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  • 99
    ATCC b cereus atcc 14579
    Overview of the glucose catabolic pathways utilized by B. cereus <t>ATCC</t> 14579 . The proteins detected in this study are indicated by their BC numbers. Protein names and functions are listed in Table S3 . The form filling indicates fold-change values that satisfied the Student's t -test statistical criteria ( p
    B Cereus Atcc 14579, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 2802 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore iodoacetamide
    LC-UV chromatograms of IgG alkylated with varying <t>IAM</t> concentrations. PS 8670 was reduced with 5 mmol/L DTT at 4 °C for 1 h, followed by alkylation with ( a ) 0 mmol/L; ( b ) 5 mmol/L; ( c ) 7.5 mmol/L; ( d ) 10 mmol/L; ( e ) 12.5 mmol/L; or ( f
    Iodoacetamide, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 12778 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher iodoacetamide
    LC-UV chromatograms of IgG alkylated with varying <t>IAM</t> concentrations. PS 8670 was reduced with 5 mmol/L DTT at 4 °C for 1 h, followed by alkylation with ( a ) 0 mmol/L; ( b ) 5 mmol/L; ( c ) 7.5 mmol/L; ( d ) 10 mmol/L; ( e ) 12.5 mmol/L; or ( f
    Iodoacetamide, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2144 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad iodoacetamide
    LC-UV chromatograms of IgG alkylated with varying <t>IAM</t> concentrations. PS 8670 was reduced with 5 mmol/L DTT at 4 °C for 1 h, followed by alkylation with ( a ) 0 mmol/L; ( b ) 5 mmol/L; ( c ) 7.5 mmol/L; ( d ) 10 mmol/L; ( e ) 12.5 mmol/L; or ( f
    Iodoacetamide, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 1118 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC s aureus atcc 12600
    LC-UV chromatograms of IgG alkylated with varying <t>IAM</t> concentrations. PS 8670 was reduced with 5 mmol/L DTT at 4 °C for 1 h, followed by alkylation with ( a ) 0 mmol/L; ( b ) 5 mmol/L; ( c ) 7.5 mmol/L; ( d ) 10 mmol/L; ( e ) 12.5 mmol/L; or ( f
    S Aureus Atcc 12600, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 737 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher n 1 pyrene iodoacetamide
    LC-UV chromatograms of IgG alkylated with varying <t>IAM</t> concentrations. PS 8670 was reduced with 5 mmol/L DTT at 4 °C for 1 h, followed by alkylation with ( a ) 0 mmol/L; ( b ) 5 mmol/L; ( c ) 7.5 mmol/L; ( d ) 10 mmol/L; ( e ) 12.5 mmol/L; or ( f
    N 1 Pyrene Iodoacetamide, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Promega iodoacetamide
    LC-UV chromatograms of IgG alkylated with varying <t>IAM</t> concentrations. PS 8670 was reduced with 5 mmol/L DTT at 4 °C for 1 h, followed by alkylation with ( a ) 0 mmol/L; ( b ) 5 mmol/L; ( c ) 7.5 mmol/L; ( d ) 10 mmol/L; ( e ) 12.5 mmol/L; or ( f
    Iodoacetamide, supplied by Promega, used in various techniques. Bioz Stars score: 94/100, based on 6637 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    ATCC bulgaricus atcc 11842
    LC-UV chromatograms of IgG alkylated with varying <t>IAM</t> concentrations. PS 8670 was reduced with 5 mmol/L DTT at 4 °C for 1 h, followed by alkylation with ( a ) 0 mmol/L; ( b ) 5 mmol/L; ( c ) 7.5 mmol/L; ( d ) 10 mmol/L; ( e ) 12.5 mmol/L; or ( f
    Bulgaricus Atcc 11842, supplied by ATCC, used in various techniques. Bioz Stars score: 97/100, based on 247 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    ATCC bacillus subtilis
    Concentration dependent assay of  T. indica  fruit pulp on  Bacillus subtilis  ATCC 6051.
