A) . The table inside the spectrum contains the predicted ion types for the modified peptide, and the ions detected in the spectrum are highlighted in red. A 32 D mass shift in Cys60 were included in the analysis of data by Maxquant software. Immunoblot analysis of recombinant LeACO1 mutation proteins (B) . NaHS-induced persulfidation of LeACO1 mutation was detected using a MBST. Recombinant proteins were pretreated with NaHS (200 μM) and NaHS plus DTT (10 mM) for 20 min, and then the immunoblot analysis carried out. Persulfidated proteins were labeled with biotin and analyzed using an anti-biotin antibody. Input is anti-his signal, which indicate the total protein amount. Analysis of recombinant LeACO1 C60S mutation protein activity (C) . Recombinant proteins were pretreated with NaHS and NaHS plus 10 mM DTT for 20 min, and then enzyme activity was analyzed. Mean values ± SE were calculated from three replicates. Within each set of experiments, bars with different letters are significant different (Duncan’s multiple range tests, P

    Article Snippet: The purified recombinant LeACO1 and LeACO2 proteins were treated with 50 to 400 μM NaHS to increase the concentration of persulfidated protein or with 10 mM DTT to reduce all of the disulfide bonds; both treatments were carried out at 4°C for 20 min. NaHS was removed using Micro BioSpinP6columns (BioRad).

    Techniques: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Recombinant, Modification, Software, Mutagenesis, Labeling, Activity Assay

    Immunoblot and activity analysis of recombinant LeACO1 and LeACO2 proteins. (A) NaHS-induced persulfidation of LeACO1 was detected using a MBST. (B) Quantification of persulfidation levels that are shown in (A) . (C) NaHS-induced persulfidation of LeACO2 was detected using a MBST. (D) Quantification of persulfidation levels that are shown in (C) . Input is anti-his signal, which indicate the total protein amount. Persulfidated proteins were labeled with biotin and analyzed using an anti-biotin antibody. Total proteins were analyzed using an anti-his antibody. Effect of NaHS-induced persulfidation on LeACO1 (E) and LeACO2 (F) activity. Recombinant proteins were pretreated with NaHS and NaHS plus DTT (10 mM) for 20 min, and then the immunoblot analysis carried out. Mean values ± SE are calculated from three replicates. Within each set of experiments, bars with different letters are significant different (Duncan’s multiple range tests, P

    Journal: Frontiers in Plant Science

    Article Title: Ethylene-Induced Hydrogen Sulfide Negatively Regulates Ethylene Biosynthesis by Persulfidation of ACO in Tomato Under Osmotic Stress

    doi: 10.3389/fpls.2018.01517

    Figure Lengend Snippet: Immunoblot and activity analysis of recombinant LeACO1 and LeACO2 proteins. (A) NaHS-induced persulfidation of LeACO1 was detected using a MBST. (B) Quantification of persulfidation levels that are shown in (A) . (C) NaHS-induced persulfidation of LeACO2 was detected using a MBST. (D) Quantification of persulfidation levels that are shown in (C) . Input is anti-his signal, which indicate the total protein amount. Persulfidated proteins were labeled with biotin and analyzed using an anti-biotin antibody. Total proteins were analyzed using an anti-his antibody. Effect of NaHS-induced persulfidation on LeACO1 (E) and LeACO2 (F) activity. Recombinant proteins were pretreated with NaHS and NaHS plus DTT (10 mM) for 20 min, and then the immunoblot analysis carried out. Mean values ± SE are calculated from three replicates. Within each set of experiments, bars with different letters are significant different (Duncan’s multiple range tests, P

    Article Snippet: The purified recombinant LeACO1 and LeACO2 proteins were treated with 50 to 400 μM NaHS to increase the concentration of persulfidated protein or with 10 mM DTT to reduce all of the disulfide bonds; both treatments were carried out at 4°C for 20 min. NaHS was removed using Micro BioSpinP6columns (BioRad).

    Techniques: Activity Assay, Recombinant, Labeling

    Potential caveats of the ProPerDP method. ( A ) In proteins with more than one free Cys that exhibit different persulfidation properties, because of a nonpersulfidated Cys residue, the protein can stay immobilized on the streptavidin beads despite containing a persulfide, leading to false-negative signals. ( B ) Intermolecular protein disulfide bonds with nonpersulfidated extra Cys residues on one of the polypeptide chains might appear as false-positive signals in the persulfide proteome. Upon reduction, P 2 proteins are cleaved off the beads and could erroneously be present in the persulfide proteome fractions (Sample 3 in Fig. 1 ). ( C ) A potential way to overcome the abovementioned caveats is to digest the alkylated proteins before the pulldown step because it is highly unlikely that both the free and persulfidated Cys [for (A)] or the disulfide and the free Cys moieties [for (B)] will end up in the same peptide using this method. With this approach, the alkylated peptide persulfides could be detected by mass spectrometry after they are cleaved off the beads by the reducing agent. This method improvement is under development in our laboratory.

