iodoacetamide  (Millipore)


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  • 99
    Name:
    Iodoacetamide
    Description:

    Catalog Number:
    i6125
    Price:
    None
    Applications:
    Alkylating reagent for cysteine residues in peptide sequencing. By virtue of reaction with cysteine, it is an irreversible inhibitor of enzymes with cysteine at the active site. It reacts much more slowly with histidine residues, but that activity is responsible for inhibition of ribonuclease.
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    Iodoacetamide

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    Images

    1) Product Images from "Disulfide Bonding among ?1 Trimers in Mammalian Reovirus Outer Capsid: a Late and Reversible Step in Virion Morphogenesis"

    Article Title: Disulfide Bonding among ?1 Trimers in Mammalian Reovirus Outer Capsid: a Late and Reversible Step in Virion Morphogenesis

    Journal: Journal of Virology

    doi: 10.1128/JVI.77.9.5389-5400.2003

    Formation of the μ1/μ1C ds bonds during the course of infection. T1L reovirus-infected cells were harvested at either 24, 48, 72, or 96 h postinfection. 50 mM IAM was added immediately upon resuspension of the infected cells in homogenization buffer. After purification, virions from each time point were mixed with nonreducing sample buffer (NR) and disrupted by boiling. The viral proteins were then resolved on a mini-sized SDS-8% PAGE gel. The amount of ds-bonded μ1/μ1C (% ds μ1/μ1C) relative to the total μ1, μ1C, and ds-bonded μ1/μ1C in each preparation was determined by densitometry of the Coomassie-stained gel. Immediately before harvesting, aliquots were taken from each time point and the proportion of dead/dying cells (% dead cells) was determined by trypan blue staining.
    Figure Legend Snippet: Formation of the μ1/μ1C ds bonds during the course of infection. T1L reovirus-infected cells were harvested at either 24, 48, 72, or 96 h postinfection. 50 mM IAM was added immediately upon resuspension of the infected cells in homogenization buffer. After purification, virions from each time point were mixed with nonreducing sample buffer (NR) and disrupted by boiling. The viral proteins were then resolved on a mini-sized SDS-8% PAGE gel. The amount of ds-bonded μ1/μ1C (% ds μ1/μ1C) relative to the total μ1, μ1C, and ds-bonded μ1/μ1C in each preparation was determined by densitometry of the Coomassie-stained gel. Immediately before harvesting, aliquots were taken from each time point and the proportion of dead/dying cells (% dead cells) was determined by trypan blue staining.

    Techniques Used: Infection, Homogenization, Purification, Polyacrylamide Gel Electrophoresis, Staining

    Analyses for ds bond formation with μ1 mutant C679S. (A) Recoated cores containing T1L wt σ3 and either T1L wt μ1 or the T1L μ1 mutant C679S were generated and purified, and particle concentrations were determined by densitometry. Equal amounts of the wt (lanes 2 and 5) or C679S (lanes 3 and 6) recoated cores were mixed with reducing (R) or nonreducing (NR) sample buffer, disrupted by boiling, and resolved on a mini-sized SDS-PAGE (8% acrylamide) gel. Virions disrupted under reducing (lane 1) or nonreducing (lane 4) conditions were included for comparison. Viral proteins were visualized by Coomassie staining. (B) Purified μ1-σ3 heterohexamers stored frozen in buffer with 10 mM DTT were thawed, and a small amount of each was diluted into nonreducing sample buffer and analyzed on a 4 to 15% acrylamide gradient native gel (Amersham Pharmacia Biotech) (lanes 1 to 3; 0 days). The remainder of each sample was passed through a PD-10 column to remove DTT and then stored at 4°C for 8 days at ambient conditions. At the end of that time, a small amount of each was diluted into sample buffer and analyzed on another 4 to 15% native gel (lanes 4 to 6). Three types of μ1-σ3 preparations were included in this analysis: complexes containing wt T1L μ1 and σ3 (lanes 1 and 4), complexes containing wt T1L μ1 and σ3 that were treated with 10 mM IAM to block free cysteines before storage (lanes 2 and 5), and complexes containing T1L C679S μ1 and wt T1L σ3 (lanes 3 and 6). The position of the native μ1-σ3 heterohexamer is indicated to the left. The positions of higher- M r forms specific to the complexes containing wt T1L μ1 and σ3 (lanes 1) are indicated (§). Positions of native gel markers (Amersham Pharmacia Biotech) are indicated in kilodaltons.
    Figure Legend Snippet: Analyses for ds bond formation with μ1 mutant C679S. (A) Recoated cores containing T1L wt σ3 and either T1L wt μ1 or the T1L μ1 mutant C679S were generated and purified, and particle concentrations were determined by densitometry. Equal amounts of the wt (lanes 2 and 5) or C679S (lanes 3 and 6) recoated cores were mixed with reducing (R) or nonreducing (NR) sample buffer, disrupted by boiling, and resolved on a mini-sized SDS-PAGE (8% acrylamide) gel. Virions disrupted under reducing (lane 1) or nonreducing (lane 4) conditions were included for comparison. Viral proteins were visualized by Coomassie staining. (B) Purified μ1-σ3 heterohexamers stored frozen in buffer with 10 mM DTT were thawed, and a small amount of each was diluted into nonreducing sample buffer and analyzed on a 4 to 15% acrylamide gradient native gel (Amersham Pharmacia Biotech) (lanes 1 to 3; 0 days). The remainder of each sample was passed through a PD-10 column to remove DTT and then stored at 4°C for 8 days at ambient conditions. At the end of that time, a small amount of each was diluted into sample buffer and analyzed on another 4 to 15% native gel (lanes 4 to 6). Three types of μ1-σ3 preparations were included in this analysis: complexes containing wt T1L μ1 and σ3 (lanes 1 and 4), complexes containing wt T1L μ1 and σ3 that were treated with 10 mM IAM to block free cysteines before storage (lanes 2 and 5), and complexes containing T1L C679S μ1 and wt T1L σ3 (lanes 3 and 6). The position of the native μ1-σ3 heterohexamer is indicated to the left. The positions of higher- M r forms specific to the complexes containing wt T1L μ1 and σ3 (lanes 1) are indicated (§). Positions of native gel markers (Amersham Pharmacia Biotech) are indicated in kilodaltons.

    Techniques Used: Mutagenesis, Generated, Purification, SDS Page, Acrylamide Gel Assay, Staining, Blocking Assay

    Effects of IAM addition on the migration of reovirus μ1 and μ1C proteins during SDS-PAGE. (A and B) Purified virions of reovirus T1L were used in these experiments. Viral proteins were resolved on a mini-sized SDS-PAGE [10% (A) or 8% (B) acrylamide] gel and visualized by Coomassie staining. The major new high- M r band observed after nonreducing disruption is indicated (*). (A) Virions were disrupted in sample buffer that contained no reducing agent (NR), and IAM was either not added (−) or added at different times after disruption (0 to 10 min) as indicated. (B) Virions were disrupted in pH 6.8 or 8.0 sample buffer that contained no reducing agent but to which 50 mM IAM was added either before (b) or immediately after (a) disruption as indicated. A sample of virions disrupted in reducing sample buffer (R; 5 mM DTT) was analyzed on the same gel (lane 1). Positions of molecular mass markers also resolved on the gel are indicated in kilodaltons. (C) Purified [ 35 S]methionine-cysteine-labeled virions of reovirus T3D were used in this experiment. The Coomassie-stained protein bands excised from the first gel (not shown; μ1C was obtained from samples of reduced virions and the high- M r band [*] was obtained from samples of IAM-treated nonreduced virions) were subjected to reduction in the gel fragments and otherwise treated as described in the text. The resulting proteins and protein fragments were resolved on a second gel and visualized by fluorography. The position of the full-length μ1C monomer is indicated. The amount of chymotrypsin added to each gel piece atop the second gel is also indicated. (D) Purified [ 35 S]methionine-cysteine- or [ 3 H]myristate-labeled virions of reovirus T3D were mixed with reducing or nonreducing sample buffer and disrupted in a boiling water bath. The viral proteins were then resolved on a full-sized 5 to 20% acrylamide gradient SDS-PAGE gel and visualized by fluorography. The major (*) and minor (**) new high- M r bands observed after nonreducing disruption of each sample are indicated.
    Figure Legend Snippet: Effects of IAM addition on the migration of reovirus μ1 and μ1C proteins during SDS-PAGE. (A and B) Purified virions of reovirus T1L were used in these experiments. Viral proteins were resolved on a mini-sized SDS-PAGE [10% (A) or 8% (B) acrylamide] gel and visualized by Coomassie staining. The major new high- M r band observed after nonreducing disruption is indicated (*). (A) Virions were disrupted in sample buffer that contained no reducing agent (NR), and IAM was either not added (−) or added at different times after disruption (0 to 10 min) as indicated. (B) Virions were disrupted in pH 6.8 or 8.0 sample buffer that contained no reducing agent but to which 50 mM IAM was added either before (b) or immediately after (a) disruption as indicated. A sample of virions disrupted in reducing sample buffer (R; 5 mM DTT) was analyzed on the same gel (lane 1). Positions of molecular mass markers also resolved on the gel are indicated in kilodaltons. (C) Purified [ 35 S]methionine-cysteine-labeled virions of reovirus T3D were used in this experiment. The Coomassie-stained protein bands excised from the first gel (not shown; μ1C was obtained from samples of reduced virions and the high- M r band [*] was obtained from samples of IAM-treated nonreduced virions) were subjected to reduction in the gel fragments and otherwise treated as described in the text. The resulting proteins and protein fragments were resolved on a second gel and visualized by fluorography. The position of the full-length μ1C monomer is indicated. The amount of chymotrypsin added to each gel piece atop the second gel is also indicated. (D) Purified [ 35 S]methionine-cysteine- or [ 3 H]myristate-labeled virions of reovirus T3D were mixed with reducing or nonreducing sample buffer and disrupted in a boiling water bath. The viral proteins were then resolved on a full-sized 5 to 20% acrylamide gradient SDS-PAGE gel and visualized by fluorography. The major (*) and minor (**) new high- M r bands observed after nonreducing disruption of each sample are indicated.

    Techniques Used: Migration, SDS Page, Purification, Acrylamide Gel Assay, Staining, Labeling

    Reversibility of the virion-associated μ1/μ1C ds bonds. For each experiment, aliquots were removed from the reaction mixture at the indicated intervals, and the reaction was quenched with 50 mM IAM. When all samples had been collected for each experiment, they were mixed with nonreducing sample buffer (NR) and disrupted by boiling. Viral proteins were resolved on mini-sized SDS-8% PAGE gels and visualized by Coomassie staining. (A) DTT at a concentration of 5 mM was added to purified T1L virions and allowed to incubate at room temperature to effect in situ reduction of the ds bonds. The 0-min aliquot (lane 1) was removed before the addition of DTT. Positions of molecular mass markers also resolved on the gel are indicated to the left. (B) The ds bonds in a sample of T1L virions were reduced by DTT as in panel A, lane 6, after which the DTT was removed by dialysis. Cystine at a concentration of 5 mM was then added to promote in situ reformation of the ds bonds. The 0-min aliquot (lane 1) was removed before the addition of cystine. A sample to which cysteine was never added was also analyzed (lane 9).
    Figure Legend Snippet: Reversibility of the virion-associated μ1/μ1C ds bonds. For each experiment, aliquots were removed from the reaction mixture at the indicated intervals, and the reaction was quenched with 50 mM IAM. When all samples had been collected for each experiment, they were mixed with nonreducing sample buffer (NR) and disrupted by boiling. Viral proteins were resolved on mini-sized SDS-8% PAGE gels and visualized by Coomassie staining. (A) DTT at a concentration of 5 mM was added to purified T1L virions and allowed to incubate at room temperature to effect in situ reduction of the ds bonds. The 0-min aliquot (lane 1) was removed before the addition of DTT. Positions of molecular mass markers also resolved on the gel are indicated to the left. (B) The ds bonds in a sample of T1L virions were reduced by DTT as in panel A, lane 6, after which the DTT was removed by dialysis. Cystine at a concentration of 5 mM was then added to promote in situ reformation of the ds bonds. The 0-min aliquot (lane 1) was removed before the addition of cystine. A sample to which cysteine was never added was also analyzed (lane 9).

    Techniques Used: Polyacrylamide Gel Electrophoresis, Staining, Concentration Assay, Purification, In Situ

    2) Product Images from "Intra- and Intermolecular Disulfide Bonds of the GP2b Glycoprotein of Equine Arteritis Virus: Relevance for Virus Assembly and Infectivity"

    Article Title: Intra- and Intermolecular Disulfide Bonds of the GP2b Glycoprotein of Equine Arteritis Virus: Relevance for Virus Assembly and Infectivity

    Journal: Journal of Virology

    doi: 10.1128/JVI.77.24.12996-13004.2003

    Conformational variants of the GP 2b monomers. vTF7.3-infected BHK-21 C13 cells were transfected with plasmids encoding the wild-type GP 2b protein or the GP 2b cysteine mutants. At 4 h p.i., the cells were labeled for 15 min with [ 35 S]methionine. Next, cell lysates were prepared in the presence of IAM and immunoprecipitations were performed with αGP 2b . The immunoprecipitates were analyzed under nonreducing conditions in an SDS-15% PAA gel. The arrowheads indicate the positions of the different GP 2b conformations. The values on the left are the molecular sizes, in kilodaltons, of marker proteins analyzed in the same gel.
    Figure Legend Snippet: Conformational variants of the GP 2b monomers. vTF7.3-infected BHK-21 C13 cells were transfected with plasmids encoding the wild-type GP 2b protein or the GP 2b cysteine mutants. At 4 h p.i., the cells were labeled for 15 min with [ 35 S]methionine. Next, cell lysates were prepared in the presence of IAM and immunoprecipitations were performed with αGP 2b . The immunoprecipitates were analyzed under nonreducing conditions in an SDS-15% PAA gel. The arrowheads indicate the positions of the different GP 2b conformations. The values on the left are the molecular sizes, in kilodaltons, of marker proteins analyzed in the same gel.

    Techniques Used: Infection, Transfection, Labeling, Marker

    3) Product Images from "Inhibition of Glutathione Biosynthesis Alters Compartmental Redox Status and the Thiol Proteome in Organogenesis-Stage Rat Conceptuses"

    Article Title: Inhibition of Glutathione Biosynthesis Alters Compartmental Redox Status and the Thiol Proteome in Organogenesis-Stage Rat Conceptuses

    Journal: Free radical biology & medicine

    doi: 10.1016/j.freeradbiomed.2013.05.040

    Gestational day 11 (GD 11) embryonic proteins covalently labeled at reduced cysteines with biotinylated iodoacetamide reagent (BIAM), separated on 2- dimensional SDS-PAGE gels and transferred to nitrocellulose membranes from embryos exposed to 1 mM BSO
    Figure Legend Snippet: Gestational day 11 (GD 11) embryonic proteins covalently labeled at reduced cysteines with biotinylated iodoacetamide reagent (BIAM), separated on 2- dimensional SDS-PAGE gels and transferred to nitrocellulose membranes from embryos exposed to 1 mM BSO

    Techniques Used: Labeling, SDS Page

    4) Product Images from "Cysteine Oxidation Regulates the RNA-Binding Activity of Iron Regulatory Protein 2 ▿Cysteine Oxidation Regulates the RNA-Binding Activity of Iron Regulatory Protein 2 ▿ †"

    Article Title: Cysteine Oxidation Regulates the RNA-Binding Activity of Iron Regulatory Protein 2 ▿Cysteine Oxidation Regulates the RNA-Binding Activity of Iron Regulatory Protein 2 ▿ †

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00004-09

    Sensitivity of IRP1 and IRP2 cysteine mutants to NEM and IAM. Flp-In TREx HEK293 cells stably expressing FLAG-tagged IRPs were treated with tetracycline overnight to induce protein expression. Cell lysates (10 μg) were treated with the indicated
    Figure Legend Snippet: Sensitivity of IRP1 and IRP2 cysteine mutants to NEM and IAM. Flp-In TREx HEK293 cells stably expressing FLAG-tagged IRPs were treated with tetracycline overnight to induce protein expression. Cell lysates (10 μg) were treated with the indicated

    Techniques Used: Stable Transfection, Expressing

    5) Product Images from "Deletion of the acid-sensing ion channel ASIC3 prevents gastritis-induced acid hyperresponsiveness of the stomach – brainstem axis"

    Article Title: Deletion of the acid-sensing ion channel ASIC3 prevents gastritis-induced acid hyperresponsiveness of the stomach – brainstem axis

    Journal:

    doi: 10.1016/j.pain.2007.04.025

    Effect of iodoacetamide pretreatment in wild-type mice
    Figure Legend Snippet: Effect of iodoacetamide pretreatment in wild-type mice

    Techniques Used: Mouse Assay

    6) Product Images from "Antibody format and drug release rate determine the therapeutic activity of non-internalizing antibody-drug conjugates"

    Article Title: Antibody format and drug release rate determine the therapeutic activity of non-internalizing antibody-drug conjugates

    Journal: Molecular cancer therapeutics

    doi: 10.1158/1535-7163.MCT-15-0480

    Characterisation of SIP(F8)-SS-DM1 and IgG(F8)-SS-DM1. (A) Schematic representation of SIP(F8)-SS-DM1, IgG(F8)-SS-DM1 and of the DM1-SH drug. (B) Biochemical characterization of the products and of reaction intermediates by SDS-PAGE, size-exclusion chromatography and ESI-MS. The calculated masses of SIP(F8)-SS-DM1 and IgG(F8)-SS-DM1 are 39464 and 24218 Dalton, respectively. M = molecular weight marker. Lanes 1 and 2 represent unmodified antibody in non-reducing and reducing conditions, lane 3 the iodoacetamide conjugate, lane 4 the Ellman intermediate, lane 5 the final DM1 conjugate. (% I = % of MS signal intensity)
    Figure Legend Snippet: Characterisation of SIP(F8)-SS-DM1 and IgG(F8)-SS-DM1. (A) Schematic representation of SIP(F8)-SS-DM1, IgG(F8)-SS-DM1 and of the DM1-SH drug. (B) Biochemical characterization of the products and of reaction intermediates by SDS-PAGE, size-exclusion chromatography and ESI-MS. The calculated masses of SIP(F8)-SS-DM1 and IgG(F8)-SS-DM1 are 39464 and 24218 Dalton, respectively. M = molecular weight marker. Lanes 1 and 2 represent unmodified antibody in non-reducing and reducing conditions, lane 3 the iodoacetamide conjugate, lane 4 the Ellman intermediate, lane 5 the final DM1 conjugate. (% I = % of MS signal intensity)