    Bacillus Subtilis, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 2060 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    FUJIFILM iodoacetamide
    Persulfidation of the peroxidative cysteine of Prx2 is modulated by the Trx system. ( A to F ) WT and TrxR1 KD HEK293 cells were exposed to 600 μM H 2 O 2 in Hanks’ balanced salt solution buffer at 37°C for 5 min and lysed in radioimmunoprecipitation assay (RIPA) buffer without SDS containing 5 mM <t>iodoacetamide.</t> The lysates were immunoprecipitated with anti-PRDX2 antibody. The eluted protein was subjected to tryptic digest in the presence of 5 mM iodoacetamide. ( G to N ) Recombinant human Prx2 (hPrx2) (12 μM) was incubated with or without 100 μM Na 2 S 2 for 10 min, followed by 1 mM H 2 O 2 for 10 min at 25°C. Samples were desalted and treated with or without TrxR1 (250 nM), TRP14 (4 μM), and NADPH (1 mM) for 1 hour at 37°C. The samples were alkylated with 1 mM iodoacetamide for 30 min, followed by tryptic digestion (37°C, 10 hours). Following LC–quadrupole time-of-flight (Q-TOF)–MS analyses, Mascot search refined the active-site Cys 51 -containing tryptic peptide (see at the top). Cys modifications were identified as shown in the panel headlines. The obtained intensities for modified peptides were normalized to the 93-EGGLGPLNIPLLADVTR-109 peptide fragment of Prx2 from the corresponding tryptic digest. Intensity values are plotted as a relative value compared to the CysSH peptide form from the untreated WT cells (A to F) or recombinant Prx2 (G to N). Data points and error bars represent means ± SD of n = 3 (A to F) and n = 9 experiments (G to N). * P
    Iodoacetamide, supplied by FUJIFILM, used in various techniques. Bioz Stars score: 92/100, based on 300 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    ATCC l plantarum atcc 14917
    Persulfidation of the peroxidative cysteine of Prx2 is modulated by the Trx system. ( A to F ) WT and TrxR1 KD HEK293 cells were exposed to 600 μM H 2 O 2 in Hanks’ balanced salt solution buffer at 37°C for 5 min and lysed in radioimmunoprecipitation assay (RIPA) buffer without SDS containing 5 mM <t>iodoacetamide.</t> The lysates were immunoprecipitated with anti-PRDX2 antibody. The eluted protein was subjected to tryptic digest in the presence of 5 mM iodoacetamide. ( G to N ) Recombinant human Prx2 (hPrx2) (12 μM) was incubated with or without 100 μM Na 2 S 2 for 10 min, followed by 1 mM H 2 O 2 for 10 min at 25°C. Samples were desalted and treated with or without TrxR1 (250 nM), TRP14 (4 μM), and NADPH (1 mM) for 1 hour at 37°C. The samples were alkylated with 1 mM iodoacetamide for 30 min, followed by tryptic digestion (37°C, 10 hours). Following LC–quadrupole time-of-flight (Q-TOF)–MS analyses, Mascot search refined the active-site Cys 51 -containing tryptic peptide (see at the top). Cys modifications were identified as shown in the panel headlines. The obtained intensities for modified peptides were normalized to the 93-EGGLGPLNIPLLADVTR-109 peptide fragment of Prx2 from the corresponding tryptic digest. Intensity values are plotted as a relative value compared to the CysSH peptide form from the untreated WT cells (A to F) or recombinant Prx2 (G to N). Data points and error bars represent means ± SD of n = 3 (A to F) and n = 9 experiments (G to N). * P
    L Plantarum Atcc 14917, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 206 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher 5 iodoacetamido fluorescein