    Journal: Science Advances

    Article Title: A novel persulfide detection method reveals protein persulfide- and polysulfide-reducing functions of thioredoxin and glutathione systems

    doi: 10.1126/sciadv.1500968

    Figure Lengend Snippet: Potential caveats of the ProPerDP method. ( A ) In proteins with more than one free Cys that exhibit different persulfidation properties, because of a nonpersulfidated Cys residue, the protein can stay immobilized on the streptavidin beads despite containing a persulfide, leading to false-negative signals. ( B ) Intermolecular protein disulfide bonds with nonpersulfidated extra Cys residues on one of the polypeptide chains might appear as false-positive signals in the persulfide proteome. Upon reduction, P 2 proteins are cleaved off the beads and could erroneously be present in the persulfide proteome fractions (Sample 3 in Fig. 1 ). ( C ) A potential way to overcome the abovementioned caveats is to digest the alkylated proteins before the pulldown step because it is highly unlikely that both the free and persulfidated Cys [for (A)] or the disulfide and the free Cys moieties [for (B)] will end up in the same peptide using this method. With this approach, the alkylated peptide persulfides could be detected by mass spectrometry after they are cleaved off the beads by the reducing agent. This method improvement is under development in our laboratory.

    Article Snippet: Biotinylated proteins were pulled down by streptavidin-coated magnetic beads (Sigma), and from this step, the same protocol was applied as for the purified protein samples, using 25 mM DTT instead of 5 mM TCEP to cleave the persulfidated proteins off the beads.

    Techniques: Mass Spectrometry

    Protein Persulfide Detection Protocol. Thiol and persulfide functional groups are alkylated by IAB to form the corresponding thioether and dialkyl disulfide derivatives, respectively (-R denotes the electrophile moiety of the alkylating agent). Oxidized Cys residues of proteins in the original sample (Sample 1) will not be derivatized by IAB. Affinity purification of alkylated proteins is achieved by pulldown with streptavidin-coated magnetic beads, leaving proteins only containing oxidized Cys residues in the supernatant (Sample 2). Resuspension of purified beads in a reducing buffer selectively cleaves the original persulfides off as thiols, which can be analyzed after separation with recovery from the beads, thus allowing the determination of protein persulfides (Sample 3). Note that some sulfenic acid (-SOH) or nitrosothiol (-SNO) derivatives might become alkylated in Sample 1, but these reactions give thioethers such as free thiols and hence do not appear as false positives in Sample 3. Native Cys residues with free thiols in the original sample will not be released from the beads by reduction but can be recovered by boiling of the beads in SDS (Sample 4). The diamond symbol (◊) denotes the biotin tag, and the pictogram in the inset refers to the biotin-streptavidin binding interaction. The order and nomenclature of the samples are maintained for all gels and blots in this study, thereby referred to as S1 to S4, respectively.

    Journal: Science Advances

    Article Title: A novel persulfide detection method reveals protein persulfide- and polysulfide-reducing functions of thioredoxin and glutathione systems

    doi: 10.1126/sciadv.1500968

    Figure Lengend Snippet: Protein Persulfide Detection Protocol. Thiol and persulfide functional groups are alkylated by IAB to form the corresponding thioether and dialkyl disulfide derivatives, respectively (-R denotes the electrophile moiety of the alkylating agent). Oxidized Cys residues of proteins in the original sample (Sample 1) will not be derivatized by IAB. Affinity purification of alkylated proteins is achieved by pulldown with streptavidin-coated magnetic beads, leaving proteins only containing oxidized Cys residues in the supernatant (Sample 2). Resuspension of purified beads in a reducing buffer selectively cleaves the original persulfides off as thiols, which can be analyzed after separation with recovery from the beads, thus allowing the determination of protein persulfides (Sample 3). Note that some sulfenic acid (-SOH) or nitrosothiol (-SNO) derivatives might become alkylated in Sample 1, but these reactions give thioethers such as free thiols and hence do not appear as false positives in Sample 3. Native Cys residues with free thiols in the original sample will not be released from the beads by reduction but can be recovered by boiling of the beads in SDS (Sample 4). The diamond symbol (◊) denotes the biotin tag, and the pictogram in the inset refers to the biotin-streptavidin binding interaction. The order and nomenclature of the samples are maintained for all gels and blots in this study, thereby referred to as S1 to S4, respectively.

    Article Snippet: Biotinylated proteins were pulled down by streptavidin-coated magnetic beads (Sigma), and from this step, the same protocol was applied as for the purified protein samples, using 25 mM DTT instead of 5 mM TCEP to cleave the persulfidated proteins off the beads.