    Techniques Used: SDS Page, Size-exclusion Chromatography, Mass Spectrometry, Molecular Weight, Marker

    7) Product Images from "A complex of novel protease inhibitor, ovostatin homolog, with its cognate proteases in immature mice uterine luminal fluid"

    Article Title: A complex of novel protease inhibitor, ovostatin homolog, with its cognate proteases in immature mice uterine luminal fluid

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-41426-4

    Characterization of mOH-associated gelatinolytic enzyme activity. ( A ) The effects of protease inhibitors on the activity of mOH-associated gelatinolytic enzymes. An aliquot (0.3 ml) isolated from gel filtration was subjected to SDS-PAGE, followed by gelatin zymographic assay. After electrophoresis, the gel was sliced, and each sliced gel was incubated without protease inhibitor (lane 1) or with different protease inhibitors. The protease inhibitors were bezamidine (2 mM, lane 2); chymostatin, (100 μM, lane 3); leupeptin (100 μM, lane 4); pepstatin (20 μM, lane 5); iodoacetamide (0.1 mM, lane 6); 1-10-phenanthroline (1 mM, lane 7). The original scans of gelatin zymograms were shown in Supplementary Fig. S5 . ( B ) The effects of pH titration on the activity of mOH-associated gelatinolytic enzymes. An aliquot (0.3 ml) isolated from gel filtration was separated by SDS-PAGE, followed by gelatin zymographic assay in the presence of different pH buffers, respectively. The buffers include 0.1 M sodium citrate (pH 5.0 and 6.0), 0.1 M Tris buffers (from pH 7.4 and 8.0) and 0.1 M glycine (pH 9, 10 and 10.5). The original scans of gelatin zymograms were shown in Supplementary Fig. S6 . ( C ) Quantitation of relative gelatinolytic activities averaged from three independent experiments. The highest activity in pH titration represent 100%. The optimal pH for the activity of 26 kDa (black bar) and 22 kDa gelatinolytic enzymes (gray bar) is 9 and 8, respectively. Means ± SD, n = 3. ( D ) The substrate specificity of mOH-associated gelatinolytic enzymes. An aliquot (2 μg) of gelatinolytic enzymes complex from purification was carried out for gelatin zymography in the presence of different substrates, type I collagen (lane 1) and type IV collagen (lane 2), respectively. ( E ) The type I collagen degraded by mOH-associated 26 kDa gelatinolytic enzyme. The free 26 and 23/22 kDa enzymes were incubated with type I collagen, respectively, as described in “Materials and Methods”. After overnight incubation, an aliquot (8 μL) of supernatant from each reaction mixture was applied to analysis by SDS-PAGE. Lane 1: incubation with blank gel. Lane 2: incubation with mOH-free 26 kDa enzyme. Lane 3: incubation with mOH-free 23/22 kDa enzymes.
    Figure Legend Snippet: Characterization of mOH-associated gelatinolytic enzyme activity. ( A ) The effects of protease inhibitors on the activity of mOH-associated gelatinolytic enzymes. An aliquot (0.3 ml) isolated from gel filtration was subjected to SDS-PAGE, followed by gelatin zymographic assay. After electrophoresis, the gel was sliced, and each sliced gel was incubated without protease inhibitor (lane 1) or with different protease inhibitors. The protease inhibitors were bezamidine (2 mM, lane 2); chymostatin, (100 μM, lane 3); leupeptin (100 μM, lane 4); pepstatin (20 μM, lane 5); iodoacetamide (0.1 mM, lane 6); 1-10-phenanthroline (1 mM, lane 7). The original scans of gelatin zymograms were shown in Supplementary Fig. S5 . ( B ) The effects of pH titration on the activity of mOH-associated gelatinolytic enzymes. An aliquot (0.3 ml) isolated from gel filtration was separated by SDS-PAGE, followed by gelatin zymographic assay in the presence of different pH buffers, respectively. The buffers include 0.1 M sodium citrate (pH 5.0 and 6.0), 0.1 M Tris buffers (from pH 7.4 and 8.0) and 0.1 M glycine (pH 9, 10 and 10.5). The original scans of gelatin zymograms were shown in Supplementary Fig. S6 . ( C ) Quantitation of relative gelatinolytic activities averaged from three independent experiments. The highest activity in pH titration represent 100%. The optimal pH for the activity of 26 kDa (black bar) and 22 kDa gelatinolytic enzymes (gray bar) is 9 and 8, respectively. Means ± SD, n = 3. ( D ) The substrate specificity of mOH-associated gelatinolytic enzymes. An aliquot (2 μg) of gelatinolytic enzymes complex from purification was carried out for gelatin zymography in the presence of different substrates, type I collagen (lane 1) and type IV collagen (lane 2), respectively. ( E ) The type I collagen degraded by mOH-associated 26 kDa gelatinolytic enzyme. The free 26 and 23/22 kDa enzymes were incubated with type I collagen, respectively, as described in “Materials and Methods”. After overnight incubation, an aliquot (8 μL) of supernatant from each reaction mixture was applied to analysis by SDS-PAGE. Lane 1: incubation with blank gel. Lane 2: incubation with mOH-free 26 kDa enzyme. Lane 3: incubation with mOH-free 23/22 kDa enzymes.

    Techniques Used: Activity Assay, Isolation, Filtration, SDS Page, Electrophoresis, Incubation, Protease Inhibitor, Titration, Quantitation Assay, Purification, Zymography

    8) Product Images from "The staying power of adhesion-associated antioxidant activity in Mytilus californianus"

    Article Title: The staying power of adhesion-associated antioxidant activity in Mytilus californianus

    Journal: Journal of the Royal Society Interface

    doi: 10.1098/rsif.2015.0614

    Cysteine contribution to antioxidant activity in mussel plaque extracts. ( a , b ) Comparing antioxidant activity in untreated plaque extracts, iodoacetamide treated (cysteine blocked) plaque extracts, buffer and buffer with iodoacetamide. Final solution
    Figure Legend Snippet: Cysteine contribution to antioxidant activity in mussel plaque extracts. ( a , b ) Comparing antioxidant activity in untreated plaque extracts, iodoacetamide treated (cysteine blocked) plaque extracts, buffer and buffer with iodoacetamide. Final solution

    Techniques Used: Antioxidant Activity Assay

    9) Product Images from "The C-Terminus of H-Ras as a Target for the Covalent Binding of Reactive Compounds Modulating Ras-Dependent Pathways"

    Article Title: The C-Terminus of H-Ras as a Target for the Covalent Binding of Reactive Compounds Modulating Ras-Dependent Pathways

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0015866

    Interaction of the bifunctional reagent DBB with H-Ras. (A) Modification of the K170-K185 peptide by DBB. The K170-K185 peptide was incubated with DBB, as described in the Methods section. When indicated the incubation mixture was subjected to reduction and alkylation with iodoacetamide to modify free cysteines. Results are representative of three independent assays. (B) Scheme of the cross-linking of two thiol groups by DBB. (C) Competition of 15d-PGJ 2 -B binding to H-Ras by DBB. H-Ras at 5 µM was incubated with 50 µM DBB for 30 min before addition of 1 µM 15d-PGJ 2 -B for 1 h. Incorporation of the biotinylated PG was assessed by SDS-PAGE, protein blot and detection with horseradish peroxidase (HRP)-streptavidin (upper panel). Incorporation of DBB (middle panel) and total H-Ras levels (lower panel) were assessed by fluorescence detection and western blot with anti-pan Ras antibody, respectively. (D) COS-7 cells transfected with the AU5-H-Ras vector were treated in the absence or presence of 50 µM DBB for 1 h. Aliquots from total cell lysates (10 µg of protein) were analyzed by SDS-PAGE. The upper component of the AU5-H-Ras doublet is indicated by an arrowhead. (E) COS-7 cells transfected with the AU5-H-Ras vector were treated with DBB and subjected to fractionation in Triton-X114 as above. In (D) and (E) levels of AU5-H-Ras were assessed by western blot with anti-AU5 antibody. The dotted line marks where lanes from the same gel have been cropped. (F) COS-7 cells transfected with YFP-RBD as above were treated with 50 µM DBB for 1 h before stimulation with 100 nM EGF, as indicated, and visualized live by confocal fluorescence microscopy. Dotted inserts show enlarged areas from the same cells for better visualization. Arrowheads mark the accumulation of YFP-RBD at defined locations of the cell periphery. Bar, 20 µm. Shown are representative images from three experiments with similar results.
    Figure Legend Snippet: Interaction of the bifunctional reagent DBB with H-Ras. (A) Modification of the K170-K185 peptide by DBB. The K170-K185 peptide was incubated with DBB, as described in the Methods section. When indicated the incubation mixture was subjected to reduction and alkylation with iodoacetamide to modify free cysteines. Results are representative of three independent assays. (B) Scheme of the cross-linking of two thiol groups by DBB. (C) Competition of 15d-PGJ 2 -B binding to H-Ras by DBB. H-Ras at 5 µM was incubated with 50 µM DBB for 30 min before addition of 1 µM 15d-PGJ 2 -B for 1 h. Incorporation of the biotinylated PG was assessed by SDS-PAGE, protein blot and detection with horseradish peroxidase (HRP)-streptavidin (upper panel). Incorporation of DBB (middle panel) and total H-Ras levels (lower panel) were assessed by fluorescence detection and western blot with anti-pan Ras antibody, respectively. (D) COS-7 cells transfected with the AU5-H-Ras vector were treated in the absence or presence of 50 µM DBB for 1 h. Aliquots from total cell lysates (10 µg of protein) were analyzed by SDS-PAGE. The upper component of the AU5-H-Ras doublet is indicated by an arrowhead. (E) COS-7 cells transfected with the AU5-H-Ras vector were treated with DBB and subjected to fractionation in Triton-X114 as above. In (D) and (E) levels of AU5-H-Ras were assessed by western blot with anti-AU5 antibody. The dotted line marks where lanes from the same gel have been cropped. (F) COS-7 cells transfected with YFP-RBD as above were treated with 50 µM DBB for 1 h before stimulation with 100 nM EGF, as indicated, and visualized live by confocal fluorescence microscopy. Dotted inserts show enlarged areas from the same cells for better visualization. Arrowheads mark the accumulation of YFP-RBD at defined locations of the cell periphery. Bar, 20 µm. Shown are representative images from three experiments with similar results.

    Techniques Used: Modification, Incubation, Binding Assay, SDS Page, Fluorescence, Western Blot, Transfection, Plasmid Preparation, Fractionation, Microscopy

    Effect of reducing agents on PAO modification of the K170-K185 peptide. (A) In the upper panels, the K170-K185 peptide was incubated with PAO and analyzed by MALDI-TOF MS. In the right panel, the incubation mixture was treated with 10 mM DTT and subsequently with 50 mM iodoacetamide. In the lower panels, the K170-K185 peptide was incubated with PAO in the presence of the indicated concentrations of GSH before analysis by MALDI-TOF MS. (B) Summary of the theoretical m/z of peptides for which compatible peaks were observed experimentally in the assays shown in (A).
    Figure Legend Snippet: Effect of reducing agents on PAO modification of the K170-K185 peptide. (A) In the upper panels, the K170-K185 peptide was incubated with PAO and analyzed by MALDI-TOF MS. In the right panel, the incubation mixture was treated with 10 mM DTT and subsequently with 50 mM iodoacetamide. In the lower panels, the K170-K185 peptide was incubated with PAO in the presence of the indicated concentrations of GSH before analysis by MALDI-TOF MS. (B) Summary of the theoretical m/z of peptides for which compatible peaks were observed experimentally in the assays shown in (A).

    Techniques Used: Modification, Incubation, Mass Spectrometry

    Modification of the K170-K185 peptide from the C-terminal region of H-Ras by cyPG. (A) The synthetic K170-K185 peptide was incubated with the indicated cyPG and the resulting adducts analyzed by MALDI-TOF MS. When indicated, incubation mixtures were treated with 50 mM iodoacetamide after addition of 10 mM DTT. Spectra presented are representative from at least three independent assays per experimental condition. Structures of the cyPG used are shown in insets. Electrophilic carbons are marked by asterisks. (B) Summary of the theoretical peptide adducts for which compatible peaks were observed experimentally. +CAM indicates that the peak is compatible with the formation of carbamidomethyl cysteine subsequent to iodoacetamide treatment. (C) Ribbon diagram for the theoretical backbone structure of the H-Ras K170-K185 peptide (in green) modified by addition of 15d-PGJ 2 to cysteines 181 and 184. The side-chains of the cysteine residues and the 15d-PGJ 2 ligand are displayed in yellow and a default atom-type color scheme, respectively.
    Figure Legend Snippet: Modification of the K170-K185 peptide from the C-terminal region of H-Ras by cyPG. (A) The synthetic K170-K185 peptide was incubated with the indicated cyPG and the resulting adducts analyzed by MALDI-TOF MS. When indicated, incubation mixtures were treated with 50 mM iodoacetamide after addition of 10 mM DTT. Spectra presented are representative from at least three independent assays per experimental condition. Structures of the cyPG used are shown in insets. Electrophilic carbons are marked by asterisks. (B) Summary of the theoretical peptide adducts for which compatible peaks were observed experimentally. +CAM indicates that the peak is compatible with the formation of carbamidomethyl cysteine subsequent to iodoacetamide treatment. (C) Ribbon diagram for the theoretical backbone structure of the H-Ras K170-K185 peptide (in green) modified by addition of 15d-PGJ 2 to cysteines 181 and 184. The side-chains of the cysteine residues and the 15d-PGJ 2 ligand are displayed in yellow and a default atom-type color scheme, respectively.

    Techniques Used: Modification, Incubation, Mass Spectrometry, Chick Chorioallantoic Membrane Assay

    10) Product Images from "Measurement of T-Cell-Derived Antigen Binding Molecules and Immunoglobulin G Specific to Candida albicans Mannan in Sera of Patients with Recurrent Vulvovaginal Candidiasis"

    Article Title: Measurement of T-Cell-Derived Antigen Binding Molecules and Immunoglobulin G Specific to Candida albicans Mannan in Sera of Patients with Recurrent Vulvovaginal Candidiasis

    Journal: Infection and Immunity

    doi:

    CTAB-TABM (1 μg) was reduced with 50 mM dithiothreitol and alkylated with 50 mM iodoacetamide. Reduced, alkylated CTAB-TABM (200 ng) was resolved on an 8 to 25% polyacrylamide gradient gel. Resolved proteins were stained by silver stain. Molecular weights were determined by comparison with the mobilities of prestained molecular weight standard proteins ( M r 200,000 to 18,000).
    Figure Legend Snippet: CTAB-TABM (1 μg) was reduced with 50 mM dithiothreitol and alkylated with 50 mM iodoacetamide. Reduced, alkylated CTAB-TABM (200 ng) was resolved on an 8 to 25% polyacrylamide gradient gel. Resolved proteins were stained by silver stain. Molecular weights were determined by comparison with the mobilities of prestained molecular weight standard proteins ( M r 200,000 to 18,000).

    Techniques Used: Staining, Silver Staining, Molecular Weight

    11) Product Images from "Redox Equivalents and Mitochondrial Bioenergetics"

    Article Title: Redox Equivalents and Mitochondrial Bioenergetics

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

    doi: 10.1007/978-1-4939-7831-1_12

    Semiquantitative analysis of fractional reduction of thioredoxin reductase-2 (TrxR2) by BIAM-blot. Cells were incubated 24 h with +Glc,+Gln, −Glc, −Gln, or −Glc, −Gln and antimycin A (30 min with 5 μM; +AA). Aliquots of cell lysates were treated with the biotinylated iodoacetamide reagent, BIAM, and parallel aliquots were reduced with TCEP and then reacted with BIAM. Following immunoprecipitation with anti-TrxR2, samples were separated by SDS-PAGE, blotted and probed with fluorescently labeled streptavidin. Controls for recovery following immunoprecipitation were performed by Western blotting with anti-TrxR2 and showed similar recovery. Although these methods are reproducible, the limiting conditions of BIAM labeling selected to maximize detection of reactive thiols in the presence of less reactive thiols do not allow strict quantification
    Figure Legend Snippet: Semiquantitative analysis of fractional reduction of thioredoxin reductase-2 (TrxR2) by BIAM-blot. Cells were incubated 24 h with +Glc,+Gln, −Glc, −Gln, or −Glc, −Gln and antimycin A (30 min with 5 μM; +AA). Aliquots of cell lysates were treated with the biotinylated iodoacetamide reagent, BIAM, and parallel aliquots were reduced with TCEP and then reacted with BIAM. Following immunoprecipitation with anti-TrxR2, samples were separated by SDS-PAGE, blotted and probed with fluorescently labeled streptavidin. Controls for recovery following immunoprecipitation were performed by Western blotting with anti-TrxR2 and showed similar recovery. Although these methods are reproducible, the limiting conditions of BIAM labeling selected to maximize detection of reactive thiols in the presence of less reactive thiols do not allow strict quantification

    Techniques Used: Incubation, Immunoprecipitation, SDS Page, Labeling, Western Blot

    12) Product Images from "Modification by covalent reaction or oxidation of cysteine residues in the Tandem-SH2 Domains of ZAP-70 and Syk Can Block Phosphopeptide Binding"

    Article Title: Modification by covalent reaction or oxidation of cysteine residues in the Tandem-SH2 Domains of ZAP-70 and Syk Can Block Phosphopeptide Binding

    Journal: The Biochemical journal

    doi: 10.1042/BJ20140793

    (A-B) Neither n-ethylmaleimide (NEM) nor iodoacetamide (IAM) inhibit ZAP-tHS2:2pY binding.
    Figure Legend Snippet: (A-B) Neither n-ethylmaleimide (NEM) nor iodoacetamide (IAM) inhibit ZAP-tHS2:2pY binding.