    Persulfidation of the peroxidative cysteine of Prx2 is modulated by the Trx system. ( A to F ) WT and TrxR1 KD HEK293 cells were exposed to 600 μM H 2 O 2 in Hanks’ balanced salt solution buffer at 37°C for 5 min and lysed in radioimmunoprecipitation assay (RIPA) buffer without SDS containing 5 mM <t>iodoacetamide.</t> The lysates were immunoprecipitated with anti-PRDX2 antibody. The eluted protein was subjected to tryptic digest in the presence of 5 mM iodoacetamide. ( G to N ) Recombinant human Prx2 (hPrx2) (12 μM) was incubated with or without 100 μM Na 2 S 2 for 10 min, followed by 1 mM H 2 O 2 for 10 min at 25°C. Samples were desalted and treated with or without TrxR1 (250 nM), TRP14 (4 μM), and NADPH (1 mM) for 1 hour at 37°C. The samples were alkylated with 1 mM iodoacetamide for 30 min, followed by tryptic digestion (37°C, 10 hours). Following LC–quadrupole time-of-flight (Q-TOF)–MS analyses, Mascot search refined the active-site Cys 51 -containing tryptic peptide (see at the top). Cys modifications were identified as shown in the panel headlines. The obtained intensities for modified peptides were normalized to the 93-EGGLGPLNIPLLADVTR-109 peptide fragment of Prx2 from the corresponding tryptic digest. Intensity values are plotted as a relative value compared to the CysSH peptide form from the untreated WT cells (A to F) or recombinant Prx2 (G to N). Data points and error bars represent means ± SD of n = 3 (A to F) and n = 9 experiments (G to N). * P
    5 Iodoacetamido Fluorescein, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 64 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    ATCC bacillus megaterium atcc 14581
    Persulfidation of the peroxidative cysteine of Prx2 is modulated by the Trx system. ( A to F ) WT and TrxR1 KD HEK293 cells were exposed to 600 μM H 2 O 2 in Hanks’ balanced salt solution buffer at 37°C for 5 min and lysed in radioimmunoprecipitation assay (RIPA) buffer without SDS containing 5 mM <t>iodoacetamide.</t> The lysates were immunoprecipitated with anti-PRDX2 antibody. The eluted protein was subjected to tryptic digest in the presence of 5 mM iodoacetamide. ( G to N ) Recombinant human Prx2 (hPrx2) (12 μM) was incubated with or without 100 μM Na 2 S 2 for 10 min, followed by 1 mM H 2 O 2 for 10 min at 25°C. Samples were desalted and treated with or without TrxR1 (250 nM), TRP14 (4 μM), and NADPH (1 mM) for 1 hour at 37°C. The samples were alkylated with 1 mM iodoacetamide for 30 min, followed by tryptic digestion (37°C, 10 hours). Following LC–quadrupole time-of-flight (Q-TOF)–MS analyses, Mascot search refined the active-site Cys 51 -containing tryptic peptide (see at the top). Cys modifications were identified as shown in the panel headlines. The obtained intensities for modified peptides were normalized to the 93-EGGLGPLNIPLLADVTR-109 peptide fragment of Prx2 from the corresponding tryptic digest. Intensity values are plotted as a relative value compared to the CysSH peptide form from the untreated WT cells (A to F) or recombinant Prx2 (G to N). Data points and error bars represent means ± SD of n = 3 (A to F) and n = 9 experiments (G to N). * P
    Bacillus Megaterium Atcc 14581, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 121 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Overview of the glucose catabolic pathways utilized by B. cereus ATCC 14579 . The proteins detected in this study are indicated by their BC numbers. Protein names and functions are listed in Table S3 . The form filling indicates fold-change values that satisfied the Student's t -test statistical criteria ( p