    Techniques: Functional Assay, Affinity Purification, Magnetic Beads, Purification, Binding Assay

    Detection of protein persulfide formation on HSA. S1 to S4 refer to sampling according to Fig. 1 . ( A ) IAB alkylated HSA-SSH samples are efficiently pulled down by streptavidin-coated magnetic beads (compare S1 to S2). Lanes S3 and S4 represent HSA-SSH that was reduced off the beads by TCEP and HSA thiol that was released during boiling, respectively. No bands were detected in S3 when HSA-SSH was reduced by TCEP before alkylation. Gels are representative of n = 9 experiments. ( A′ ) Incubation of HSA-SSH with 5 mM sulfide for 30 min before alkylation reduces some of the HSA-SSH by shifting the equilibrium of reaction 1 (see text). ( B and B′ ) HSA-SSH formation was observed in HS x − -treated plasma samples (B), but no endogenous HSA-SSH was detectable in untreated plasma (B′) by Colloidal Coomassie Blue staining. Gels are representative of n = 3 experiments. ( C and C′ ) Concentration-dependent (C) HS x − or (C′) H 2 S treatment induced HSA-SSH formation in plasma detected by immunoblot analyses against HSA. Blots only show S3. Plasma protein (100 ng) and pure HSA (20 ng) were applied as loading controls. Blots are representative of n = 3 experiments. The observed mobility shifts on each gels are due to the reduction of structural disulfide bonds in HSA during the final reduction step. Mass spectrometry confirmed that the shifted bands indeed represent HSA.

    Journal: Science Advances

    Article Title: A novel persulfide detection method reveals protein persulfide- and polysulfide-reducing functions of thioredoxin and glutathione systems

    doi: 10.1126/sciadv.1500968

    Figure Lengend Snippet: Detection of protein persulfide formation on HSA. S1 to S4 refer to sampling according to Fig. 1 . ( A ) IAB alkylated HSA-SSH samples are efficiently pulled down by streptavidin-coated magnetic beads (compare S1 to S2). Lanes S3 and S4 represent HSA-SSH that was reduced off the beads by TCEP and HSA thiol that was released during boiling, respectively. No bands were detected in S3 when HSA-SSH was reduced by TCEP before alkylation. Gels are representative of n = 9 experiments. ( A′ ) Incubation of HSA-SSH with 5 mM sulfide for 30 min before alkylation reduces some of the HSA-SSH by shifting the equilibrium of reaction 1 (see text). ( B and B′ ) HSA-SSH formation was observed in HS x − -treated plasma samples (B), but no endogenous HSA-SSH was detectable in untreated plasma (B′) by Colloidal Coomassie Blue staining. Gels are representative of n = 3 experiments. ( C and C′ ) Concentration-dependent (C) HS x − or (C′) H 2 S treatment induced HSA-SSH formation in plasma detected by immunoblot analyses against HSA. Blots only show S3. Plasma protein (100 ng) and pure HSA (20 ng) were applied as loading controls. Blots are representative of n = 3 experiments. The observed mobility shifts on each gels are due to the reduction of structural disulfide bonds in HSA during the final reduction step. Mass spectrometry confirmed that the shifted bands indeed represent HSA.

    Article Snippet: Biotinylated proteins were pulled down by streptavidin-coated magnetic beads (Sigma), and from this step, the same protocol was applied as for the purified protein samples, using 25 mM DTT instead of 5 mM TCEP to cleave the persulfidated proteins off the beads.

    Techniques: Sampling, Magnetic Beads, Incubation, Staining, Concentration Assay, Mass Spectrometry

    TrxR1 and TRP14 counteract intracellular protein persulfide accumulation. S3 refers to sampling according to Fig. 1 . ( A ) Western blot showing the knockdown of TRP14 and TrxR1 in stably transfected HEK293 cells, compared to control cells. GAPDH is applied as a loading control. ( B ) Representative silver-stained gel (of n = 4 experiments) shows that more protein persulfides are detected in TrxR1 and TRP14 knockdown HEK293 cells than in the control (control cells have been transfected with a plasmid that transcribes a scramble shRNA) under normal growth conditions. ( C ) Increases in protein persulfide levels in TrxR1 and TRP14 knockdown HEK293 cells compared to control reached statistical significance (* P

    Journal: Science Advances

    Article Title: A novel persulfide detection method reveals protein persulfide- and polysulfide-reducing functions of thioredoxin and glutathione systems

    doi: 10.1126/sciadv.1500968

    Figure Lengend Snippet: TrxR1 and TRP14 counteract intracellular protein persulfide accumulation. S3 refers to sampling according to Fig. 1 . ( A ) Western blot showing the knockdown of TRP14 and TrxR1 in stably transfected HEK293 cells, compared to control cells. GAPDH is applied as a loading control. ( B ) Representative silver-stained gel (of n = 4 experiments) shows that more protein persulfides are detected in TrxR1 and TRP14 knockdown HEK293 cells than in the control (control cells have been transfected with a plasmid that transcribes a scramble shRNA) under normal growth conditions. ( C ) Increases in protein persulfide levels in TrxR1 and TRP14 knockdown HEK293 cells compared to control reached statistical significance (* P

    Article Snippet: Protein persulfide detection in intact cells by ProPerDP A549 or HEK293 cells were incubated in the presence or absence of 200 μM polysulfide in Hanks’ balanced salt solution (HBSS; Lonza) for 30 min or 2 hours (in ) at 37°C, after which cells were rinsed with phosphate-buffered saline (PBS) or HBSS and incubated with 1 mM IAB (Thermo Scientific) in HBSS for 3 hours at 37°C.

    Techniques: Sampling, Western Blot, Stable Transfection, Transfection, Staining, Plasmid Preparation, shRNA