    Techniques Used: Binding Assay

    13) Product Images from "Evaluation and optimization of reduction and alkylation methods to maximize peptide identification with MS-based proteomics"

    Article Title: Evaluation and optimization of reduction and alkylation methods to maximize peptide identification with MS-based proteomics

    Journal: Molecular bioSystems

    doi: 10.1039/c7mb00393e

    Experimental Procedure. (A) Comparison of the reduction with DTT, 2-ME, TCEP, or THPP. (B) Comparison of the alkylation with iodoacetamide, acrylamide, N-EM, or 4-VP. The reduction and alkylation were performed at the peptide level.
    Figure Legend Snippet: Experimental Procedure. (A) Comparison of the reduction with DTT, 2-ME, TCEP, or THPP. (B) Comparison of the alkylation with iodoacetamide, acrylamide, N-EM, or 4-VP. The reduction and alkylation were performed at the peptide level.

    Techniques Used:

    Possible alkylation reactions with iodoacetamide. Ideal alkylation is on the sulfhydryl group of cysteine (A). Side reactions may occur, and the alkylation reactions at the peptide N-terminus and the side chains of lysine and aspartic acid are shown here as examples (B).
    Figure Legend Snippet: Possible alkylation reactions with iodoacetamide. Ideal alkylation is on the sulfhydryl group of cysteine (A). Side reactions may occur, and the alkylation reactions at the peptide N-terminus and the side chains of lysine and aspartic acid are shown here as examples (B).

    Techniques Used:

    Optimization of alkylation conditions. (A) Effects of the iodoacetamide concentration on the identification of proteins and peptides. (B) The number of identified peptides with alkylated cysteine, free cysteine (due to incomplete reaction), or the side reactions on the peptide N-terminus and the side chains of lysine and aspartic acid as a function of the iodoacetamide concentration. (C) Effects of the alkylation temperature on the identification of proteins and peptides. (D) The number of identified peptides with alkylated cysteine, free cysteine, or the side reactions on the peptide N-terminus and the side chains of lysine and aspartic acid at different alkylation temperatures.
    Figure Legend Snippet: Optimization of alkylation conditions. (A) Effects of the iodoacetamide concentration on the identification of proteins and peptides. (B) The number of identified peptides with alkylated cysteine, free cysteine (due to incomplete reaction), or the side reactions on the peptide N-terminus and the side chains of lysine and aspartic acid as a function of the iodoacetamide concentration. (C) Effects of the alkylation temperature on the identification of proteins and peptides. (D) The number of identified peptides with alkylated cysteine, free cysteine, or the side reactions on the peptide N-terminus and the side chains of lysine and aspartic acid at different alkylation temperatures.

    Techniques Used: Concentration Assay

    Enrichment of peptides containing cysteine using Thiopropyl Sepharose 6B resin. Peptides from the yeast whole-cell lysate are incubated with the resin. Peptides containing cysteine are bound to the resin through disulfide bonds while unbound peptides are removed. Enriched peptides are cleaved with DTT, and then alkylated with iodoacetamide prior to LC-MS/MS analysis.
    Figure Legend Snippet: Enrichment of peptides containing cysteine using Thiopropyl Sepharose 6B resin. Peptides from the yeast whole-cell lysate are incubated with the resin. Peptides containing cysteine are bound to the resin through disulfide bonds while unbound peptides are removed. Enriched peptides are cleaved with DTT, and then alkylated with iodoacetamide prior to LC-MS/MS analysis.

    Techniques Used: Incubation, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    14) Product Images from "Improving Limits of Detection for B-type Natriuretic Peptide Using PC-IDMS: An Application of the ALiPHAT Strategy"

    Article Title: Improving Limits of Detection for B-type Natriuretic Peptide Using PC-IDMS: An Application of the ALiPHAT Strategy

    Journal:

    doi: 10.1039/b919484c

    (A) Plotted are the average peak areas (n=3) for the natural version of the modified peptides loaded on column in different amounts. (B) The average abundances of each modified peptide are normalized to that of the IAM-modified peptide (A X /A IAM ) to determine
    Figure Legend Snippet: (A) Plotted are the average peak areas (n=3) for the natural version of the modified peptides loaded on column in different amounts. (B) The average abundances of each modified peptide are normalized to that of the IAM-modified peptide (A X /A IAM ) to determine

    Techniques Used: Modification

    The LOD for each modified peptide is displayed in the bar graph as attomoles of modified peptide loaded on column. To determine the fold improvements, the LOD for each species was normalized to the IAM-modified peptide (LOD IAM /LOD X ). In terms of the total
    Figure Legend Snippet: The LOD for each modified peptide is displayed in the bar graph as attomoles of modified peptide loaded on column. To determine the fold improvements, the LOD for each species was normalized to the IAM-modified peptide (LOD IAM /LOD X ). In terms of the total

    Techniques Used: Modification

    15) Product Images from "Subcritical Water Hydrolysis of Peptides: Amino Acid Side-Chain Modifications"

    Article Title: Subcritical Water Hydrolysis of Peptides: Amino Acid Side-Chain Modifications

    Journal: Journal of the American Society for Mass Spectrometry

    doi: 10.1007/s13361-017-1676-1

    Direct infusion electrospray mass spectrum of peptide VQSIKCADFLHYMENPTWGR following iodoacetamide treatment treated with SCW at 140 °C for 10 min
    Figure Legend Snippet: Direct infusion electrospray mass spectrum of peptide VQSIKCADFLHYMENPTWGR following iodoacetamide treatment treated with SCW at 140 °C for 10 min

    Techniques Used:

    16) Product Images from "The tumor-infiltrating B cell response in medullary breast cancer is oligoclonal and directed against the autoantigen actin exposed on the surface of apoptotic cancer cells"

    Article Title: The tumor-infiltrating B cell response in medullary breast cancer is oligoclonal and directed against the autoantigen actin exposed on the surface of apoptotic cancer cells

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

    doi: 10.1073/pnas.171460798

    MCB contain actin fragments similar to those obtained following cleavage with granzyme B. Western blots of tumor lysate obtained from fresh-frozen MCB tumors (lanes 1 and 2) or purified nonmuscle actin either uncut (lane 3), cleaved with 50 mM granzyme B in the presence (lane 4) or absence (lane 5) of iodoacetamide, or caspase-3 (lane 6) were stained for actin. Caspase-3 in the absence of actin (lane 7) yielded some higher molecular weigh background staining, whereas no staining was observed for granzyme B in the absence of actin (lane 8).
    Figure Legend Snippet: MCB contain actin fragments similar to those obtained following cleavage with granzyme B. Western blots of tumor lysate obtained from fresh-frozen MCB tumors (lanes 1 and 2) or purified nonmuscle actin either uncut (lane 3), cleaved with 50 mM granzyme B in the presence (lane 4) or absence (lane 5) of iodoacetamide, or caspase-3 (lane 6) were stained for actin. Caspase-3 in the absence of actin (lane 7) yielded some higher molecular weigh background staining, whereas no staining was observed for granzyme B in the absence of actin (lane 8).

    Techniques Used: Western Blot, Purification, Staining

    17) Product Images from "Effect of Niacin on Inflammation and Angiogenesis in a Murine Model of Ulcerative Colitis"

    Article Title: Effect of Niacin on Inflammation and Angiogenesis in a Murine Model of Ulcerative Colitis

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-07280-y

    Effect of niacin on proangiogenic and antiangiogenic factors in colonic tissues of rats with iodoacetamide-induced colitis. (a) Vascular endothelial growth factor (VEGF), (b) angiostatin, (c) endostatin. Data are expressed as means ± SEM of 8 animals. # P ≤ 0.05 vs. normal control, * P ≤ 0.05 vs. colitis control.
    Figure Legend Snippet: Effect of niacin on proangiogenic and antiangiogenic factors in colonic tissues of rats with iodoacetamide-induced colitis. (a) Vascular endothelial growth factor (VEGF), (b) angiostatin, (c) endostatin. Data are expressed as means ± SEM of 8 animals. # P ≤ 0.05 vs. normal control, * P ≤ 0.05 vs. colitis control.

    Techniques Used:

    Effect of niacin on colon wet weight in rats with iodoacetamide-induced colitis. Data are expressed as means ± SEM of 8 animals. # P ≤ 0.05 vs. normal control, * P ≤ 0.05 vs. colitis control.
    Figure Legend Snippet: Effect of niacin on colon wet weight in rats with iodoacetamide-induced colitis. Data are expressed as means ± SEM of 8 animals. # P ≤ 0.05 vs. normal control, * P ≤ 0.05 vs. colitis control.

    Techniques Used:

    Effect of niacin on inflammatory and anti-inflammatory parameters in colonic tissues of rats with iodoacetamide-induced colitis. ( a ) Myeloperoxidase (MPO) activity, ( b ) tumour necrosis factor (TNF)-α levels, ( c ) IL-10 levels. Data are expressed as means ± SEM of 8 animals. # P ≤ 0.05 vs. normal control, * P ≤ 0.05 vs. colitis control.
    Figure Legend Snippet: Effect of niacin on inflammatory and anti-inflammatory parameters in colonic tissues of rats with iodoacetamide-induced colitis. ( a ) Myeloperoxidase (MPO) activity, ( b ) tumour necrosis factor (TNF)-α levels, ( c ) IL-10 levels. Data are expressed as means ± SEM of 8 animals. # P ≤ 0.05 vs. normal control, * P ≤ 0.05 vs. colitis control.

    Techniques Used: Activity Assay

    Effect of niacin on histopathological changes of rat colon in iodoacetamide model of colitis. ( a ) Normal control rat: normal histological structure of mucosa, ( b ) Colitis control rat showing necrosis of epithelium, inflammatory infiltrate in mucosa and submucosa as well as submucosal oedema, ( c ) Niacin (80 mg/kg) pretreated rat showing moderate inflammatory infiltrate in lamina propria and submucosa, (d) Rat pretreated with niacin (320 mg/kg) showing minimal changes. (H E staining, ×100 original magnification). ( e ) Total histology score, data are expressed as box plots of the median of at least six animals. # P ≤ 0.05 vs. normal control.
    Figure Legend Snippet: Effect of niacin on histopathological changes of rat colon in iodoacetamide model of colitis. ( a ) Normal control rat: normal histological structure of mucosa, ( b ) Colitis control rat showing necrosis of epithelium, inflammatory infiltrate in mucosa and submucosa as well as submucosal oedema, ( c ) Niacin (80 mg/kg) pretreated rat showing moderate inflammatory infiltrate in lamina propria and submucosa, (d) Rat pretreated with niacin (320 mg/kg) showing minimal changes. (H E staining, ×100 original magnification). ( e ) Total histology score, data are expressed as box plots of the median of at least six animals. # P ≤ 0.05 vs. normal control.

    Techniques Used: Staining

    Effect of pretreatment with niacin on the increase in body weight of animals with iodoacetamide-induced colitis measured from the time of induction of colitis until sacrifice. Data are expressed as means ± SEM of 8 animals. # P ≤ 0.05 vs. normal control.
    Figure Legend Snippet: Effect of pretreatment with niacin on the increase in body weight of animals with iodoacetamide-induced colitis measured from the time of induction of colitis until sacrifice. Data are expressed as means ± SEM of 8 animals. # P ≤ 0.05 vs. normal control.

    Techniques Used:

    Role of GPR109A in the protective effect of niacin against iodoacetamide-induced colitis in rats. ( a ) increase in body weight of animals measured from the time of induction of colitis until sacrifice, (b ) colon wet weight, ( c ) Myeloperoxidase (MPO) activity, ( d ) vascular endothelial growth factor (VEGF). Data are expressed as means ± SEM of 5 animals. # P ≤ 0.05 vs. normal control, * P ≤ 0.05 vs. colitis control.
    Figure Legend Snippet: Role of GPR109A in the protective effect of niacin against iodoacetamide-induced colitis in rats. ( a ) increase in body weight of animals measured from the time of induction of colitis until sacrifice, (b ) colon wet weight, ( c ) Myeloperoxidase (MPO) activity, ( d ) vascular endothelial growth factor (VEGF). Data are expressed as means ± SEM of 5 animals. # P ≤ 0.05 vs. normal control, * P ≤ 0.05 vs. colitis control.

    Techniques Used: Activity Assay

    18) Product Images from "An Angiotensin I-Converting Enzyme Mutation (Y465D) Causes a Dramatic Increase in Blood ACE via Accelerated ACE Shedding"

    Article Title: An Angiotensin I-Converting Enzyme Mutation (Y465D) Causes a Dramatic Increase in Blood ACE via Accelerated ACE Shedding

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0025952

    MALDI TOF/TOF spectrum of mutant (Y465D) ACE after tryptic digest. An in-gel tryptic digest was performed on mutant (Y465D) ACE, the cysteines were protected using iodoacetamide, and the total digest subjected to MALDI TOF/TOF using the matrix α-cyano-4-hydroxycinmanic acid. Masses corresponding to predicted ACE peptides are labeled. The masses corresponding to peptide containing Y465D and the C-terminal cleavage peptide are indicated in bold.
    Figure Legend Snippet: MALDI TOF/TOF spectrum of mutant (Y465D) ACE after tryptic digest. An in-gel tryptic digest was performed on mutant (Y465D) ACE, the cysteines were protected using iodoacetamide, and the total digest subjected to MALDI TOF/TOF using the matrix α-cyano-4-hydroxycinmanic acid. Masses corresponding to predicted ACE peptides are labeled. The masses corresponding to peptide containing Y465D and the C-terminal cleavage peptide are indicated in bold.

    Techniques Used: Mutagenesis, Labeling

    19) Product Images from "Extraordinarily stable disulfide-linked homodimer of human growth hormone"

    Article Title: Extraordinarily stable disulfide-linked homodimer of human growth hormone

    Journal: Protein Science : A Publication of the Protein Society

    doi: 10.1110/ps.041048805

    Formation of MER-45-kDa hGH by reassociation of 22-kDa hGH subunits and prevention of their reassociation by modification with iodoacetamide. An analytical 13.5% SDS polyacrylamide gel in the absence of reductants was used to separate samples and to assess the degree of 22-kDa hGH subunit association. ( Section I ) MER-45-kDa hGH was dissociated into 22-kDa hGH subunits by incubation in 10% mercaptoethanol at 100°C for 5 h (lane 1 ). The dissociated 22-kDa hGH samples were then dialyzed against 5 mM ammonium bicarbonate buffer containing 20 mM 2-mercaptoethanol (lane 2 ) or with buffer in absence of reductant (lane 3 ). ( Section II ) MER-45-kDa hGH was dissociated into 22-kDa hGH subunits by incubation in 10% mercaptoethanol at 100°C for 5 h, and 22-kDa hGH subunits were treated with 7 M iodoacetamide for 15 min at room temperature (lane 1 ). The dissociated and modified 22-kDa hGH samples were then dialyzed against 10 mM Tris-HCl buffer (lane 2 ).
    Figure Legend Snippet: Formation of MER-45-kDa hGH by reassociation of 22-kDa hGH subunits and prevention of their reassociation by modification with iodoacetamide. An analytical 13.5% SDS polyacrylamide gel in the absence of reductants was used to separate samples and to assess the degree of 22-kDa hGH subunit association. ( Section I ) MER-45-kDa hGH was dissociated into 22-kDa hGH subunits by incubation in 10% mercaptoethanol at 100°C for 5 h (lane 1 ). The dissociated 22-kDa hGH samples were then dialyzed against 5 mM ammonium bicarbonate buffer containing 20 mM 2-mercaptoethanol (lane 2 ) or with buffer in absence of reductant (lane 3 ). ( Section II ) MER-45-kDa hGH was dissociated into 22-kDa hGH subunits by incubation in 10% mercaptoethanol at 100°C for 5 h, and 22-kDa hGH subunits were treated with 7 M iodoacetamide for 15 min at room temperature (lane 1 ). The dissociated and modified 22-kDa hGH samples were then dialyzed against 10 mM Tris-HCl buffer (lane 2 ).