    Journal: Frontiers in Microbiology

    Article Title: Proteomics identifies Bacillus cereus EntD as a pivotal protein for the production of numerous virulence factors

    doi: 10.3389/fmicb.2015.01004

    Figure Lengend Snippet: Overview of the glucose catabolic pathways utilized by B. cereus ATCC 14579 . The proteins detected in this study are indicated by their BC numbers. Protein names and functions are listed in Table S3 . The form filling indicates fold-change values that satisfied the Student's t -test statistical criteria ( p

    Article Snippet: The aim of this study was to evaluate the role of EntD, an exoprotein identified by proteogenomics of B. cereus ATCC 14579 (Ivanova et al., ), in the virulence of B. cereus ATCC 14579 by in-depth characterization of an entD knockout mutant.

    Techniques:

    Identification of the  entD  gene, based on peptide mapping to the BC_3716 locus .  (A)  Genetic organization of the BC_3716 chromosomal region of  B. cereus  ATCC 14579. Large arrows represent the open reading frame identified in strain 14579, and their orientation shows the transcriptional direction. Only the names of ORF-encoding proteins with predicted metabolic functions are indicated.  (B)  Amino acid sequence of the  entD  product. Peptides detected by nanoLC-MS/MS are in red and boldfaced. The signal peptide is underlined. The SH3 domains located at the N terminus and the 3D domain located at the C terminus are colored in gray.

    Journal: Frontiers in Microbiology

    Article Title: Proteomics identifies Bacillus cereus EntD as a pivotal protein for the production of numerous virulence factors

    doi: 10.3389/fmicb.2015.01004

    Figure Lengend Snippet: Identification of the entD gene, based on peptide mapping to the BC_3716 locus . (A) Genetic organization of the BC_3716 chromosomal region of B. cereus ATCC 14579. Large arrows represent the open reading frame identified in strain 14579, and their orientation shows the transcriptional direction. Only the names of ORF-encoding proteins with predicted metabolic functions are indicated. (B) Amino acid sequence of the entD product. Peptides detected by nanoLC-MS/MS are in red and boldfaced. The signal peptide is underlined. The SH3 domains located at the N terminus and the 3D domain located at the C terminus are colored in gray.

    Article Snippet: The aim of this study was to evaluate the role of EntD, an exoprotein identified by proteogenomics of B. cereus ATCC 14579 (Ivanova et al., ), in the virulence of B. cereus ATCC 14579 by in-depth characterization of an entD knockout mutant.

    Techniques: Sequencing, Mass Spectrometry

    Colony morphotypes. Colonies of B . cereus ATCC 14579 WT (a), its Δ rpoN mutant derivative (b) and the complemented mutant (Δ rpoN -comp) (c) cultivated on BHI plates for 24 h at 30°C.

    Journal: PLoS ONE

    Article Title: Bacillus cereus ATCC 14579 RpoN (Sigma 54) Is a Pleiotropic Regulator of Growth, Carbohydrate Metabolism, Motility, Biofilm Formation and Toxin Production

    doi: 10.1371/journal.pone.0134872

    Figure Lengend Snippet: Colony morphotypes. Colonies of B . cereus ATCC 14579 WT (a), its Δ rpoN mutant derivative (b) and the complemented mutant (Δ rpoN -comp) (c) cultivated on BHI plates for 24 h at 30°C.

    Article Snippet: Microarray design and data analysis Custom-made array design for B . cereus ATCC 14579 was based on the 8 x 15K platform of Agilent Technologies (GEO accession number GPL9493, 3rd design) and the genome sequence of B . cereus ATCC 14579 (NCBI accession number NC_004722).

    Techniques: Mutagenesis

    Cells of the WT and its Δ rpoN mutant. SEM images of aerobically grown overnight cultures of B . cereus ATCC 14579 WT (a) and its Δ rpoN mutant derivative (b) in BHI at 30°C.

    Journal: PLoS ONE

    Article Title: Bacillus cereus ATCC 14579 RpoN (Sigma 54) Is a Pleiotropic Regulator of Growth, Carbohydrate Metabolism, Motility, Biofilm Formation and Toxin Production

    doi: 10.1371/journal.pone.0134872

    Figure Lengend Snippet: Cells of the WT and its Δ rpoN mutant. SEM images of aerobically grown overnight cultures of B . cereus ATCC 14579 WT (a) and its Δ rpoN mutant derivative (b) in BHI at 30°C.

