pierce high sensitivity streptavidin horseradish peroxidase  (Thermo Fisher)


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    Structured Review

    Thermo Fisher pierce high sensitivity streptavidin horseradish peroxidase
    Pierce High Sensitivity Streptavidin Horseradish Peroxidase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pierce high sensitivity streptavidin horseradish peroxidase/product/Thermo Fisher
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pierce high sensitivity streptavidin horseradish peroxidase - by Bioz Stars, 2020-03
    86/100 stars

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    Related Articles

    Bicinchoninic Acid Protein Assay:

    Article Title: Synaptic Amyloid-β Oligomers Precede p-Tau and Differentiate High Pathology Control Cases
    Article Snippet: .. Pierce high-sensitivity streptavidin-horseradish peroxidase, Supersignal West Femto substrate, and bicinchoninic acid protein assay kit were from Thermo Scientific, (Rockford, IL). .. 3,3′,5,5′-Tetramethylbenzidine substrate was purchased from Sigma-Aldrich (St. Louis, MO).

    Labeling:

    Article Title: Novel Cell Wall Hydrolase CwlC from Bacillus thuringiensis Is Essential for Mother Cell Lysis
    Article Snippet: CwlC protein (1 mg) was labeled with EZ-Link Sulfo-NHS-SS-Biotin in accordance with the manufacturer's instructions (Thermo Fisher). .. The pellets containing the cell wall were analyzed by Western blot analysis with Pierce high-sensitivity streptavidin-horseradish peroxidase (Thermo Fisher) to detect the bound biotinylated CwlC.

    Article Title: Synaptic Amyloid-β Oligomers Precede p-Tau and Differentiate High Pathology Control Cases
    Article Snippet: Zenon isotype-specific IgG labeling kits, Alexa 488-conjugated chicken anti-mouse and Alexa 594-conjugated chicken anti-rabbit IgG were purchased from Molecular Probes (Eugene, OR). .. Pierce high-sensitivity streptavidin-horseradish peroxidase, Supersignal West Femto substrate, and bicinchoninic acid protein assay kit were from Thermo Scientific, (Rockford, IL).

    Incubation:

    Article Title: Novel Cell Wall Hydrolase CwlC from Bacillus thuringiensis Is Essential for Mother Cell Lysis
    Article Snippet: After that, excess unlabeled CwlC protein (10,000 to 320,000 nM) was added to the above mixture for incubation for another 30 min. .. The pellets containing the cell wall were analyzed by Western blot analysis with Pierce high-sensitivity streptavidin-horseradish peroxidase (Thermo Fisher) to detect the bound biotinylated CwlC.

    Western Blot:

    Article Title: Novel Cell Wall Hydrolase CwlC from Bacillus thuringiensis Is Essential for Mother Cell Lysis
    Article Snippet: .. The pellets containing the cell wall were analyzed by Western blot analysis with Pierce high-sensitivity streptavidin-horseradish peroxidase (Thermo Fisher) to detect the bound biotinylated CwlC. ..

    Recombinant:

    Article Title: Synaptic Amyloid-β Oligomers Precede p-Tau and Differentiate High Pathology Control Cases
    Article Snippet: Recombinant Aβ-40 peptide was purchased from US PEPTIde (Rancho Cucamonga, CA). .. Pierce high-sensitivity streptavidin-horseradish peroxidase, Supersignal West Femto substrate, and bicinchoninic acid protein assay kit were from Thermo Scientific, (Rockford, IL).

    Competitive Binding Assay:

    Article Title: Novel Cell Wall Hydrolase CwlC from Bacillus thuringiensis Is Essential for Mother Cell Lysis
    Article Snippet: Paragraph title: Homologous competition assay. ... The pellets containing the cell wall were analyzed by Western blot analysis with Pierce high-sensitivity streptavidin-horseradish peroxidase (Thermo Fisher) to detect the bound biotinylated CwlC.