    Techniques Used: Modification, Incubation

    20) Product Images from "Covalent Targeting of the Vacuolar H+-ATPase Activates Autophagy Via mTORC1 Inhibition"

    Article Title: Covalent Targeting of the Vacuolar H+-ATPase Activates Autophagy Via mTORC1 Inhibition

    Journal: Nature chemical biology

    doi: 10.1038/s41589-019-0308-4

    Target identification and validation of EN6. (a) isoTOP-ABPP analysis of EN6 in MEF cells in situ . MEF cells were pre-treated with DMSO or EN6 (50 μM, 4 h in situ ) prior to labeling of proteomes in vitro with IA-alkyne (100 μM, 1 h), followed by appendage of isotopically light (for DMSO-treated) or heavy (for EN6-treated) TEV protease cleavable biotin-azide tags by copper-catalyzed azide-alkyne cycloaddition (CuAAC), followed by the isoTOP-ABPP procedure. Shown are average light/heavy ratios for n=3 biologically independent samples/group. More detailed data and individual replicate ratios can be found in Supplementary Dataset 2 . (b) Gel-based ABPP analysis of EN6 interactions with recombinant ATP6V1A. Vehicle DMSO or EN6 were pre-incubated with recombinant human ATP6V1A (1 h) followed by labeling with a rhodamine-functionalized iodoacetamide probe (IA-rhodamine) (1 μM, 1 h) after which probe-labeled proteins were read-out by SDS/PAGE and in-gel fluorescence. Data were quantified by densitometry using Image J. Original gel images can be found in Supplementary Fig. 8a . (c) mTORC1 signaling with EN6 treatment in HEK293A cells. HEK293A cells, starved or stimulated with amino acids, were treated with vehicle DMSO or EN6 (25 μM) for 1 h and mTORC1 signaling was assessed by Western blotting. Original gel images are in Supplementary Fig. 7b . (d) Autophagy markers (LC3 and p62) and mTORC1 signaling in Hela cells treated with EN6. Endogenous ATP6V1A was knocked down with shRNA and replaced with a Flag-tagged wild-type or C277A mutant ATP6V1A and these cells were treated with EN6 (25 μM) for 4 h. Original gel images can be found in Supplementary Fig. 8b . Validation of ATP6V1A knockdown can be found in Supplementary Fig. 2e . Data shown in (b) are average ± sem from n=3 biologically independent samples/group. Gels shown in (b, c, d) are representative gels from n=3 biologically independent samples/group.
    Figure Legend Snippet: Target identification and validation of EN6. (a) isoTOP-ABPP analysis of EN6 in MEF cells in situ . MEF cells were pre-treated with DMSO or EN6 (50 μM, 4 h in situ ) prior to labeling of proteomes in vitro with IA-alkyne (100 μM, 1 h), followed by appendage of isotopically light (for DMSO-treated) or heavy (for EN6-treated) TEV protease cleavable biotin-azide tags by copper-catalyzed azide-alkyne cycloaddition (CuAAC), followed by the isoTOP-ABPP procedure. Shown are average light/heavy ratios for n=3 biologically independent samples/group. More detailed data and individual replicate ratios can be found in Supplementary Dataset 2 . (b) Gel-based ABPP analysis of EN6 interactions with recombinant ATP6V1A. Vehicle DMSO or EN6 were pre-incubated with recombinant human ATP6V1A (1 h) followed by labeling with a rhodamine-functionalized iodoacetamide probe (IA-rhodamine) (1 μM, 1 h) after which probe-labeled proteins were read-out by SDS/PAGE and in-gel fluorescence. Data were quantified by densitometry using Image J. Original gel images can be found in Supplementary Fig. 8a . (c) mTORC1 signaling with EN6 treatment in HEK293A cells. HEK293A cells, starved or stimulated with amino acids, were treated with vehicle DMSO or EN6 (25 μM) for 1 h and mTORC1 signaling was assessed by Western blotting. Original gel images are in Supplementary Fig. 7b . (d) Autophagy markers (LC3 and p62) and mTORC1 signaling in Hela cells treated with EN6. Endogenous ATP6V1A was knocked down with shRNA and replaced with a Flag-tagged wild-type or C277A mutant ATP6V1A and these cells were treated with EN6 (25 μM) for 4 h. Original gel images can be found in Supplementary Fig. 8b . Validation of ATP6V1A knockdown can be found in Supplementary Fig. 2e . Data shown in (b) are average ± sem from n=3 biologically independent samples/group. Gels shown in (b, c, d) are representative gels from n=3 biologically independent samples/group.

    Techniques Used: In Situ, Labeling, In Vitro, Recombinant, Incubation, SDS Page, Fluorescence, Western Blot, shRNA, Mutagenesis

    21) Product Images from "Inter-domain dynamics drive cholesterol transport by NPC1 and NPC1L1 proteins"

    Article Title: Inter-domain dynamics drive cholesterol transport by NPC1 and NPC1L1 proteins

    Journal: bioRxiv

    doi: 10.1101/2020.03.26.009217

    Engineered cysteines C251 and C929 form a disulfide bond in NPC1. (A) Extracted ion chromatograms from LC-MS analysis of synthetic peptides corresponding to engineered cysteines in NPC1. In both samples, blue traces represent m/z = 526.2569 (corresponding to carbamidomethylated CQPPPPPMK from NPC1 P251C), red traces represent m/z = 943.3462 (corresponding to carbamidomethylated NAAECDTY from NPC1 L929C), and green traces represent m/z = 626.6005 (corresponding to the disulfide formed between CQPPPPPMK and NAAECDTY). The peptide standards CQPPPPPMK and NAAECDTY were produced by solid phase synthesis and either reduced and carbamidomethylated or oxidized to the disulfide using Ellman’s reagent. (B) Extracted ion chromatograms from LC-MS analysis of proteolyzed NPC1. Colors as in A. NPC1 protein was carbamidomethylated in the presence or absence of reducing agent prior to proteolysis. (C) EThcD mass spectrum from NPC1 sample in part B, demonstrating the reductive fragmentation of the putative disulfide precursor into ions with masses corresponding to the two constituent peptides. Peaks matching the mass of the peptide CQPPPPPMK (monoisotopic mass of thiol = 994.49 Da) are colored in blue; peaks matching the masses of the peptides NAAECDTY (monoisotopic mass of thiol radical = 885.32 Da) are colored in red; peaks matching multiple charge states of the spectrum’s parent ions (m/z = 626.60, z = 3, corresponding to the disulfide) are colored in green. (D) MS1 mass spectra of NPC1 peptides whose disulfide content has been quantified using isotope-labeled iodoacetamide. Free thiols in purified NPC1 were labeled with 13 C 2 D 2 -iodoacetamide (“heavy”). Then the disulfides were reduced and the resulting reactive cysteines were labeled with iodoacetamide lacking isotope labels (“light”) followed by proteolysis and LC-MS analysis. Control samples were labeled with the same reagent before and after reduction (“light/reduce/light” and “heavy/reduce/heavy”) to identify isotope distributions in the limiting cases. Colored peaks fall within 10 ppm of expected masses in the isotope envelope of carbamidomethylated CQPPPPPMK (monoisotopic m/z = 526.2569) or carbamidomethylated NAAECDTY (monoisotopic m/z = 943.3462); black peaks correspond to unrelated ions. Dashed gray lines indicate the expected m/z of the monoisotopic peak and the +4 Da peak of the labeled peptides.
    Figure Legend Snippet: Engineered cysteines C251 and C929 form a disulfide bond in NPC1. (A) Extracted ion chromatograms from LC-MS analysis of synthetic peptides corresponding to engineered cysteines in NPC1. In both samples, blue traces represent m/z = 526.2569 (corresponding to carbamidomethylated CQPPPPPMK from NPC1 P251C), red traces represent m/z = 943.3462 (corresponding to carbamidomethylated NAAECDTY from NPC1 L929C), and green traces represent m/z = 626.6005 (corresponding to the disulfide formed between CQPPPPPMK and NAAECDTY). The peptide standards CQPPPPPMK and NAAECDTY were produced by solid phase synthesis and either reduced and carbamidomethylated or oxidized to the disulfide using Ellman’s reagent. (B) Extracted ion chromatograms from LC-MS analysis of proteolyzed NPC1. Colors as in A. NPC1 protein was carbamidomethylated in the presence or absence of reducing agent prior to proteolysis. (C) EThcD mass spectrum from NPC1 sample in part B, demonstrating the reductive fragmentation of the putative disulfide precursor into ions with masses corresponding to the two constituent peptides. Peaks matching the mass of the peptide CQPPPPPMK (monoisotopic mass of thiol = 994.49 Da) are colored in blue; peaks matching the masses of the peptides NAAECDTY (monoisotopic mass of thiol radical = 885.32 Da) are colored in red; peaks matching multiple charge states of the spectrum’s parent ions (m/z = 626.60, z = 3, corresponding to the disulfide) are colored in green. (D) MS1 mass spectra of NPC1 peptides whose disulfide content has been quantified using isotope-labeled iodoacetamide. Free thiols in purified NPC1 were labeled with 13 C 2 D 2 -iodoacetamide (“heavy”). Then the disulfides were reduced and the resulting reactive cysteines were labeled with iodoacetamide lacking isotope labels (“light”) followed by proteolysis and LC-MS analysis. Control samples were labeled with the same reagent before and after reduction (“light/reduce/light” and “heavy/reduce/heavy”) to identify isotope distributions in the limiting cases. Colored peaks fall within 10 ppm of expected masses in the isotope envelope of carbamidomethylated CQPPPPPMK (monoisotopic m/z = 526.2569) or carbamidomethylated NAAECDTY (monoisotopic m/z = 943.3462); black peaks correspond to unrelated ions. Dashed gray lines indicate the expected m/z of the monoisotopic peak and the +4 Da peak of the labeled peptides.

    Techniques Used: Liquid Chromatography with Mass Spectroscopy, Produced, Labeling, Purification

    22) Product Images from "Inter-domain dynamics drive cholesterol transport by NPC1 and NPC1L1 proteins"

    Article Title: Inter-domain dynamics drive cholesterol transport by NPC1 and NPC1L1 proteins

    Journal: bioRxiv

    doi: 10.1101/2020.03.26.009217

    Engineered cysteines C251 and C929 form a disulfide bond in NPC1. (A) Extracted ion chromatograms from LC-MS analysis of synthetic peptides corresponding to engineered cysteines in NPC1. In both samples, blue traces represent m/z = 526.2569 (corresponding to carbamidomethylated CQPPPPPMK from NPC1 P251C), red traces represent m/z = 943.3462 (corresponding to carbamidomethylated NAAECDTY from NPC1 L929C), and green traces represent m/z = 626.6005 (corresponding to the disulfide formed between CQPPPPPMK and NAAECDTY). The peptide standards CQPPPPPMK and NAAECDTY were produced by solid phase synthesis and either reduced and carbamidomethylated or oxidized to the disulfide using Ellman’s reagent. (B) Extracted ion chromatograms from LC-MS analysis of proteolyzed NPC1. Colors as in A. NPC1 protein was carbamidomethylated in the presence or absence of reducing agent prior to proteolysis. (C) EThcD mass spectrum from NPC1 sample in part B, demonstrating the reductive fragmentation of the putative disulfide precursor into ions with masses corresponding to the two constituent peptides. Peaks matching the mass of the peptide CQPPPPPMK (monoisotopic mass of thiol = 994.49 Da) are colored in blue; peaks matching the masses of the peptides NAAECDTY (monoisotopic mass of thiol radical = 885.32 Da) are colored in red; peaks matching multiple charge states of the spectrum’s parent ions (m/z = 626.60, z = 3, corresponding to the disulfide) are colored in green. (D) MS1 mass spectra of NPC1 peptides whose disulfide content has been quantified using isotope-labeled iodoacetamide. Free thiols in purified NPC1 were labeled with 13 C 2 D 2 -iodoacetamide (“heavy”). Then the disulfides were reduced and the resulting reactive cysteines were labeled with iodoacetamide lacking isotope labels (“light”) followed by proteolysis and LC-MS analysis. Control samples were labeled with the same reagent before and after reduction (“light/reduce/light” and “heavy/reduce/heavy”) to identify isotope distributions in the limiting cases. Colored peaks fall within 10 ppm of expected masses in the isotope envelope of carbamidomethylated CQPPPPPMK (monoisotopic m/z = 526.2569) or carbamidomethylated NAAECDTY (monoisotopic m/z = 943.3462); black peaks correspond to unrelated ions. Dashed gray lines indicate the expected m/z of the monoisotopic peak and the +4 Da peak of the labeled peptides.
    Figure Legend Snippet: Engineered cysteines C251 and C929 form a disulfide bond in NPC1. (A) Extracted ion chromatograms from LC-MS analysis of synthetic peptides corresponding to engineered cysteines in NPC1. In both samples, blue traces represent m/z = 526.2569 (corresponding to carbamidomethylated CQPPPPPMK from NPC1 P251C), red traces represent m/z = 943.3462 (corresponding to carbamidomethylated NAAECDTY from NPC1 L929C), and green traces represent m/z = 626.6005 (corresponding to the disulfide formed between CQPPPPPMK and NAAECDTY). The peptide standards CQPPPPPMK and NAAECDTY were produced by solid phase synthesis and either reduced and carbamidomethylated or oxidized to the disulfide using Ellman’s reagent. (B) Extracted ion chromatograms from LC-MS analysis of proteolyzed NPC1. Colors as in A. NPC1 protein was carbamidomethylated in the presence or absence of reducing agent prior to proteolysis. (C) EThcD mass spectrum from NPC1 sample in part B, demonstrating the reductive fragmentation of the putative disulfide precursor into ions with masses corresponding to the two constituent peptides. Peaks matching the mass of the peptide CQPPPPPMK (monoisotopic mass of thiol = 994.49 Da) are colored in blue; peaks matching the masses of the peptides NAAECDTY (monoisotopic mass of thiol radical = 885.32 Da) are colored in red; peaks matching multiple charge states of the spectrum’s parent ions (m/z = 626.60, z = 3, corresponding to the disulfide) are colored in green. (D) MS1 mass spectra of NPC1 peptides whose disulfide content has been quantified using isotope-labeled iodoacetamide. Free thiols in purified NPC1 were labeled with 13 C 2 D 2 -iodoacetamide (“heavy”). Then the disulfides were reduced and the resulting reactive cysteines were labeled with iodoacetamide lacking isotope labels (“light”) followed by proteolysis and LC-MS analysis. Control samples were labeled with the same reagent before and after reduction (“light/reduce/light” and “heavy/reduce/heavy”) to identify isotope distributions in the limiting cases. Colored peaks fall within 10 ppm of expected masses in the isotope envelope of carbamidomethylated CQPPPPPMK (monoisotopic m/z = 526.2569) or carbamidomethylated NAAECDTY (monoisotopic m/z = 943.3462); black peaks correspond to unrelated ions. Dashed gray lines indicate the expected m/z of the monoisotopic peak and the +4 Da peak of the labeled peptides.

    Techniques Used: Liquid Chromatography with Mass Spectroscopy, Produced, Labeling, Purification

    23) Product Images from "Membrane Association and Dimerization of a Cysteine-Rich, 16-Kilodalton Polypeptide Released from the C-Terminal Region of the Coronavirus Infectious Bronchitis Virus 1a Polyprotein"

    Article Title: Membrane Association and Dimerization of a Cysteine-Rich, 16-Kilodalton Polypeptide Released from the C-Terminal Region of the Coronavirus Infectious Bronchitis Virus 1a Polyprotein

    Journal: Journal of Virology

    doi: 10.1128/JVI.76.12.6257-6267.2002

    (a) Comparison of the amino acid sequence of the 16-kDa protein with those of the equivalent proteins of other coronaviruses. The products included in this comparison are the HcoV-229E 15-kDa protein, the MHV-JHM 15-kDa protein, the porcine transmissible gastroenteritis virus (TGEV) 14-kDa protein, and the bovine coronavirus (BCoV-ENT) 15-kDa protein. The conserved regions are in bold and outlined, and the cysteine residues are in bold and underlined. The residue (Ser-136) putatively involved in phosphorylation is outlined. (b) Dimerization of the 16-kDa protein. Plasmid pT716K was transiently expressed in Cos-7 cells with the vaccinia virus-T7 system. Cells were labeled with [ 35 S]methionine-cysteine, lysates were prepared in TGP buffer (1% Triton X-100, 10% glycerol, 50 mM HEPES [pH 7.4], 1 mM sodium vanadate, 10 μg of aprotinin per ml, 10 μg of leupeptin per ml) with (lanes 1 and 2) or without (lanes 3 and 4) 10 mM iodoacetamide (Sigma), and proteins were immunoprecipitated with anti-T7 antibody in duplicate. Gel electrophoresis of the polypeptides was performed on an SDS-15% polyacrylamide gel in the presence [R (+DTT)] or absence [NR (−DTT)] of the reducing agent dithiothreitol. Polypeptides were detected by fluorography. The values on the left are molecular sizes in kilodaltons.
    Figure Legend Snippet: (a) Comparison of the amino acid sequence of the 16-kDa protein with those of the equivalent proteins of other coronaviruses. The products included in this comparison are the HcoV-229E 15-kDa protein, the MHV-JHM 15-kDa protein, the porcine transmissible gastroenteritis virus (TGEV) 14-kDa protein, and the bovine coronavirus (BCoV-ENT) 15-kDa protein. The conserved regions are in bold and outlined, and the cysteine residues are in bold and underlined. The residue (Ser-136) putatively involved in phosphorylation is outlined. (b) Dimerization of the 16-kDa protein. Plasmid pT716K was transiently expressed in Cos-7 cells with the vaccinia virus-T7 system. Cells were labeled with [ 35 S]methionine-cysteine, lysates were prepared in TGP buffer (1% Triton X-100, 10% glycerol, 50 mM HEPES [pH 7.4], 1 mM sodium vanadate, 10 μg of aprotinin per ml, 10 μg of leupeptin per ml) with (lanes 1 and 2) or without (lanes 3 and 4) 10 mM iodoacetamide (Sigma), and proteins were immunoprecipitated with anti-T7 antibody in duplicate. Gel electrophoresis of the polypeptides was performed on an SDS-15% polyacrylamide gel in the presence [R (+DTT)] or absence [NR (−DTT)] of the reducing agent dithiothreitol. Polypeptides were detected by fluorography. The values on the left are molecular sizes in kilodaltons.