    Article Snippet: Microarray design and data analysis Custom-made array design for B . cereus ATCC 14579 was based on the 8 x 15K platform of Agilent Technologies (GEO accession number GPL9493, 3rd design) and the genome sequence of B . cereus ATCC 14579 (NCBI accession number NC_004722).

    Techniques: Mutagenesis

    Motility and biofilm formation. Motility, presence of flagella and biofilm formation by B . cereus ATCC 14579 WT, Δ rpoN and Δ rpoN -comp. (a-c) Motility on swimming BHI plates with 0.3% agar after 48 h, (d-f) flagella staining of cells taken from the swimming plate, (g-i) Static biofilm formation on stainless steel coupons partly submerged in BHI after 48 h at 30°C. Biofilms were stained with crystal violet.

    Journal: PLoS ONE

    Article Title: Bacillus cereus ATCC 14579 RpoN (Sigma 54) Is a Pleiotropic Regulator of Growth, Carbohydrate Metabolism, Motility, Biofilm Formation and Toxin Production

    doi: 10.1371/journal.pone.0134872

    Figure Lengend Snippet: Motility and biofilm formation. Motility, presence of flagella and biofilm formation by B . cereus ATCC 14579 WT, Δ rpoN and Δ rpoN -comp. (a-c) Motility on swimming BHI plates with 0.3% agar after 48 h, (d-f) flagella staining of cells taken from the swimming plate, (g-i) Static biofilm formation on stainless steel coupons partly submerged in BHI after 48 h at 30°C. Biofilms were stained with crystal violet.

    Article Snippet: Microarray design and data analysis Custom-made array design for B . cereus ATCC 14579 was based on the 8 x 15K platform of Agilent Technologies (GEO accession number GPL9493, 3rd design) and the genome sequence of B . cereus ATCC 14579 (NCBI accession number NC_004722).

    Techniques: Staining

    Spore formation. Number of spores and total viable counts in BHI formed by B . cereus ATCC 14579 WT, Δ rpoN and Δ rpoN -comp in BHI following aerated growth at 30°C for 48 h. The asterisk indicates significant difference (student’s t test, p

    Journal: PLoS ONE

    Article Title: Bacillus cereus ATCC 14579 RpoN (Sigma 54) Is a Pleiotropic Regulator of Growth, Carbohydrate Metabolism, Motility, Biofilm Formation and Toxin Production

    doi: 10.1371/journal.pone.0134872

    Figure Lengend Snippet: Spore formation. Number of spores and total viable counts in BHI formed by B . cereus ATCC 14579 WT, Δ rpoN and Δ rpoN -comp in BHI following aerated growth at 30°C for 48 h. The asterisk indicates significant difference (student’s t test, p

    Article Snippet: Microarray design and data analysis Custom-made array design for B . cereus ATCC 14579 was based on the 8 x 15K platform of Agilent Technologies (GEO accession number GPL9493, 3rd design) and the genome sequence of B . cereus ATCC 14579 (NCBI accession number NC_004722).

    Techniques:

    Growth. Growth of B . cereus ATCC 14579 WT, Δ rpoN and Δ rpoN -comp in BHI in three conditions with different oxygen availability a) aerated, b) static and c) anaerobic at 30°C.

    Journal: PLoS ONE

    Article Title: Bacillus cereus ATCC 14579 RpoN (Sigma 54) Is a Pleiotropic Regulator of Growth, Carbohydrate Metabolism, Motility, Biofilm Formation and Toxin Production

    doi: 10.1371/journal.pone.0134872

    Figure Lengend Snippet: Growth. Growth of B . cereus ATCC 14579 WT, Δ rpoN and Δ rpoN -comp in BHI in three conditions with different oxygen availability a) aerated, b) static and c) anaerobic at 30°C.