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  • 99
    Thermo Fisher streptavidin hrp
    Characterization of the BRWD2/PHIP CryptoTudor domain. ( A ) Diagram depicting the recombinant BRWD2/PHIP CryptoTudor–bromodomain (BRWD2/PHIP–Crypt–bromo) construct used for binding studies. N-terminal 10X-Histidine (red box) and C-terminal 1XFlag (green box) tags were included for protein purification. ( B ) Coomassie stained SDS-PAGE gel of in vitro chromatin capture experiment using the BRWD2/PHIP–Crypt–bromo module. (Lane 1 ) Input MNase-digested chromatin. (Lane 2 ) Chromatin subjected to anti-Flag immunoprecipitation without BRWD2/PHIP–Crypt–bromo. (Lane 3 ) Chromatin incubated with recombinant BRWD2/PHIP–Crypt–bromo and subjected to anti-Flag immunoprecipitation. ( C ) Quantitative MS analysis of histones captured in B . Bars indicate the fraction of total histone represented by each modification state in the input (gray) or Flag immunoprecipitation material (red). (Un) Unmodified; (me1) monomethylated; (me2) dimethylated; (me3) trimethylated; (Ac) acetylated. ( D ) Coomassie-stained SDS-PAGE gel of histone peptide pull-downs performed with human ( top ) or Drosophila ( bottom ) Crypt–bromo constructs. Recombinant protein was incubated with <t>streptavidin</t> beads alone (SA-beads) or the biotinylated histone peptides indicated. Ten percent of input and 20% of each pull-down sample were loaded. ( Bottom ) Eluted proteins were subjected to dot blotting using <t>streptavidin-HRP</t> (SA-HRP). ( E ) ITC experiments with human BRWD2–Crypt–bromo titrated with H3 unmodified ( left ), H3K4me1 ( middle ), and H3K4me3 ( right ) peptides. ( F ) Coomassie-stained SDS-PAGE gel of pull-downs performed with human BRWD2/PHIP–Crypt–bromo and a panel of histone peptides. Ten percent of input and 20% of each pull-down sample were loaded. Streptavidin-HRP dot blot loading control is shown below the gel image. ( G ) Coomassie-stained SDS-PAGE gel of a panel of histone peptide pull-downs performed with human BRWD2–Crypt–bromo ( top ), the isolated CryptoTudor domain ( middle ), or the isolated tandem bromodomains ( bottom ). Ten percent of input and 20% of each pull-down sample were loaded. Dot blot loading control is shown below each gel image. ( H ) Coomassie-stained gels of histone peptide pull-downs performed with point mutations of the BRWD2 CryptoTudor domain. Dot blot loading controls are shown below the gels. ( I ). Positions of residues predicted to be involved in methyl-lysine binding are highlighted in red.
    Streptavidin Hrp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 189 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/streptavidin hrp/product/Thermo Fisher
    Average 99 stars, based on 189 article reviews
    Price from $9.99 to $1999.99
    streptavidin hrp - by Bioz Stars, 2020-03
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      Buy from Supplier

    99
    Thermo Fisher pierce high sensitivity streptavidin hrp
    ECs have higher levels of dimeric rsFcγRIIIa-binding antibodies than viremic subjects, (A) Dimeric rsFcγRIIIa-binding assay setup. HIV antigen (50 ng/well) was applied to a 96-well MaxiSorp plate, followed by addition of 1:10 diluted serum (containing IgGs). Subsequently, biotinylated dimeric rsFcγRIIIa was added, binding between antibodies and dimeric rsFcγRIIIa was measured using <t>HRP-conjugated</t> <t>streptavidin</t> and TMB, and reaction stopped with HCl. Absorbance was measured at 450 nm. (B) Dimeric rsFcγRIIIa V158-binding antibodies detected in ECs ( n = 22) compared to those of viremic subjects ( n = 44) against HIV Env (left panel), Vpu protein (middle panel), and Vpu19 peptide (right panel) using a 1:10 serum dilution; for Env, the dotted line at OD = 0.455 represent a cutoff of dimeric rsFcγRIIIa-binding activity calculated using ROC analyses. All data were normalized to 5 μg/ml HIV IgG immunoglobulin. Bars represent medians ( P values from Mann-Whitney U test). (C) Comparison of dimeric rsFcγRIIIa V158-binding antibody responses in ECs carrying protective versus nonprotective HLA I to those in viremic subjects. Bars represent medians ( P values, Kruskal-Wallis test). For both panels B and C, the dashed lines represent three times the mean OD obtained using five different HIV-negative serum samples against each antigen tested. (D) Endpoint titration curves of dimeric rsFcγRIIIa V158 binding to half-log dilutions of sera against HIV Env for 10 ECs (left panel) and case-matched 20 viremic subjects (right panel); the dotted lines at OD = 0.455 represent a cutoff for dimeric rsFcγRIIIa binding activity calculated using ROC analyses of the data shown in panel B. (E) Dimeric rsFcγRIIIa V158-binding antibody endpoint titer comparison of ECs with viremic subjects. Bars represent medians ( P values, Mann-Whitney U test).
    Pierce High Sensitivity Streptavidin Hrp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pierce high sensitivity streptavidin hrp/product/Thermo Fisher
    Average 99 stars, based on 9 article reviews
    Price from $9.99 to $1999.99
    pierce high sensitivity streptavidin hrp - by Bioz Stars, 2020-03
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      Buy from Supplier