    Techniques Used: Sequencing, Plasmid Preparation, Labeling, Immunoprecipitation, Nucleic Acid Electrophoresis

    24) Product Images from "Proteome-wide analysis of cysteine oxidation reveals metabolic sensitivity to redox stress"

    Article Title: Proteome-wide analysis of cysteine oxidation reveals metabolic sensitivity to redox stress

    Journal: Nature Communications

    doi: 10.1038/s41467-018-04003-3

    Schematic overview of the Stable Isotope Cysteine Labelling with IodoAcetamide (SICyLIA) methodology. a Samples are extracted in presence of either light ( 12 C 2 H 4 INO) or heavy ( 13 C 2 D 2 H 2 INO) iodoacetamide (IAM) to alkylate free cysteine thiols, introducing a carbamidomethyl (CAM) group. Equal amounts of modified protein extracts are mixed, reversibly oxidised thiols are reduced with DTT and subsequently alkylated with NEM. Protein extracts are digested and peptides are fractionated prior to UHPLC-MS/MS analysis. b In parallel, labelled proteome extracts are trypsin digested and dimethylated using light (H 12 CHO/NaBH 3 CN) or heavy (D 13 CDO/NaBD 3 CN) formaldehyde/sodium cyanoborohydride, peptides are fractioned, and analysed using UHPLC-MS/MS similarly to IAM-modified peptides
    Figure Legend Snippet: Schematic overview of the Stable Isotope Cysteine Labelling with IodoAcetamide (SICyLIA) methodology. a Samples are extracted in presence of either light ( 12 C 2 H 4 INO) or heavy ( 13 C 2 D 2 H 2 INO) iodoacetamide (IAM) to alkylate free cysteine thiols, introducing a carbamidomethyl (CAM) group. Equal amounts of modified protein extracts are mixed, reversibly oxidised thiols are reduced with DTT and subsequently alkylated with NEM. Protein extracts are digested and peptides are fractionated prior to UHPLC-MS/MS analysis. b In parallel, labelled proteome extracts are trypsin digested and dimethylated using light (H 12 CHO/NaBH 3 CN) or heavy (D 13 CDO/NaBD 3 CN) formaldehyde/sodium cyanoborohydride, peptides are fractioned, and analysed using UHPLC-MS/MS similarly to IAM-modified peptides

    Techniques Used: Chick Chorioallantoic Membrane Assay, Modification, Mass Spectrometry

    25) Product Images from "Inter-domain dynamics drive cholesterol transport by NPC1 and NPC1L1 proteins"

    Article Title: Inter-domain dynamics drive cholesterol transport by NPC1 and NPC1L1 proteins

    Journal: bioRxiv

    doi: 10.1101/2020.03.26.009217

    Engineered cysteines C251 and C929 form a disulfide bond in NPC1. (A) Extracted ion chromatograms from LC-MS analysis of synthetic peptides corresponding to engineered cysteines in NPC1. In both samples, blue traces represent m/z = 526.2569 (corresponding to carbamidomethylated CQPPPPPMK from NPC1 P251C), red traces represent m/z = 943.3462 (corresponding to carbamidomethylated NAAECDTY from NPC1 L929C), and green traces represent m/z = 626.6005 (corresponding to the disulfide formed between CQPPPPPMK and NAAECDTY). The peptide standards CQPPPPPMK and NAAECDTY were produced by solid phase synthesis and either reduced and carbamidomethylated or oxidized to the disulfide using Ellman’s reagent. (B) Extracted ion chromatograms from LC-MS analysis of proteolyzed NPC1. Colors as in A. NPC1 protein was carbamidomethylated in the presence or absence of reducing agent prior to proteolysis. (C) EThcD mass spectrum from NPC1 sample in part B, demonstrating the reductive fragmentation of the putative disulfide precursor into ions with masses corresponding to the two constituent peptides. Peaks matching the mass of the peptide CQPPPPPMK (monoisotopic mass of thiol = 994.49 Da) are colored in blue; peaks matching the masses of the peptides NAAECDTY (monoisotopic mass of thiol radical = 885.32 Da) are colored in red; peaks matching multiple charge states of the spectrum’s parent ions (m/z = 626.60, z = 3, corresponding to the disulfide) are colored in green. (D) MS1 mass spectra of NPC1 peptides whose disulfide content has been quantified using isotope-labeled iodoacetamide. Free thiols in purified NPC1 were labeled with 13 C 2 D 2 -iodoacetamide (“heavy”). Then the disulfides were reduced and the resulting reactive cysteines were labeled with iodoacetamide lacking isotope labels (“light”) followed by proteolysis and LC-MS analysis. Control samples were labeled with the same reagent before and after reduction (“light/reduce/light” and “heavy/reduce/heavy”) to identify isotope distributions in the limiting cases. Colored peaks fall within 10 ppm of expected masses in the isotope envelope of carbamidomethylated CQPPPPPMK (monoisotopic m/z = 526.2569) or carbamidomethylated NAAECDTY (monoisotopic m/z = 943.3462); black peaks correspond to unrelated ions. Dashed gray lines indicate the expected m/z of the monoisotopic peak and the +4 Da peak of the labeled peptides.
    Figure Legend Snippet: Engineered cysteines C251 and C929 form a disulfide bond in NPC1. (A) Extracted ion chromatograms from LC-MS analysis of synthetic peptides corresponding to engineered cysteines in NPC1. In both samples, blue traces represent m/z = 526.2569 (corresponding to carbamidomethylated CQPPPPPMK from NPC1 P251C), red traces represent m/z = 943.3462 (corresponding to carbamidomethylated NAAECDTY from NPC1 L929C), and green traces represent m/z = 626.6005 (corresponding to the disulfide formed between CQPPPPPMK and NAAECDTY). The peptide standards CQPPPPPMK and NAAECDTY were produced by solid phase synthesis and either reduced and carbamidomethylated or oxidized to the disulfide using Ellman’s reagent. (B) Extracted ion chromatograms from LC-MS analysis of proteolyzed NPC1. Colors as in A. NPC1 protein was carbamidomethylated in the presence or absence of reducing agent prior to proteolysis. (C) EThcD mass spectrum from NPC1 sample in part B, demonstrating the reductive fragmentation of the putative disulfide precursor into ions with masses corresponding to the two constituent peptides. Peaks matching the mass of the peptide CQPPPPPMK (monoisotopic mass of thiol = 994.49 Da) are colored in blue; peaks matching the masses of the peptides NAAECDTY (monoisotopic mass of thiol radical = 885.32 Da) are colored in red; peaks matching multiple charge states of the spectrum’s parent ions (m/z = 626.60, z = 3, corresponding to the disulfide) are colored in green. (D) MS1 mass spectra of NPC1 peptides whose disulfide content has been quantified using isotope-labeled iodoacetamide. Free thiols in purified NPC1 were labeled with 13 C 2 D 2 -iodoacetamide (“heavy”). Then the disulfides were reduced and the resulting reactive cysteines were labeled with iodoacetamide lacking isotope labels (“light”) followed by proteolysis and LC-MS analysis. Control samples were labeled with the same reagent before and after reduction (“light/reduce/light” and “heavy/reduce/heavy”) to identify isotope distributions in the limiting cases. Colored peaks fall within 10 ppm of expected masses in the isotope envelope of carbamidomethylated CQPPPPPMK (monoisotopic m/z = 526.2569) or carbamidomethylated NAAECDTY (monoisotopic m/z = 943.3462); black peaks correspond to unrelated ions. Dashed gray lines indicate the expected m/z of the monoisotopic peak and the +4 Da peak of the labeled peptides.

    Techniques Used: Liquid Chromatography with Mass Spectroscopy, Produced, Labeling, Purification

    26) Product Images from "Persistent and Transient Replication of Full-Length Hepatitis C Virus Genomes in Cell Culture"

    Article Title: Persistent and Transient Replication of Full-Length Hepatitis C Virus Genomes in Cell Culture

    Journal: Journal of Virology

    doi: 10.1128/JVI.76.8.4008-4021.2002

    Analysis of structural protein expression in cell lines carrying sfl genomes. (A) Detection of HCV core protein by Western blotting. Lysates, each corresponding to 2 × 10 5 cells of the given cell lines, were loaded onto a gel, separated by electrophoreses, blotted, and probed with an HCV core-specific monoclonal antibody. (B) Pulse-chase and endoglycosidase H analysis of HCV glycoprotein complexes. Cells were incubated with [ 35 S]methionine/cysteine-containing medium for 1 h, and after the cells were washed extensively, nonradioactive medium was added for the times specified above the lanes. Glycoprotein complexes were isolated by immunoprecipitations from cell lysates under nondenaturing conditions and were either mock treated (− EndoH) or deglycosylated by endoglycosidase H digestion (+ EndoH). After SDS-PAGE, proteins were detected by autoradiography. The deglycosylated forms of the proteins are labeled with asterisks. (C) Analysis of HCV glycoprotein complexes under reducing and nonreducing conditions. Immunoprecipitations were performed in the presence of 20 mM iodoacetamide in the lysis and wash buffer. Prior to loading, half of each sample was boiled in denaturing buffer with or without 2-ME as indicated. Proteins were detected as described above.
    Figure Legend Snippet: Analysis of structural protein expression in cell lines carrying sfl genomes. (A) Detection of HCV core protein by Western blotting. Lysates, each corresponding to 2 × 10 5 cells of the given cell lines, were loaded onto a gel, separated by electrophoreses, blotted, and probed with an HCV core-specific monoclonal antibody. (B) Pulse-chase and endoglycosidase H analysis of HCV glycoprotein complexes. Cells were incubated with [ 35 S]methionine/cysteine-containing medium for 1 h, and after the cells were washed extensively, nonradioactive medium was added for the times specified above the lanes. Glycoprotein complexes were isolated by immunoprecipitations from cell lysates under nondenaturing conditions and were either mock treated (− EndoH) or deglycosylated by endoglycosidase H digestion (+ EndoH). After SDS-PAGE, proteins were detected by autoradiography. The deglycosylated forms of the proteins are labeled with asterisks. (C) Analysis of HCV glycoprotein complexes under reducing and nonreducing conditions. Immunoprecipitations were performed in the presence of 20 mM iodoacetamide in the lysis and wash buffer. Prior to loading, half of each sample was boiled in denaturing buffer with or without 2-ME as indicated. Proteins were detected as described above.

    Techniques Used: Expressing, Western Blot, Pulse Chase, Incubation, Isolation, SDS Page, Autoradiography, Labeling, Lysis

    27) Product Images from "Identification of multiple serine to asparagine sequence variation sites in an intended copy product of LUCENTIS® by mass spectrometry"

    Article Title: Identification of multiple serine to asparagine sequence variation sites in an intended copy product of LUCENTIS® by mass spectrometry

    Journal: mAbs

    doi: 10.1080/19420862.2017.1366395

    Fab subunit LC-UV/ESI-MS analysis. (A) Overlay of UV chromatograms at 214 nm of light and heavy chains of LUCENTIS® and RAZUMAB batches 1 and 2 after reduction and carbamidomethylation. Signal offset: 10%. (B) Time-resolved deconvolution for the light chain (LC). LC, and LC + 27 Da (in RAZUMAB samples), are annotated. ║: In-source dehydration, ◊: Guanidine adduct, §: Possible LC + (2 × 27 Da) in RAZUMAB batches, ‡: Sodium adduct. (C) Time-resolved deconvolution for the heavy chain (HC) species. HC, oxidized HC (HCox) and N-terminal pyroglutamate formation (HC(pE)) are annotated. *major sample preparation artifact is overalkylation with iodoacetamide as shown by the addition of +57 Da. Heat maps were generated from time-resolved deconvolution performed in parallel for all samples. Intensity is color-coded ranging from less intense (black) to most intense (red).
    Figure Legend Snippet: Fab subunit LC-UV/ESI-MS analysis. (A) Overlay of UV chromatograms at 214 nm of light and heavy chains of LUCENTIS® and RAZUMAB batches 1 and 2 after reduction and carbamidomethylation. Signal offset: 10%. (B) Time-resolved deconvolution for the light chain (LC). LC, and LC + 27 Da (in RAZUMAB samples), are annotated. ║: In-source dehydration, ◊: Guanidine adduct, §: Possible LC + (2 × 27 Da) in RAZUMAB batches, ‡: Sodium adduct. (C) Time-resolved deconvolution for the heavy chain (HC) species. HC, oxidized HC (HCox) and N-terminal pyroglutamate formation (HC(pE)) are annotated. *major sample preparation artifact is overalkylation with iodoacetamide as shown by the addition of +57 Da. Heat maps were generated from time-resolved deconvolution performed in parallel for all samples. Intensity is color-coded ranging from less intense (black) to most intense (red).

    Techniques Used: Mass Spectrometry, Sample Prep, Generated

    28) Product Images from "?-Macroglobulins Are Present in Some Gram-negative Bacteria"

    Article Title: ?-Macroglobulins Are Present in Some Gram-negative Bacteria

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M803127200

    Fluorescence and light microscopy of E. coli overexpressing full-length ECAM. Panel A , incubation with IAF alone; panel B , reaction with methylamine for 2 h followed by reaction with IAF; panel C , control reaction in which iodoacetamide was used to block new
    Figure Legend Snippet: Fluorescence and light microscopy of E. coli overexpressing full-length ECAM. Panel A , incubation with IAF alone; panel B , reaction with methylamine for 2 h followed by reaction with IAF; panel C , control reaction in which iodoacetamide was used to block new

    Techniques Used: Fluorescence, Light Microscopy, Incubation, Blocking Assay

    29) Product Images from "Role of S-Palmitoylation by ZDHHC13 in Mitochondrial function and Metabolism in Liver"

    Article Title: Role of S-Palmitoylation by ZDHHC13 in Mitochondrial function and Metabolism in Liver

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-02159-4

    Identification and characterization of liver S-palmitoylome. ( a ) A schematic overview for the site-specific identification of the S-palmitoylome by a modified alkylating RAC assay. Free thiols are first blocked with NEM. Disulfide bond was cleaved by TCEP and further blocked with IAM. Thioesters are then cleaved with neutral HA and the newly liberated thiols are purified with thiol sepharose 4B. The capture proteins on sepharoses were digested with trypsin. The supernatant of digested samples was removed and the captured peptides on sepharoses were eluted with TCEP and subjected to downstream MS analysis for S-palmitoylated site identification. Peptide alignment was applied to increase the coverage based on the retention time and m/z of identified peptides. Extracted ion chromatogram of identified peptides and the same m/z in different LC samples were used to quantify the relative abundance. The protein levels were quantified by TMT10-plex labelling. ( b ) Pie diagram illustrating the subcellular localization of identified S-palmitoylated proteins. ( c ) Pie diagram illustrating the functions of identified S-palmitoylated proteins. NEM, N-ethylmaleimide; IAM, Iodoacetamide; HA, hydroxylamine.
    Figure Legend Snippet: Identification and characterization of liver S-palmitoylome. ( a ) A schematic overview for the site-specific identification of the S-palmitoylome by a modified alkylating RAC assay. Free thiols are first blocked with NEM. Disulfide bond was cleaved by TCEP and further blocked with IAM. Thioesters are then cleaved with neutral HA and the newly liberated thiols are purified with thiol sepharose 4B. The capture proteins on sepharoses were digested with trypsin. The supernatant of digested samples was removed and the captured peptides on sepharoses were eluted with TCEP and subjected to downstream MS analysis for S-palmitoylated site identification. Peptide alignment was applied to increase the coverage based on the retention time and m/z of identified peptides. Extracted ion chromatogram of identified peptides and the same m/z in different LC samples were used to quantify the relative abundance. The protein levels were quantified by TMT10-plex labelling. ( b ) Pie diagram illustrating the subcellular localization of identified S-palmitoylated proteins. ( c ) Pie diagram illustrating the functions of identified S-palmitoylated proteins. NEM, N-ethylmaleimide; IAM, Iodoacetamide; HA, hydroxylamine.

    Techniques Used: Modification, Purification, Mass Spectrometry

    30) Product Images from "de novo Sequencing and Disulfide Mapping of a Bromotryptophan-Containing Conotoxin by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry"

    Article Title: de novo Sequencing and Disulfide Mapping of a Bromotryptophan-Containing Conotoxin by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

    Journal: Analytical chemistry

    doi: 10.1021/ac0607764

    MALDI MS/MS comparision of reduced and alkylated peptides: native Mo 1274 (top) and synthetic Mo 1274 (bottom). The reduction and alkylation were effected by use of dithiothreitol (DTT) and iodoacetamide.
    Figure Legend Snippet: MALDI MS/MS comparision of reduced and alkylated peptides: native Mo 1274 (top) and synthetic Mo 1274 (bottom). The reduction and alkylation were effected by use of dithiothreitol (DTT) and iodoacetamide.

    Techniques Used: Mass Spectrometry

    31) Product Images from "SUMOylation Is Required for Optimal TRAF3 Signaling Capacity"

    Article Title: SUMOylation Is Required for Optimal TRAF3 Signaling Capacity

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0080470

    TRAF3 SUMOylation affects its CD40-interacting capacity. ( A ) The binding of TRAF3 to GST-CD40CT increases when SUMO modification is maintained. HEK293 cells were lysed in the presence or absence of iodoacetamide (IDO) and lysates were incubated with bacterially produced GST-CD40 C-terminus (CT) or, as control GST-CD40CTA, carrying a T 254 →A mutation (GST-CD40CTA) which abolishes interaction with TRAF3, bound to glutathione sepharose beads. Interacting proteins were fractionated by SDS-PAGE and immunoblotted ( I.B .) with anti-TRAF3 Ab. Whole cell lysates ( WCL ; 30 µg) were analyzed by immunoblot for TRAF3 expression levels. Lower right panel: Coomassie-stained gel showing GST-CD40CT and GST-CD40CTA produced in bacteria. ( B ) Over-expression of “dominant-negative” Ubc9 C93A reduces binding of TRAF3 to CD40. HEK293 cells were transfected with Ubc9 C93A or control vector ( CV ), lysates were obtained using an IDO-containing lysis buffer and incubated with GST-CD40CT or GST-CD40CTA bound to glutathione sepharose beads. Interacting proteins were fractionated by SDS-PAGE and immunoblotted ( I.B .) with anti-TRAF3. Whole cell lysates ( WCL ; 30 µg) were analyzed for TRAF3 and Ubc9 C93A expression levels by immunoblotting using anti-TRAF3 and Myc tag Abs, respectively. Results in (A) (B) are representative of 3 independent experiments.
    Figure Legend Snippet: TRAF3 SUMOylation affects its CD40-interacting capacity. ( A ) The binding of TRAF3 to GST-CD40CT increases when SUMO modification is maintained. HEK293 cells were lysed in the presence or absence of iodoacetamide (IDO) and lysates were incubated with bacterially produced GST-CD40 C-terminus (CT) or, as control GST-CD40CTA, carrying a T 254 →A mutation (GST-CD40CTA) which abolishes interaction with TRAF3, bound to glutathione sepharose beads. Interacting proteins were fractionated by SDS-PAGE and immunoblotted ( I.B .) with anti-TRAF3 Ab. Whole cell lysates ( WCL ; 30 µg) were analyzed by immunoblot for TRAF3 expression levels. Lower right panel: Coomassie-stained gel showing GST-CD40CT and GST-CD40CTA produced in bacteria. ( B ) Over-expression of “dominant-negative” Ubc9 C93A reduces binding of TRAF3 to CD40. HEK293 cells were transfected with Ubc9 C93A or control vector ( CV ), lysates were obtained using an IDO-containing lysis buffer and incubated with GST-CD40CT or GST-CD40CTA bound to glutathione sepharose beads. Interacting proteins were fractionated by SDS-PAGE and immunoblotted ( I.B .) with anti-TRAF3. Whole cell lysates ( WCL ; 30 µg) were analyzed for TRAF3 and Ubc9 C93A expression levels by immunoblotting using anti-TRAF3 and Myc tag Abs, respectively. Results in (A) (B) are representative of 3 independent experiments.