    Article Snippet: Microarray design and data analysis Custom-made array design for B . cereus ATCC 14579 was based on the 8 x 15K platform of Agilent Technologies (GEO accession number GPL9493, 3rd design) and the genome sequence of B . cereus ATCC 14579 (NCBI accession number NC_004722).

    Techniques:

    Growth at low temperature. Growth of B . cereus ATCC 14579 WT, Δ rpoN and Δ rpoN -comp at 12°C. a) Aerated growth in BHI monitored up to 96 h, b) growth on BHI agar plate, picture was taken on day 12.

    Journal: PLoS ONE

    Article Title: Bacillus cereus ATCC 14579 RpoN (Sigma 54) Is a Pleiotropic Regulator of Growth, Carbohydrate Metabolism, Motility, Biofilm Formation and Toxin Production

    doi: 10.1371/journal.pone.0134872

    Figure Lengend Snippet: Growth at low temperature. Growth of B . cereus ATCC 14579 WT, Δ rpoN and Δ rpoN -comp at 12°C. a) Aerated growth in BHI monitored up to 96 h, b) growth on BHI agar plate, picture was taken on day 12.

    Article Snippet: Microarray design and data analysis Custom-made array design for B . cereus ATCC 14579 was based on the 8 x 15K platform of Agilent Technologies (GEO accession number GPL9493, 3rd design) and the genome sequence of B . cereus ATCC 14579 (NCBI accession number NC_004722).

    Techniques:

    LC-UV chromatograms of IgG alkylated with varying IAM concentrations. PS 8670 was reduced with 5 mmol/L DTT at 4 °C for 1 h, followed by alkylation with ( a ) 0 mmol/L; ( b ) 5 mmol/L; ( c ) 7.5 mmol/L; ( d ) 10 mmol/L; ( e ) 12.5 mmol/L; or ( f

    Journal: Analytical and Bioanalytical Chemistry

    Article Title: Development of an LC-MS/MS peptide mapping protocol for the NISTmAb

    doi: 10.1007/s00216-018-0848-6

    Figure Lengend Snippet: LC-UV chromatograms of IgG alkylated with varying IAM concentrations. PS 8670 was reduced with 5 mmol/L DTT at 4 °C for 1 h, followed by alkylation with ( a ) 0 mmol/L; ( b ) 5 mmol/L; ( c ) 7.5 mmol/L; ( d ) 10 mmol/L; ( e ) 12.5 mmol/L; or ( f

    Article Snippet: Guanidine HCl (Cat #RDD001), Tris(hydroxymethyl)aminomethane (Cat #T6066), Tris(hydroxymethyl)aminomethane HCl (Cat #T5941), ethylenediaminetetraacetic acid (EDTA) (Cat #39692), urea (Cat #U0631), acetic acid (Cat #695084), iodoacetamide (IAM) (Cat #A3221), recombinant porcine trypsin expressed in P. pastoris (Cat #03708985001), trypsin purified from bovine pancreas (Cat #TRYPSEQM-RO) and trypsin purified from porcine pancreas (Cat #T6567) were purchased from Sigma Aldrich.

    Techniques:

    Concentration dependent assay of  T. indica  fruit pulp on  Bacillus subtilis  ATCC 6051.

    Journal: International Journal of Molecular Sciences

    Article Title: Assessment of Tamarindus indica Extracts for Antibacterial Activity

    doi: 10.3390/ijms12106385

    Figure Lengend Snippet: Concentration dependent assay of T. indica fruit pulp on Bacillus subtilis ATCC 6051.

    Article Snippet: The minimum inhibitory concentrations (MIC) ranged from 7.81 mg/mL against Bacillus subtilis ATCC 6051 to 31.25 mg/mL against Escherichia coli ATCC 11775; and the minimum bactericidal concentration (MBC) ranged from 125 mg/mL against Pseudomonas aeruginosa ATCC 10145 to 250 mg/mL against Bacillus subtilis ATCC 6051.