    Techniques Used: Binding Assay, Modification, Incubation, Produced, Mutagenesis, SDS Page, Expressing, Staining, Over Expression, Transfection, Plasmid Preparation, Lysis

    TRAF3 is post-translationally modified by SUMO. ( A ) HeLa cells stably expressing His-tagged SUMO-1 or SUMO-2 and parental cells were lysed in a protein-denaturing buffer and lysates were subjected to enrichment of SUMOylated proteins on nickel-nitrilotriacetic acid (Ni-NTA) columns. Eluates were immunoblotted with a TRAF3 polyclonal antibody. ( B C ) SUMO-1 modification of TRAF3. Protein lysates were obtained from EJ bladder carcinoma, BJAB lymphoma and HeLa cervical carcinoma cells stably transfected with SUMO-1 (B) or mouse splenocytes (C) in the presence or absence of iodocetamide (IDO) and immunoprecipitated ( I.P ) with anti-TRAF3 C20 antibody. The SUMO-1 conjugates were detected by anti-SUMO-1 specific antibody. ( D ) SUMO-2/3 modification of TRAF3. Protein lysates were obtained from EJ cells in the presence orabsence of IDA and TRAF3 immunoprecipitates ( I.P ) were immunoblotted ( I.B ) with an anti-SUMO-2/3 specific antibody.
    Figure Legend Snippet: TRAF3 is post-translationally modified by SUMO. ( A ) HeLa cells stably expressing His-tagged SUMO-1 or SUMO-2 and parental cells were lysed in a protein-denaturing buffer and lysates were subjected to enrichment of SUMOylated proteins on nickel-nitrilotriacetic acid (Ni-NTA) columns. Eluates were immunoblotted with a TRAF3 polyclonal antibody. ( B C ) SUMO-1 modification of TRAF3. Protein lysates were obtained from EJ bladder carcinoma, BJAB lymphoma and HeLa cervical carcinoma cells stably transfected with SUMO-1 (B) or mouse splenocytes (C) in the presence or absence of iodocetamide (IDO) and immunoprecipitated ( I.P ) with anti-TRAF3 C20 antibody. The SUMO-1 conjugates were detected by anti-SUMO-1 specific antibody. ( D ) SUMO-2/3 modification of TRAF3. Protein lysates were obtained from EJ cells in the presence orabsence of IDA and TRAF3 immunoprecipitates ( I.P ) were immunoblotted ( I.B ) with an anti-SUMO-2/3 specific antibody.

    Techniques Used: Modification, Stable Transfection, Expressing, Transfection, Immunoprecipitation

    32) Product Images from "Simultaneous Activation of Complement and Coagulation by MBL-Associated Serine Protease 2"

    Article Title: Simultaneous Activation of Complement and Coagulation by MBL-Associated Serine Protease 2

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0000623

    Fibrin deposition on MBL target surface. Fig 5A shows 125 I-fibrin deposition on a mannan/fibrinogen coated surface to which rMBL/rMASP2K complexes are bound. The background (a control in which rMBL/rMASP2K complexes were prevented from binding to the surface due to the presence of EDTA) has been subtracted from the remaining samples. The figure shows that deposition above background level only occurs if rMASP2K is present and if the incubation with fibrinogen is in the presence of prothrombin. Incubation was for 4h at 37°C. Fig 5B 125 I-fibrin deposition on S. aureus -derivatized beads. S. aureus -derivatized beads were incubated at 4°C with rMBL/rMASP2 complexes and subsequently with prothrombin and 125 I-fibrinogen for 7 h at 37°C. The figure shows that fibrin gets deposited on the beads upon activation of prothrombin by rMBL/rMASP2 complexes (columns 2 and 5, closed bars). The fibrin bound to the surface also gets covalently cross-linked to the bacteria since it was not removed by washing the beads with urea (column 2, open bar). If the deposition was done in the presence of iodoacetamide (IAM), which inhibits factor XIIIa, the radioactivity associated with the beads was reduced to background level upon urea extraction (column 5, open bar). In the controls in which no rMASP2 was added or in the presence of protease inhibitors (C1 inhibitor or Pefabloc SC) no deposition on the beads could be observed. Each sample was tested twice and the error bars represent one standard deviation from the mean.
    Figure Legend Snippet: Fibrin deposition on MBL target surface. Fig 5A shows 125 I-fibrin deposition on a mannan/fibrinogen coated surface to which rMBL/rMASP2K complexes are bound. The background (a control in which rMBL/rMASP2K complexes were prevented from binding to the surface due to the presence of EDTA) has been subtracted from the remaining samples. The figure shows that deposition above background level only occurs if rMASP2K is present and if the incubation with fibrinogen is in the presence of prothrombin. Incubation was for 4h at 37°C. Fig 5B 125 I-fibrin deposition on S. aureus -derivatized beads. S. aureus -derivatized beads were incubated at 4°C with rMBL/rMASP2 complexes and subsequently with prothrombin and 125 I-fibrinogen for 7 h at 37°C. The figure shows that fibrin gets deposited on the beads upon activation of prothrombin by rMBL/rMASP2 complexes (columns 2 and 5, closed bars). The fibrin bound to the surface also gets covalently cross-linked to the bacteria since it was not removed by washing the beads with urea (column 2, open bar). If the deposition was done in the presence of iodoacetamide (IAM), which inhibits factor XIIIa, the radioactivity associated with the beads was reduced to background level upon urea extraction (column 5, open bar). In the controls in which no rMASP2 was added or in the presence of protease inhibitors (C1 inhibitor or Pefabloc SC) no deposition on the beads could be observed. Each sample was tested twice and the error bars represent one standard deviation from the mean.

    Techniques Used: Binding Assay, Incubation, Activation Assay, Radioactivity, Standard Deviation

    33) Product Images from "Impact of acid secretion, gastritis, and mucus thickness on gastric transfer of antibiotics in rats"

    Article Title: Impact of acid secretion, gastritis, and mucus thickness on gastric transfer of antibiotics in rats

    Journal: Gut

    doi:

    Effect of Helicobacter pylori , iodoacetamide (iodo), and pronase on gastric mucus thickness. Hp −ve, H pylori negative control (no surgery); Hp +ve Op, H pylori infected (had surgery); Hp +ve Unop, H pylori infected control (no surgery).
    Figure Legend Snippet: Effect of Helicobacter pylori , iodoacetamide (iodo), and pronase on gastric mucus thickness. Hp −ve, H pylori negative control (no surgery); Hp +ve Op, H pylori infected (had surgery); Hp +ve Unop, H pylori infected control (no surgery).

    Techniques Used: Negative Control, Infection

    34) Product Images from "Heterologous Biosynthesis, Modifications and Structural Characterization of Ruminococcin-A, a Lanthipeptide From the Gut Bacterium Ruminococcus gnavus E1, in Escherichia coli"

    Article Title: Heterologous Biosynthesis, Modifications and Structural Characterization of Ruminococcin-A, a Lanthipeptide From the Gut Bacterium Ruminococcus gnavus E1, in Escherichia coli

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.01688

    Iodoacetamide derivatization and trypsin cleavage of preRumA ∗ . (A) Iodoacetamide reaction with the sulfhydryl group of cysteine side chain. (B) Schematic representation of preRumA ∗ showing five different fragments expected from the tryptic digestion. (C) Charged ions peaks observed in the Orbitrap Fusion nLC-MS spectrum of the digested preRumA ∗ products. The predicted exact masses for each of the fragments are labeled in their respective spectra. (D) MS 2 experiments and assignment of trypsin-activated RumA fragment ion peaks. Major product ions in the spectrum were identified to fit successive loss of Gln18, Trp17 and Dhb16. ( M ’ +3H) 3+ = 892.40 ∗ .
    Figure Legend Snippet: Iodoacetamide derivatization and trypsin cleavage of preRumA ∗ . (A) Iodoacetamide reaction with the sulfhydryl group of cysteine side chain. (B) Schematic representation of preRumA ∗ showing five different fragments expected from the tryptic digestion. (C) Charged ions peaks observed in the Orbitrap Fusion nLC-MS spectrum of the digested preRumA ∗ products. The predicted exact masses for each of the fragments are labeled in their respective spectra. (D) MS 2 experiments and assignment of trypsin-activated RumA fragment ion peaks. Major product ions in the spectrum were identified to fit successive loss of Gln18, Trp17 and Dhb16. ( M ’ +3H) 3+ = 892.40 ∗ .

    Techniques Used: Mass Spectrometry, Labeling

    35) Product Images from "In vitro and in vivo evidence for shear-induced activation of latent transforming growth factor-?1"

    Article Title: In vitro and in vivo evidence for shear-induced activation of latent transforming growth factor-?1

    Journal: Blood

    doi: 10.1182/blood-2008-04-151753

    Thiol-disulfide exchange contributes to latent TGF-β1 activation. (A) Platelet releasates were stirred in the presence or absence of the indicated reagents for 120 minutes at 37°C and TGF-β1 activity was measured by ELISA. The concentrations used were as follows: N -ethylmaleimide (NEM) 1 mM, iodoacetamide (IODO) 1 mM, N -acetyl cysteine (NAC) 1 mM, MPB 100 μM, and glutathione (GSH) 200 μM. Stirring increased active TGF-β1 from approximately 0.2 to 5 ng/mL; total TGF-β1 was approximately 56 ng/mL. Data are expressed as percentage of the control value of active TGF-β1 in the absence of a thiol-reactive reagent (CON). Error bars represent SD. (B) MPB (100 μM) labeled a number of proteins in platelet releasates and the intensity of labeling increased when samples were stirred for 2 hours after adding the MPB. One of the labeled proteins was TGF-β1 itself (Mr, ∼ 25 kD) as judged by immunoprecipitation (middle panel) with a combination of anti-LAP and anti–TGF-β1 antibodies. (C) MPB labeling of the approximately 25-kD band corresponding to the migration of TGF-β1 (arrow), as well as some other proteins, was dramatically reduced when the MPB was added after 2 hours of stirring. Pretreatment with NEM (1 mM) prevented MPB labeling. (D) Densitometric quantification of biotin-MPB incorporation into the approximately 25-kD protein ( ) in panels B and C (left). Error bars represent SD. (E) Platelet releasates were subjected to shear at 1800 s −1 for the indicated time periods and then labeled with MPB (100 μM) for 2 hours at 37°C. MPB-labeled proteins in platelet releasates were detected with streptavidin-HRP. The sample sheared for 120 minutes from the same gel was also immunoblotted separately to identify TGF-β1. The vertical line separates the membranes reacted with streptavidin-HRP from the membrane reacted with the antibody to TGF-β1. (F) Thrombin-stimulated platelet releasates were incubated with the indicated thiol-reactive compounds followed by labeling with biotin-MPB (100 μM) for 2 hours. MPB-labeled proteins were detected with streptavidin-HRP (left panel) and immunoblotted to identify TGF-β1 (right panel). Vertical lines indicate deletion of intermediate lanes from the same gel.
    Figure Legend Snippet: Thiol-disulfide exchange contributes to latent TGF-β1 activation. (A) Platelet releasates were stirred in the presence or absence of the indicated reagents for 120 minutes at 37°C and TGF-β1 activity was measured by ELISA. The concentrations used were as follows: N -ethylmaleimide (NEM) 1 mM, iodoacetamide (IODO) 1 mM, N -acetyl cysteine (NAC) 1 mM, MPB 100 μM, and glutathione (GSH) 200 μM. Stirring increased active TGF-β1 from approximately 0.2 to 5 ng/mL; total TGF-β1 was approximately 56 ng/mL. Data are expressed as percentage of the control value of active TGF-β1 in the absence of a thiol-reactive reagent (CON). Error bars represent SD. (B) MPB (100 μM) labeled a number of proteins in platelet releasates and the intensity of labeling increased when samples were stirred for 2 hours after adding the MPB. One of the labeled proteins was TGF-β1 itself (Mr, ∼ 25 kD) as judged by immunoprecipitation (middle panel) with a combination of anti-LAP and anti–TGF-β1 antibodies. (C) MPB labeling of the approximately 25-kD band corresponding to the migration of TGF-β1 (arrow), as well as some other proteins, was dramatically reduced when the MPB was added after 2 hours of stirring. Pretreatment with NEM (1 mM) prevented MPB labeling. (D) Densitometric quantification of biotin-MPB incorporation into the approximately 25-kD protein ( ) in panels B and C (left). Error bars represent SD. (E) Platelet releasates were subjected to shear at 1800 s −1 for the indicated time periods and then labeled with MPB (100 μM) for 2 hours at 37°C. MPB-labeled proteins in platelet releasates were detected with streptavidin-HRP. The sample sheared for 120 minutes from the same gel was also immunoblotted separately to identify TGF-β1. The vertical line separates the membranes reacted with streptavidin-HRP from the membrane reacted with the antibody to TGF-β1. (F) Thrombin-stimulated platelet releasates were incubated with the indicated thiol-reactive compounds followed by labeling with biotin-MPB (100 μM) for 2 hours. MPB-labeled proteins were detected with streptavidin-HRP (left panel) and immunoblotted to identify TGF-β1 (right panel). Vertical lines indicate deletion of intermediate lanes from the same gel.

    Techniques Used: Activation Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Labeling, Immunoprecipitation, Migration, Incubation

    36) Product Images from "Fab-dsFv: A bispecific antibody format with extended serum half-life through albumin binding"

    Article Title: Fab-dsFv: A bispecific antibody format with extended serum half-life through albumin binding

    Journal: mAbs

    doi: 10.1080/19420862.2016.1210747

    Isoelectric focusing of Fab-dsFv. The pI of non-reduced (black line) and reduced and alkylated (blue line) of purified (A) Fab' fragment and (B) Fab-dsFv was determined by iCE280 capillary isoelectric focusing. Samples were mixed with methylcellulose, pharmalytes (pH3-10) and synthetic pI markers prior to separation. For mild reduced and alkylated samples, iodoacetamide was used as the alkylating agent after reduction with Tris(3-hydroxypropyl)phosphine (THPP). Profiles were continuously detected at an absorbance of 280 nm. The pI values of the non-reduced sample are indicated on each graph. Calibrated electropherograms were analyzed using Empower 2 (Waters).
    Figure Legend Snippet: Isoelectric focusing of Fab-dsFv. The pI of non-reduced (black line) and reduced and alkylated (blue line) of purified (A) Fab' fragment and (B) Fab-dsFv was determined by iCE280 capillary isoelectric focusing. Samples were mixed with methylcellulose, pharmalytes (pH3-10) and synthetic pI markers prior to separation. For mild reduced and alkylated samples, iodoacetamide was used as the alkylating agent after reduction with Tris(3-hydroxypropyl)phosphine (THPP). Profiles were continuously detected at an absorbance of 280 nm. The pI values of the non-reduced sample are indicated on each graph. Calibrated electropherograms were analyzed using Empower 2 (Waters).

    Techniques Used: Purification

    37) Product Images from "Mouse Antibody of IgM Class is Prone to Non-Enzymatic Cleavage between CH1 and CH2 Domains"

    Article Title: Mouse Antibody of IgM Class is Prone to Non-Enzymatic Cleavage between CH1 and CH2 Domains

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-19003-4

    Legumain does not cleave mouse IgM. ( a ) IgM cleavage in serum treated with alkylating agents. Alkylating agents, such as iodoacetamide or NEM, are potent inhibitors of legumain – a cysteine protease specific to Asn at P1 site. Activity of legumain present in FBS was tested using fluorogenic substrate Z-Ala-Ala-Asn-AMC. Samples were analyzed using western blotting with anti-mouse IgMκ antibody. The full-length blot is presented in Supplementary Figure S2 . ( b ) Recombinant legumain did not cleave IgMs. The gel was stained with Coomassie BB. ( a and b ) Representatives of two independent experiments are shown.
    Figure Legend Snippet: Legumain does not cleave mouse IgM. ( a ) IgM cleavage in serum treated with alkylating agents. Alkylating agents, such as iodoacetamide or NEM, are potent inhibitors of legumain – a cysteine protease specific to Asn at P1 site. Activity of legumain present in FBS was tested using fluorogenic substrate Z-Ala-Ala-Asn-AMC. Samples were analyzed using western blotting with anti-mouse IgMκ antibody. The full-length blot is presented in Supplementary Figure S2 . ( b ) Recombinant legumain did not cleave IgMs. The gel was stained with Coomassie BB. ( a and b ) Representatives of two independent experiments are shown.

    Techniques Used: Activity Assay, Western Blot, Recombinant, Staining

    38) Product Images from "Preventing Disulfide Bond Formation Weakens Non-Covalent Forces among Lysozyme Aggregates"

    Article Title: Preventing Disulfide Bond Formation Weakens Non-Covalent Forces among Lysozyme Aggregates

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0087012

    Force-versus-extension (FX) traces of HEWL aggregates from single-molecule force spectroscopy. A) FX traces obtained by repeated approach and retraction of AFM cantilever tip onto the gold coverslip on which 40 µl of freshly prepared HEWL at pH 12.2 was added. Each trace is offset by 150 pN, with respect to the previous trace. B) Representative FX traces obtained by pulling HEWL aggregates formed after 4 days of incubation at pH 12.2. Each trace is offset by 1000 pN with respect to the previous one. See Figure S3 for more traces under this condition. C) FX traces of HEWL at pH 12.2 incubated with iodoacetamide at a final concentration of 12 mM for 3 days.
    Figure Legend Snippet: Force-versus-extension (FX) traces of HEWL aggregates from single-molecule force spectroscopy. A) FX traces obtained by repeated approach and retraction of AFM cantilever tip onto the gold coverslip on which 40 µl of freshly prepared HEWL at pH 12.2 was added. Each trace is offset by 150 pN, with respect to the previous trace. B) Representative FX traces obtained by pulling HEWL aggregates formed after 4 days of incubation at pH 12.2. Each trace is offset by 1000 pN with respect to the previous one. See Figure S3 for more traces under this condition. C) FX traces of HEWL at pH 12.2 incubated with iodoacetamide at a final concentration of 12 mM for 3 days.

    Techniques Used: Spectroscopy, Incubation, Concentration Assay

    39) Product Images from "Time-gated detection of protein-protein interactions with transcriptional readout"

    Article Title: Time-gated detection of protein-protein interactions with transcriptional readout

    Journal: eLife

    doi: 10.7554/eLife.30233

    Further characterization of SPARK tool. ( A ) HA-β2AR construct recruits β-arrestin2 -EGFP to the plasma membrane. GFP images of HEK293T cells transiently expressing rat β-arrestin2-EGFP along with one of the following: HA-β2AR, β2AR SPARK TF component (from Figure 1B ), or TM SPARK TF component (TM from CD4, used in Figure 1F ). Live cell GFP images were acquired before and after incubation with 10 μM isoproterenol to activate β2AR. Arrowheads point to regions showing re-localization of β-arrestin2-GFP. Scale bar, 10 μm. ( B ) Additional fields of view for the experiment shown in Figure 1F . Scale bar, 100 μm. ( C ) HEK293T cells were transiently transfected (using PEI max) with the SPARK constructs shown in Figure 1B . 18 hr post-transfection, cells were stimulated with 10 μM isoproterenol and blue light (467 nm, 60 mW/cm 2 , 10% duty cycle) for 5 or 30 min total. Cells were then immediately lysed in the presence of 20 mM iodoacetamide TEVp inhibitor and run on 8% SDS-PAGE. Anti-V5 blot visualizes the SPARK TF component, which is 97 kD before cleavage and 32 kD after cleavage at the TEVcs. Negative controls omit isoproterenol or light. ( D ) HEK293T cells were prepared as in Figure 1D . 15 hr post-transfection, cells were stimulated with 5 min of either ambient room light or blue LED light (467 nm, 60 mW/cm 2 , 10% duty cycle) concurrently with 10 μM isoproterenol. Nine hours later, cells were analyzed for luciferse activity. Each condition was replicated four times.
    Figure Legend Snippet: Further characterization of SPARK tool. ( A ) HA-β2AR construct recruits β-arrestin2 -EGFP to the plasma membrane. GFP images of HEK293T cells transiently expressing rat β-arrestin2-EGFP along with one of the following: HA-β2AR, β2AR SPARK TF component (from Figure 1B ), or TM SPARK TF component (TM from CD4, used in Figure 1F ). Live cell GFP images were acquired before and after incubation with 10 μM isoproterenol to activate β2AR. Arrowheads point to regions showing re-localization of β-arrestin2-GFP. Scale bar, 10 μm. ( B ) Additional fields of view for the experiment shown in Figure 1F . Scale bar, 100 μm. ( C ) HEK293T cells were transiently transfected (using PEI max) with the SPARK constructs shown in Figure 1B . 18 hr post-transfection, cells were stimulated with 10 μM isoproterenol and blue light (467 nm, 60 mW/cm 2 , 10% duty cycle) for 5 or 30 min total. Cells were then immediately lysed in the presence of 20 mM iodoacetamide TEVp inhibitor and run on 8% SDS-PAGE. Anti-V5 blot visualizes the SPARK TF component, which is 97 kD before cleavage and 32 kD after cleavage at the TEVcs. Negative controls omit isoproterenol or light. ( D ) HEK293T cells were prepared as in Figure 1D . 15 hr post-transfection, cells were stimulated with 5 min of either ambient room light or blue LED light (467 nm, 60 mW/cm 2 , 10% duty cycle) concurrently with 10 μM isoproterenol. Nine hours later, cells were analyzed for luciferse activity. Each condition was replicated four times.

    Techniques Used: Construct, Expressing, Incubation, Transfection, SDS Page, Activity Assay

    40) Product Images from "Persulfide Dioxygenase From Acidithiobacillus caldus: Variable Roles of Cysteine Residues and Hydrogen Bond Networks of the Active Site"

    Article Title: Persulfide Dioxygenase From Acidithiobacillus caldus: Variable Roles of Cysteine Residues and Hydrogen Bond Networks of the Active Site

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.01610

    Effect of Inhibitors on the Ac PDO. (A) Residual PDO activity with 1 mM GSH, sulfur and N -Ethylmaleimide (NEM) or iodoacetamide (IAA) after adding the substance directly to the enzyme assay mixture. (B) Same as in A only that the GSH concentration was varied. (C) Residual PDO activity after pre-incubation of the Ac PDO with NEM, IAA, or Tris(2-carboxyethyl)phosphine (TCEP). Error bars represent the standard deviation from triplicate measurements.
    Figure Legend Snippet: Effect of Inhibitors on the Ac PDO. (A) Residual PDO activity with 1 mM GSH, sulfur and N -Ethylmaleimide (NEM) or iodoacetamide (IAA) after adding the substance directly to the enzyme assay mixture. (B) Same as in A only that the GSH concentration was varied. (C) Residual PDO activity after pre-incubation of the Ac PDO with NEM, IAA, or Tris(2-carboxyethyl)phosphine (TCEP). Error bars represent the standard deviation from triplicate measurements.

    Techniques Used: Activity Assay, Enzymatic Assay, Concentration Assay, Incubation, Standard Deviation

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    Protease Inhibitor:

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    Article Snippet: .. All cultures were harvested by the addition of 1 ml ice-cold sterile double-distilled water (ddH2 O) containing 150 μM iodoacetamide (IAM) (Sigma) and protease inhibitor cocktail (Roche, Indianapolis, IN) and incubation on ice for 5 min. Material was transferred to a microcentrifuge tube and trichloroacetate (TCA) precipitated overnight at 4°C before being processed for immunoblotting using SDS-PAGE buffer without beta-mercaptoethanol (β-ME). .. For ampicillin treatment, cultures were supplemented with a final concentration of 20 μg/ml ampicillin dissolved in ddH2 O at 15 h postinfection (p.i.) and harvested 7 h later as described above.

    Labeling:

    Article Title: Intra- and Intermolecular Disulfide Bonds of the GP2b Glycoprotein of Equine Arteritis Virus: Relevance for Virus Assembly and Infectivity
    Article Snippet: .. After the labeling period, the cells were put on ice and washed with ice-cold PBS containing 50 mM CaCl2 , 50 mM MgCl2 , and, to block reactive thiol groups, 20 mM N -ethylmaleimide (NEM; Sigma-Aldrich) or 50 mM iodoacetamide (IAM; Sigma-Aldrich) as indicated in the figure legends. .. Next, the cells were lysed in ice-cold lysis buffer (20 mM Tris-HCl [pH 7.6], 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS]) supplemented with 1 μg each of aprotinin, leupeptin, and pepstatin A per ml and containing either 20 mM NEM or 50 mM IAM.

    Concentration Assay:

    Article Title: Deletion of the acid-sensing ion channel ASIC3 prevents gastritis-induced acid hyperresponsiveness of the stomach – brainstem axis
    Article Snippet: .. To induce gastritis, iodoacetamide (Sigma, Vienna, Austria) was added to the drinking water at a concentration of 0.1 % (w/w) for 7 days ( ; ). ..

    Article Title: Cysteine Oxidation Regulates the RNA-Binding Activity of Iron Regulatory Protein 2 ▿Cysteine Oxidation Regulates the RNA-Binding Activity of Iron Regulatory Protein 2 ▿ †
    Article Snippet: .. Samples were incubated with diamide (Sigma), N -ethylmaleimide (NEM; Sigma), or iodoacetamide (IAM; Sigma) for 10 min at 4°C prior to or after the addition of the 32 P-labeled IRE probe. β-ME (Sigma) was added to lysates at a final concentration of 1% or 0.5% to activate latent IRP1 or IRP2, respectively, prior to the addition of the 32 P-labeled IRE ( ). ..

    Article Title: Disulfide Bonding among ?1 Trimers in Mammalian Reovirus Outer Capsid: a Late and Reversible Step in Virion Morphogenesis
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    Incubation:

    Article Title: Disulfide Bonding within Components of the Chlamydia Type III Secretion Apparatus Correlates with Development ▿
    Article Snippet: .. All cultures were harvested by the addition of 1 ml ice-cold sterile double-distilled water (ddH2 O) containing 150 μM iodoacetamide (IAM) (Sigma) and protease inhibitor cocktail (Roche, Indianapolis, IN) and incubation on ice for 5 min. Material was transferred to a microcentrifuge tube and trichloroacetate (TCA) precipitated overnight at 4°C before being processed for immunoblotting using SDS-PAGE buffer without beta-mercaptoethanol (β-ME). .. For ampicillin treatment, cultures were supplemented with a final concentration of 20 μg/ml ampicillin dissolved in ddH2 O at 15 h postinfection (p.i.) and harvested 7 h later as described above.

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    Article Snippet: .. Samples were incubated with diamide (Sigma), N -ethylmaleimide (NEM; Sigma), or iodoacetamide (IAM; Sigma) for 10 min at 4°C prior to or after the addition of the 32 P-labeled IRE probe. β-ME (Sigma) was added to lysates at a final concentration of 1% or 0.5% to activate latent IRP1 or IRP2, respectively, prior to the addition of the 32 P-labeled IRE ( ). ..

    other:

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    Blocking Assay:

    Article Title: Intra- and Intermolecular Disulfide Bonds of the GP2b Glycoprotein of Equine Arteritis Virus: Relevance for Virus Assembly and Infectivity
    Article Snippet: .. After the labeling period, the cells were put on ice and washed with ice-cold PBS containing 50 mM CaCl2 , 50 mM MgCl2 , and, to block reactive thiol groups, 20 mM N -ethylmaleimide (NEM; Sigma-Aldrich) or 50 mM iodoacetamide (IAM; Sigma-Aldrich) as indicated in the figure legends. .. Next, the cells were lysed in ice-cold lysis buffer (20 mM Tris-HCl [pH 7.6], 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS]) supplemented with 1 μg each of aprotinin, leupeptin, and pepstatin A per ml and containing either 20 mM NEM or 50 mM IAM.

    SDS Page:

    Article Title: Disulfide Bonding within Components of the Chlamydia Type III Secretion Apparatus Correlates with Development ▿
    Article Snippet: .. All cultures were harvested by the addition of 1 ml ice-cold sterile double-distilled water (ddH2 O) containing 150 μM iodoacetamide (IAM) (Sigma) and protease inhibitor cocktail (Roche, Indianapolis, IN) and incubation on ice for 5 min. Material was transferred to a microcentrifuge tube and trichloroacetate (TCA) precipitated overnight at 4°C before being processed for immunoblotting using SDS-PAGE buffer without beta-mercaptoethanol (β-ME). .. For ampicillin treatment, cultures were supplemented with a final concentration of 20 μg/ml ampicillin dissolved in ddH2 O at 15 h postinfection (p.i.) and harvested 7 h later as described above.

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  • 99
    Millipore iodoacetamide
    Interaction of the bifunctional reagent DBB with H-Ras. (A) Modification of the K170-K185 peptide by DBB. The K170-K185 peptide was incubated with DBB, as described in the Methods section. When indicated the incubation mixture was subjected to reduction and alkylation with <t>iodoacetamide</t> to modify free cysteines. Results are representative of three independent assays. (B) Scheme of the cross-linking of two thiol groups by DBB. (C) Competition of 15d-PGJ 2 -B binding to H-Ras by DBB. H-Ras at 5 µM was incubated with 50 µM DBB for 30 min before addition of 1 µM 15d-PGJ 2 -B for 1 h. Incorporation of the biotinylated PG was assessed by SDS-PAGE, protein blot and detection with horseradish peroxidase (HRP)-streptavidin (upper panel). Incorporation of DBB (middle panel) and total H-Ras levels (lower panel) were assessed by fluorescence detection and western blot with anti-pan Ras antibody, respectively. (D) COS-7 cells transfected with the AU5-H-Ras vector were treated in the absence or presence of 50 µM DBB for 1 h. Aliquots from total cell lysates (10 µg of protein) were analyzed by SDS-PAGE. The upper component of the AU5-H-Ras doublet is indicated by an arrowhead. (E) COS-7 cells transfected with the AU5-H-Ras vector were treated with DBB and subjected to fractionation in Triton-X114 as above. In (D) and (E) levels of AU5-H-Ras were assessed by western blot with anti-AU5 antibody. The dotted line marks where lanes from the same gel have been cropped. (F) COS-7 cells transfected with YFP-RBD as above were treated with 50 µM DBB for 1 h before stimulation with 100 nM EGF, as indicated, and visualized live by confocal fluorescence microscopy. Dotted inserts show enlarged areas from the same cells for better visualization. Arrowheads mark the accumulation of YFP-RBD at defined locations of the cell periphery. Bar, 20 µm. Shown are representative images from three experiments with similar results.
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    ClpA–ClpP substrate degradation. ( A ) Top, SDS-PAGE assay of the kinetics of λ cI N -ssrA degradation by the uncrosslinked A•P control (containing equivalent concentrations of E613C ClpA ‡ and ClpP +C to the crosslinked pool) and the A–P pool Bottom, quantification of λ cI-ssrA degradation. Values are means ± 1 SD (n ≥ 3). ( B ) Degradation of substrates of varying thermodynamic stability (18µM FITC-casein, 5µM <t>5-IAF</t> titin V13P -ssrA, 20µM cp7 GFP-ssrA, 15 µM λ cl N -ssrA by the purified A–P pool. Fraction crosslinked activity was calculated by normalizing to the activity of the A•P sample. Values are means ± 1 SD (n ≥ 3). ( C ) Degradation of FITC-casein (18 µM) by the purified A–P pool in the presence of ATP or ATPγS. FITC-casein degraded was quantified by normalizing the relative fluorescence units to the total FITC-casein degraded upon porcine elastase addition at the endpoint of the assay and subtracting the contributions of photobleaching from the buffer-only control. Values are means ± 1 SD (n ≥ 3). The inset shows representative kinetics of FITC-casein degradation assay. ( D ) Michaelis-Menten analysis of cp7 GFP-ssrA degradation kinetics by the A–P pool and A•P control. Values are means ± 1 SD (n ≥ 3). For the A–P pool, Vmax was 1.4 ± 0.07 min -1 ClpA6 -1 , K M was 13 ± 1.6 µM, and R 2 was 0.96; for the A•P control, the Vmax was 3.0 ± 0.10 min -1 ClpA6 -1 , K M was 3.7 ± 0.5 µM, and R 2 was 0.96, where the errors are those of non-linear least-squares fitting to the Michaelis-Menten equation.
    5 Iodoacetamido Fluorescein, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Interaction of the bifunctional reagent DBB with H-Ras. (A) Modification of the K170-K185 peptide by DBB. The K170-K185 peptide was incubated with DBB, as described in the Methods section. When indicated the incubation mixture was subjected to reduction and alkylation with iodoacetamide to modify free cysteines. Results are representative of three independent assays. (B) Scheme of the cross-linking of two thiol groups by DBB. (C) Competition of 15d-PGJ 2 -B binding to H-Ras by DBB. H-Ras at 5 µM was incubated with 50 µM DBB for 30 min before addition of 1 µM 15d-PGJ 2 -B for 1 h. Incorporation of the biotinylated PG was assessed by SDS-PAGE, protein blot and detection with horseradish peroxidase (HRP)-streptavidin (upper panel). Incorporation of DBB (middle panel) and total H-Ras levels (lower panel) were assessed by fluorescence detection and western blot with anti-pan Ras antibody, respectively. (D) COS-7 cells transfected with the AU5-H-Ras vector were treated in the absence or presence of 50 µM DBB for 1 h. Aliquots from total cell lysates (10 µg of protein) were analyzed by SDS-PAGE. The upper component of the AU5-H-Ras doublet is indicated by an arrowhead. (E) COS-7 cells transfected with the AU5-H-Ras vector were treated with DBB and subjected to fractionation in Triton-X114 as above. In (D) and (E) levels of AU5-H-Ras were assessed by western blot with anti-AU5 antibody. The dotted line marks where lanes from the same gel have been cropped. (F) COS-7 cells transfected with YFP-RBD as above were treated with 50 µM DBB for 1 h before stimulation with 100 nM EGF, as indicated, and visualized live by confocal fluorescence microscopy. Dotted inserts show enlarged areas from the same cells for better visualization. Arrowheads mark the accumulation of YFP-RBD at defined locations of the cell periphery. Bar, 20 µm. Shown are representative images from three experiments with similar results.