    Techniques: Concentration Assay

    Persulfidation of the peroxidative cysteine of Prx2 is modulated by the Trx system. ( A to F ) WT and TrxR1 KD HEK293 cells were exposed to 600 μM H 2 O 2 in Hanks’ balanced salt solution buffer at 37°C for 5 min and lysed in radioimmunoprecipitation assay (RIPA) buffer without SDS containing 5 mM iodoacetamide. The lysates were immunoprecipitated with anti-PRDX2 antibody. The eluted protein was subjected to tryptic digest in the presence of 5 mM iodoacetamide. ( G to N ) Recombinant human Prx2 (hPrx2) (12 μM) was incubated with or without 100 μM Na 2 S 2 for 10 min, followed by 1 mM H 2 O 2 for 10 min at 25°C. Samples were desalted and treated with or without TrxR1 (250 nM), TRP14 (4 μM), and NADPH (1 mM) for 1 hour at 37°C. The samples were alkylated with 1 mM iodoacetamide for 30 min, followed by tryptic digestion (37°C, 10 hours). Following LC–quadrupole time-of-flight (Q-TOF)–MS analyses, Mascot search refined the active-site Cys 51 -containing tryptic peptide (see at the top). Cys modifications were identified as shown in the panel headlines. The obtained intensities for modified peptides were normalized to the 93-EGGLGPLNIPLLADVTR-109 peptide fragment of Prx2 from the corresponding tryptic digest. Intensity values are plotted as a relative value compared to the CysSH peptide form from the untreated WT cells (A to F) or recombinant Prx2 (G to N). Data points and error bars represent means ± SD of n = 3 (A to F) and n = 9 experiments (G to N). * P

    Journal: Science Advances

    Article Title: Control of protein function through oxidation and reduction of persulfidated states

    doi: 10.1126/sciadv.aax8358

    Figure Lengend Snippet: Persulfidation of the peroxidative cysteine of Prx2 is modulated by the Trx system. ( A to F ) WT and TrxR1 KD HEK293 cells were exposed to 600 μM H 2 O 2 in Hanks’ balanced salt solution buffer at 37°C for 5 min and lysed in radioimmunoprecipitation assay (RIPA) buffer without SDS containing 5 mM iodoacetamide. The lysates were immunoprecipitated with anti-PRDX2 antibody. The eluted protein was subjected to tryptic digest in the presence of 5 mM iodoacetamide. ( G to N ) Recombinant human Prx2 (hPrx2) (12 μM) was incubated with or without 100 μM Na 2 S 2 for 10 min, followed by 1 mM H 2 O 2 for 10 min at 25°C. Samples were desalted and treated with or without TrxR1 (250 nM), TRP14 (4 μM), and NADPH (1 mM) for 1 hour at 37°C. The samples were alkylated with 1 mM iodoacetamide for 30 min, followed by tryptic digestion (37°C, 10 hours). Following LC–quadrupole time-of-flight (Q-TOF)–MS analyses, Mascot search refined the active-site Cys 51 -containing tryptic peptide (see at the top). Cys modifications were identified as shown in the panel headlines. The obtained intensities for modified peptides were normalized to the 93-EGGLGPLNIPLLADVTR-109 peptide fragment of Prx2 from the corresponding tryptic digest. Intensity values are plotted as a relative value compared to the CysSH peptide form from the untreated WT cells (A to F) or recombinant Prx2 (G to N). Data points and error bars represent means ± SD of n = 3 (A to F) and n = 9 experiments (G to N). * P

    Article Snippet: After peroxide treatment, cells were washed in PBS three times and lysed in 1 ml of lysis buffer [10 mM tris-HCl (pH 7.4), 150 mM NaCl, 0.1% NP-40, 0.1% sodium deoxycholate, deoxyribonuclease I (10 μg/ml), ribonuclease A (10 μg/ml), and protease inhibitor cocktail] without SDS containing 5 mM iodoacetamide (Fujifilm Wako Pure Chemical Corporation).