    Journal: PLoS ONE

    Article Title: The C-Terminus of H-Ras as a Target for the Covalent Binding of Reactive Compounds Modulating Ras-Dependent Pathways

    doi: 10.1371/journal.pone.0015866

    Figure Lengend Snippet: Interaction of the bifunctional reagent DBB with H-Ras. (A) Modification of the K170-K185 peptide by DBB. The K170-K185 peptide was incubated with DBB, as described in the Methods section. When indicated the incubation mixture was subjected to reduction and alkylation with iodoacetamide to modify free cysteines. Results are representative of three independent assays. (B) Scheme of the cross-linking of two thiol groups by DBB. (C) Competition of 15d-PGJ 2 -B binding to H-Ras by DBB. H-Ras at 5 µM was incubated with 50 µM DBB for 30 min before addition of 1 µM 15d-PGJ 2 -B for 1 h. Incorporation of the biotinylated PG was assessed by SDS-PAGE, protein blot and detection with horseradish peroxidase (HRP)-streptavidin (upper panel). Incorporation of DBB (middle panel) and total H-Ras levels (lower panel) were assessed by fluorescence detection and western blot with anti-pan Ras antibody, respectively. (D) COS-7 cells transfected with the AU5-H-Ras vector were treated in the absence or presence of 50 µM DBB for 1 h. Aliquots from total cell lysates (10 µg of protein) were analyzed by SDS-PAGE. The upper component of the AU5-H-Ras doublet is indicated by an arrowhead. (E) COS-7 cells transfected with the AU5-H-Ras vector were treated with DBB and subjected to fractionation in Triton-X114 as above. In (D) and (E) levels of AU5-H-Ras were assessed by western blot with anti-AU5 antibody. The dotted line marks where lanes from the same gel have been cropped. (F) COS-7 cells transfected with YFP-RBD as above were treated with 50 µM DBB for 1 h before stimulation with 100 nM EGF, as indicated, and visualized live by confocal fluorescence microscopy. Dotted inserts show enlarged areas from the same cells for better visualization. Arrowheads mark the accumulation of YFP-RBD at defined locations of the cell periphery. Bar, 20 µm. Shown are representative images from three experiments with similar results.

    Article Snippet: When indicated, aliquots of H-Ras, tryptic digests or K170-K185 peptide were subsequently incubated with 10 mM DTT for 30 min followed by 50 mM iodoacetamide for 30 min at r.t. before purification on ZipTip C18 (Millipore) and MALDI-TOF MS analysis on an AUTOFLEX III MALDI-TOF-TOF instrument (Bruker-Franzen Analytik, Bremen, FRG) operated in the positive mode, as reported in detail .

    Techniques: Modification, Incubation, Binding Assay, SDS Page, Fluorescence, Western Blot, Transfection, Plasmid Preparation, Fractionation, Microscopy

    Effect of reducing agents on PAO modification of the K170-K185 peptide. (A) In the upper panels, the K170-K185 peptide was incubated with PAO and analyzed by MALDI-TOF MS. In the right panel, the incubation mixture was treated with 10 mM DTT and subsequently with 50 mM iodoacetamide. In the lower panels, the K170-K185 peptide was incubated with PAO in the presence of the indicated concentrations of GSH before analysis by MALDI-TOF MS. (B) Summary of the theoretical m/z of peptides for which compatible peaks were observed experimentally in the assays shown in (A).

    Journal: PLoS ONE

    Article Title: The C-Terminus of H-Ras as a Target for the Covalent Binding of Reactive Compounds Modulating Ras-Dependent Pathways

    doi: 10.1371/journal.pone.0015866

    Figure Lengend Snippet: Effect of reducing agents on PAO modification of the K170-K185 peptide. (A) In the upper panels, the K170-K185 peptide was incubated with PAO and analyzed by MALDI-TOF MS. In the right panel, the incubation mixture was treated with 10 mM DTT and subsequently with 50 mM iodoacetamide. In the lower panels, the K170-K185 peptide was incubated with PAO in the presence of the indicated concentrations of GSH before analysis by MALDI-TOF MS. (B) Summary of the theoretical m/z of peptides for which compatible peaks were observed experimentally in the assays shown in (A).

    Article Snippet: When indicated, aliquots of H-Ras, tryptic digests or K170-K185 peptide were subsequently incubated with 10 mM DTT for 30 min followed by 50 mM iodoacetamide for 30 min at r.t. before purification on ZipTip C18 (Millipore) and MALDI-TOF MS analysis on an AUTOFLEX III MALDI-TOF-TOF instrument (Bruker-Franzen Analytik, Bremen, FRG) operated in the positive mode, as reported in detail .

    Techniques: Modification, Incubation, Mass Spectrometry

    Modification of the K170-K185 peptide from the C-terminal region of H-Ras by cyPG. (A) The synthetic K170-K185 peptide was incubated with the indicated cyPG and the resulting adducts analyzed by MALDI-TOF MS. When indicated, incubation mixtures were treated with 50 mM iodoacetamide after addition of 10 mM DTT. Spectra presented are representative from at least three independent assays per experimental condition. Structures of the cyPG used are shown in insets. Electrophilic carbons are marked by asterisks. (B) Summary of the theoretical peptide adducts for which compatible peaks were observed experimentally. +CAM indicates that the peak is compatible with the formation of carbamidomethyl cysteine subsequent to iodoacetamide treatment. (C) Ribbon diagram for the theoretical backbone structure of the H-Ras K170-K185 peptide (in green) modified by addition of 15d-PGJ 2 to cysteines 181 and 184. The side-chains of the cysteine residues and the 15d-PGJ 2 ligand are displayed in yellow and a default atom-type color scheme, respectively.

    Journal: PLoS ONE

    Article Title: The C-Terminus of H-Ras as a Target for the Covalent Binding of Reactive Compounds Modulating Ras-Dependent Pathways

    doi: 10.1371/journal.pone.0015866

    Figure Lengend Snippet: Modification of the K170-K185 peptide from the C-terminal region of H-Ras by cyPG. (A) The synthetic K170-K185 peptide was incubated with the indicated cyPG and the resulting adducts analyzed by MALDI-TOF MS. When indicated, incubation mixtures were treated with 50 mM iodoacetamide after addition of 10 mM DTT. Spectra presented are representative from at least three independent assays per experimental condition. Structures of the cyPG used are shown in insets. Electrophilic carbons are marked by asterisks. (B) Summary of the theoretical peptide adducts for which compatible peaks were observed experimentally. +CAM indicates that the peak is compatible with the formation of carbamidomethyl cysteine subsequent to iodoacetamide treatment. (C) Ribbon diagram for the theoretical backbone structure of the H-Ras K170-K185 peptide (in green) modified by addition of 15d-PGJ 2 to cysteines 181 and 184. The side-chains of the cysteine residues and the 15d-PGJ 2 ligand are displayed in yellow and a default atom-type color scheme, respectively.

    Article Snippet: When indicated, aliquots of H-Ras, tryptic digests or K170-K185 peptide were subsequently incubated with 10 mM DTT for 30 min followed by 50 mM iodoacetamide for 30 min at r.t. before purification on ZipTip C18 (Millipore) and MALDI-TOF MS analysis on an AUTOFLEX III MALDI-TOF-TOF instrument (Bruker-Franzen Analytik, Bremen, FRG) operated in the positive mode, as reported in detail .

    Techniques: Modification, Incubation, Mass Spectrometry, Chick Chorioallantoic Membrane Assay

    Engineered cysteines C251 and C929 form a disulfide bond in NPC1. (A) Extracted ion chromatograms from LC-MS analysis of synthetic peptides corresponding to engineered cysteines in NPC1. In both samples, blue traces represent m/z = 526.2569 (corresponding to carbamidomethylated CQPPPPPMK from NPC1 P251C), red traces represent m/z = 943.3462 (corresponding to carbamidomethylated NAAECDTY from NPC1 L929C), and green traces represent m/z = 626.6005 (corresponding to the disulfide formed between CQPPPPPMK and NAAECDTY). The peptide standards CQPPPPPMK and NAAECDTY were produced by solid phase synthesis and either reduced and carbamidomethylated or oxidized to the disulfide using Ellman’s reagent. (B) Extracted ion chromatograms from LC-MS analysis of proteolyzed NPC1. Colors as in A. NPC1 protein was carbamidomethylated in the presence or absence of reducing agent prior to proteolysis. (C) EThcD mass spectrum from NPC1 sample in part B, demonstrating the reductive fragmentation of the putative disulfide precursor into ions with masses corresponding to the two constituent peptides. Peaks matching the mass of the peptide CQPPPPPMK (monoisotopic mass of thiol = 994.49 Da) are colored in blue; peaks matching the masses of the peptides NAAECDTY (monoisotopic mass of thiol radical = 885.32 Da) are colored in red; peaks matching multiple charge states of the spectrum’s parent ions (m/z = 626.60, z = 3, corresponding to the disulfide) are colored in green. (D) MS1 mass spectra of NPC1 peptides whose disulfide content has been quantified using isotope-labeled iodoacetamide. Free thiols in purified NPC1 were labeled with 13 C 2 D 2 -iodoacetamide (“heavy”). Then the disulfides were reduced and the resulting reactive cysteines were labeled with iodoacetamide lacking isotope labels (“light”) followed by proteolysis and LC-MS analysis. Control samples were labeled with the same reagent before and after reduction (“light/reduce/light” and “heavy/reduce/heavy”) to identify isotope distributions in the limiting cases. Colored peaks fall within 10 ppm of expected masses in the isotope envelope of carbamidomethylated CQPPPPPMK (monoisotopic m/z = 526.2569) or carbamidomethylated NAAECDTY (monoisotopic m/z = 943.3462); black peaks correspond to unrelated ions. Dashed gray lines indicate the expected m/z of the monoisotopic peak and the +4 Da peak of the labeled peptides.

    Journal: bioRxiv

    Article Title: Inter-domain dynamics drive cholesterol transport by NPC1 and NPC1L1 proteins

    doi: 10.1101/2020.03.26.009217

    Figure Lengend Snippet: Engineered cysteines C251 and C929 form a disulfide bond in NPC1. (A) Extracted ion chromatograms from LC-MS analysis of synthetic peptides corresponding to engineered cysteines in NPC1. In both samples, blue traces represent m/z = 526.2569 (corresponding to carbamidomethylated CQPPPPPMK from NPC1 P251C), red traces represent m/z = 943.3462 (corresponding to carbamidomethylated NAAECDTY from NPC1 L929C), and green traces represent m/z = 626.6005 (corresponding to the disulfide formed between CQPPPPPMK and NAAECDTY). The peptide standards CQPPPPPMK and NAAECDTY were produced by solid phase synthesis and either reduced and carbamidomethylated or oxidized to the disulfide using Ellman’s reagent. (B) Extracted ion chromatograms from LC-MS analysis of proteolyzed NPC1. Colors as in A. NPC1 protein was carbamidomethylated in the presence or absence of reducing agent prior to proteolysis. (C) EThcD mass spectrum from NPC1 sample in part B, demonstrating the reductive fragmentation of the putative disulfide precursor into ions with masses corresponding to the two constituent peptides. Peaks matching the mass of the peptide CQPPPPPMK (monoisotopic mass of thiol = 994.49 Da) are colored in blue; peaks matching the masses of the peptides NAAECDTY (monoisotopic mass of thiol radical = 885.32 Da) are colored in red; peaks matching multiple charge states of the spectrum’s parent ions (m/z = 626.60, z = 3, corresponding to the disulfide) are colored in green. (D) MS1 mass spectra of NPC1 peptides whose disulfide content has been quantified using isotope-labeled iodoacetamide. Free thiols in purified NPC1 were labeled with 13 C 2 D 2 -iodoacetamide (“heavy”). Then the disulfides were reduced and the resulting reactive cysteines were labeled with iodoacetamide lacking isotope labels (“light”) followed by proteolysis and LC-MS analysis. Control samples were labeled with the same reagent before and after reduction (“light/reduce/light” and “heavy/reduce/heavy”) to identify isotope distributions in the limiting cases. Colored peaks fall within 10 ppm of expected masses in the isotope envelope of carbamidomethylated CQPPPPPMK (monoisotopic m/z = 526.2569) or carbamidomethylated NAAECDTY (monoisotopic m/z = 943.3462); black peaks correspond to unrelated ions. Dashed gray lines indicate the expected m/z of the monoisotopic peak and the +4 Da peak of the labeled peptides.

    Article Snippet: For reduced standards, peptides were suspended in 180 mM borate pH 8.6 and 90 mM NaCl, reduced with 1 mM TCEP for 5 min, and alkylated with 10 mM iodoacetamide at room temperature in the dark for 45 min, when formic acid was added to 2.5%.

    Techniques: Liquid Chromatography with Mass Spectroscopy, Produced, Labeling, Purification

    Cysteines involved in disulfide bridges, as shown by secondly modified with iodoacetamide. Reflectron MALDI-TOF MS spectrum of one HPLC fraction from the combination of trypsin, Asp-N, and Glu-C triple-enzyme digest of OMC demonstrates modified cysteine

    Journal:

    Article Title: Identification of surface-exposed components of MOMP of Chlamydia trachomatis serovar F

    doi: 10.1110/ps.051616206

    Figure Lengend Snippet: Cysteines involved in disulfide bridges, as shown by secondly modified with iodoacetamide. Reflectron MALDI-TOF MS spectrum of one HPLC fraction from the combination of trypsin, Asp-N, and Glu-C triple-enzyme digest of OMC demonstrates modified cysteine

    Article Snippet: The nonreduced proteolytic OMC digests were pyridylethylated with 1 M VP before RP-HPLC separation, half of each RP-HPLC collected fraction was reduced with 20 mM DTT and carboxymethylated again with 100 mM iodoacetamide (IAM) ( ) in darkness for half an hour before C18 Ziptip (Millipore) cleanup.

    Techniques: Modification, Mass Spectrometry, High Performance Liquid Chromatography

    ClpA–ClpP substrate degradation. ( A ) Top, SDS-PAGE assay of the kinetics of λ cI N -ssrA degradation by the uncrosslinked A•P control (containing equivalent concentrations of E613C ClpA ‡ and ClpP +C to the crosslinked pool) and the A–P pool Bottom, quantification of λ cI-ssrA degradation. Values are means ± 1 SD (n ≥ 3). ( B ) Degradation of substrates of varying thermodynamic stability (18µM FITC-casein, 5µM 5-IAF titin V13P -ssrA, 20µM cp7 GFP-ssrA, 15 µM λ cl N -ssrA by the purified A–P pool. Fraction crosslinked activity was calculated by normalizing to the activity of the A•P sample. Values are means ± 1 SD (n ≥ 3). ( C ) Degradation of FITC-casein (18 µM) by the purified A–P pool in the presence of ATP or ATPγS. FITC-casein degraded was quantified by normalizing the relative fluorescence units to the total FITC-casein degraded upon porcine elastase addition at the endpoint of the assay and subtracting the contributions of photobleaching from the buffer-only control. Values are means ± 1 SD (n ≥ 3). The inset shows representative kinetics of FITC-casein degradation assay. ( D ) Michaelis-Menten analysis of cp7 GFP-ssrA degradation kinetics by the A–P pool and A•P control. Values are means ± 1 SD (n ≥ 3). For the A–P pool, Vmax was 1.4 ± 0.07 min -1 ClpA6 -1 , K M was 13 ± 1.6 µM, and R 2 was 0.96; for the A•P control, the Vmax was 3.0 ± 0.10 min -1 ClpA6 -1 , K M was 3.7 ± 0.5 µM, and R 2 was 0.96, where the errors are those of non-linear least-squares fitting to the Michaelis-Menten equation.

    Journal: bioRxiv

    Article Title: ClpAP proteolysis does not require rotation of the ClpA unfoldase relative to ClpP

    doi: 10.1101/2020.07.26.221812

    Figure Lengend Snippet: ClpA–ClpP substrate degradation. ( A ) Top, SDS-PAGE assay of the kinetics of λ cI N -ssrA degradation by the uncrosslinked A•P control (containing equivalent concentrations of E613C ClpA ‡ and ClpP +C to the crosslinked pool) and the A–P pool Bottom, quantification of λ cI-ssrA degradation. Values are means ± 1 SD (n ≥ 3). ( B ) Degradation of substrates of varying thermodynamic stability (18µM FITC-casein, 5µM 5-IAF titin V13P -ssrA, 20µM cp7 GFP-ssrA, 15 µM λ cl N -ssrA by the purified A–P pool. Fraction crosslinked activity was calculated by normalizing to the activity of the A•P sample. Values are means ± 1 SD (n ≥ 3). ( C ) Degradation of FITC-casein (18 µM) by the purified A–P pool in the presence of ATP or ATPγS. FITC-casein degraded was quantified by normalizing the relative fluorescence units to the total FITC-casein degraded upon porcine elastase addition at the endpoint of the assay and subtracting the contributions of photobleaching from the buffer-only control. Values are means ± 1 SD (n ≥ 3). The inset shows representative kinetics of FITC-casein degradation assay. ( D ) Michaelis-Menten analysis of cp7 GFP-ssrA degradation kinetics by the A–P pool and A•P control. Values are means ± 1 SD (n ≥ 3). For the A–P pool, Vmax was 1.4 ± 0.07 min -1 ClpA6 -1 , K M was 13 ± 1.6 µM, and R 2 was 0.96; for the A•P control, the Vmax was 3.0 ± 0.10 min -1 ClpA6 -1 , K M was 3.7 ± 0.5 µM, and R 2 was 0.96, where the errors are those of non-linear least-squares fitting to the Michaelis-Menten equation.

    Article Snippet: V13P titinI27 -ssrA was labeled with 5-iodoacetamidofluorescein (5-IAF) for fluorescent assays by an established protocol ( ).

    Techniques: SDS Page, Purification, Activity Assay, Fluorescence, Degradation Assay