    Techniques: Radio Immunoprecipitation, Immunoprecipitation, Recombinant, Incubation, Mass Spectrometry, Modification

    Reversible inhibition of PTP1B and concomitant formation of active-site CysSSO 1-3 H derivatives upon inorganic polysulfide and H 2 O 2 treatment. Recombinant human PTP1B [( A and B ), 13.5 μM; ( C to R ), 20 μM] was incubated with or without 50 μM Na 2 S 2 for 10 min followed by 100 μM H 2 O 2 for 10 min at 25°C. Samples were then treated with or without Trx1 (15 μM), TRP14 (15 μM), or TRP32 (15 μM) with TrxR1 (250 nM) in NADPH [(A and B), 500 μM; (C to R), 1 mM] or 20 mM DTT for 30 min (A and C to J) or 60 min (B and K to R) at 37°C. (A and B) Phosphatase activity of PTP1B was measured in spectrophotometric assays using para -nitrophenyl phosphate (pNPP) as substrate. The control activity (PTP1B stock; last bar to the right) represents the untreated sample. (C to R) Protein samples were alkylated with 1 mM iodoacetamide for 30 min, digested with trypsin (37°C, 10 hours), and analyzed by LC-Q-TOF-MS. Peptides with modified active-site Cys derivatives were determined using Mascot; their levels were normalized to the intensities of the corresponding 157-QLELENLTTQETR-169 tryptic peptides and shown as relative values to the Cys thiol form in nontreated PTP1B set as 1.0. Data values are means ± SD of n = 3 (A to J) or n = 6 (K to R) experiments; * P

    Journal: Science Advances

    Article Title: Control of protein function through oxidation and reduction of persulfidated states

    doi: 10.1126/sciadv.aax8358

    Figure Lengend Snippet: Reversible inhibition of PTP1B and concomitant formation of active-site CysSSO 1-3 H derivatives upon inorganic polysulfide and H 2 O 2 treatment. Recombinant human PTP1B [( A and B ), 13.5 μM; ( C to R ), 20 μM] was incubated with or without 50 μM Na 2 S 2 for 10 min followed by 100 μM H 2 O 2 for 10 min at 25°C. Samples were then treated with or without Trx1 (15 μM), TRP14 (15 μM), or TRP32 (15 μM) with TrxR1 (250 nM) in NADPH [(A and B), 500 μM; (C to R), 1 mM] or 20 mM DTT for 30 min (A and C to J) or 60 min (B and K to R) at 37°C. (A and B) Phosphatase activity of PTP1B was measured in spectrophotometric assays using para -nitrophenyl phosphate (pNPP) as substrate. The control activity (PTP1B stock; last bar to the right) represents the untreated sample. (C to R) Protein samples were alkylated with 1 mM iodoacetamide for 30 min, digested with trypsin (37°C, 10 hours), and analyzed by LC-Q-TOF-MS. Peptides with modified active-site Cys derivatives were determined using Mascot; their levels were normalized to the intensities of the corresponding 157-QLELENLTTQETR-169 tryptic peptides and shown as relative values to the Cys thiol form in nontreated PTP1B set as 1.0. Data values are means ± SD of n = 3 (A to J) or n = 6 (K to R) experiments; * P

    Article Snippet: After peroxide treatment, cells were washed in PBS three times and lysed in 1 ml of lysis buffer [10 mM tris-HCl (pH 7.4), 150 mM NaCl, 0.1% NP-40, 0.1% sodium deoxycholate, deoxyribonuclease I (10 μg/ml), ribonuclease A (10 μg/ml), and protease inhibitor cocktail] without SDS containing 5 mM iodoacetamide (Fujifilm Wako Pure Chemical Corporation).

    Techniques: Inhibition, Recombinant, Incubation, Activity Assay, Mass Spectrometry, Modification