    Image Search Results


    Characterization of the BRWD2/PHIP CryptoTudor domain. ( A ) Diagram depicting the recombinant BRWD2/PHIP CryptoTudor–bromodomain (BRWD2/PHIP–Crypt–bromo) construct used for binding studies. N-terminal 10X-Histidine (red box) and C-terminal 1XFlag (green box) tags were included for protein purification. ( B ) Coomassie stained SDS-PAGE gel of in vitro chromatin capture experiment using the BRWD2/PHIP–Crypt–bromo module. (Lane 1 ) Input MNase-digested chromatin. (Lane 2 ) Chromatin subjected to anti-Flag immunoprecipitation without BRWD2/PHIP–Crypt–bromo. (Lane 3 ) Chromatin incubated with recombinant BRWD2/PHIP–Crypt–bromo and subjected to anti-Flag immunoprecipitation. ( C ) Quantitative MS analysis of histones captured in B . Bars indicate the fraction of total histone represented by each modification state in the input (gray) or Flag immunoprecipitation material (red). (Un) Unmodified; (me1) monomethylated; (me2) dimethylated; (me3) trimethylated; (Ac) acetylated. ( D ) Coomassie-stained SDS-PAGE gel of histone peptide pull-downs performed with human ( top ) or Drosophila ( bottom ) Crypt–bromo constructs. Recombinant protein was incubated with streptavidin beads alone (SA-beads) or the biotinylated histone peptides indicated. Ten percent of input and 20% of each pull-down sample were loaded. ( Bottom ) Eluted proteins were subjected to dot blotting using streptavidin-HRP (SA-HRP). ( E ) ITC experiments with human BRWD2–Crypt–bromo titrated with H3 unmodified ( left ), H3K4me1 ( middle ), and H3K4me3 ( right ) peptides. ( F ) Coomassie-stained SDS-PAGE gel of pull-downs performed with human BRWD2/PHIP–Crypt–bromo and a panel of histone peptides. Ten percent of input and 20% of each pull-down sample were loaded. Streptavidin-HRP dot blot loading control is shown below the gel image. ( G ) Coomassie-stained SDS-PAGE gel of a panel of histone peptide pull-downs performed with human BRWD2–Crypt–bromo ( top ), the isolated CryptoTudor domain ( middle ), or the isolated tandem bromodomains ( bottom ). Ten percent of input and 20% of each pull-down sample were loaded. Dot blot loading control is shown below each gel image. ( H ) Coomassie-stained gels of histone peptide pull-downs performed with point mutations of the BRWD2 CryptoTudor domain. Dot blot loading controls are shown below the gels. ( I ). Positions of residues predicted to be involved in methyl-lysine binding are highlighted in red.

    Journal: Genes & Development

    Article Title: A cryptic Tudor domain links BRWD2/PHIP to COMPASS-mediated histone H3K4 methylation

    doi: 10.1101/gad.305201.117

    Figure Lengend Snippet: Characterization of the BRWD2/PHIP CryptoTudor domain. ( A ) Diagram depicting the recombinant BRWD2/PHIP CryptoTudor–bromodomain (BRWD2/PHIP–Crypt–bromo) construct used for binding studies. N-terminal 10X-Histidine (red box) and C-terminal 1XFlag (green box) tags were included for protein purification. ( B ) Coomassie stained SDS-PAGE gel of in vitro chromatin capture experiment using the BRWD2/PHIP–Crypt–bromo module. (Lane 1 ) Input MNase-digested chromatin. (Lane 2 ) Chromatin subjected to anti-Flag immunoprecipitation without BRWD2/PHIP–Crypt–bromo. (Lane 3 ) Chromatin incubated with recombinant BRWD2/PHIP–Crypt–bromo and subjected to anti-Flag immunoprecipitation. ( C ) Quantitative MS analysis of histones captured in B . Bars indicate the fraction of total histone represented by each modification state in the input (gray) or Flag immunoprecipitation material (red). (Un) Unmodified; (me1) monomethylated; (me2) dimethylated; (me3) trimethylated; (Ac) acetylated. ( D ) Coomassie-stained SDS-PAGE gel of histone peptide pull-downs performed with human ( top ) or Drosophila ( bottom ) Crypt–bromo constructs. Recombinant protein was incubated with streptavidin beads alone (SA-beads) or the biotinylated histone peptides indicated. Ten percent of input and 20% of each pull-down sample were loaded. ( Bottom ) Eluted proteins were subjected to dot blotting using streptavidin-HRP (SA-HRP). ( E ) ITC experiments with human BRWD2–Crypt–bromo titrated with H3 unmodified ( left ), H3K4me1 ( middle ), and H3K4me3 ( right ) peptides. ( F ) Coomassie-stained SDS-PAGE gel of pull-downs performed with human BRWD2/PHIP–Crypt–bromo and a panel of histone peptides. Ten percent of input and 20% of each pull-down sample were loaded. Streptavidin-HRP dot blot loading control is shown below the gel image. ( G ) Coomassie-stained SDS-PAGE gel of a panel of histone peptide pull-downs performed with human BRWD2–Crypt–bromo ( top ), the isolated CryptoTudor domain ( middle ), or the isolated tandem bromodomains ( bottom ). Ten percent of input and 20% of each pull-down sample were loaded. Dot blot loading control is shown below each gel image. ( H ) Coomassie-stained gels of histone peptide pull-downs performed with point mutations of the BRWD2 CryptoTudor domain. Dot blot loading controls are shown below the gels. ( I ). Positions of residues predicted to be involved in methyl-lysine binding are highlighted in red.

    Article Snippet: As a loading control, 0.25 µL of the eluted proteins was spotted onto a nitrocellulose membrane and detected using streptavidin-HRP (Thermo Scientific, no. 21130).

    Techniques: Recombinant, Construct, Binding Assay, Protein Purification, Staining, SDS Page, In Vitro, Immunoprecipitation, Incubation, Mass Spectrometry, Modification, Dot Blot, Isolation

    The enrichment of Anl-labeled Bt-MetRS NLL from infected host cells via affinity purification. (A) A549 cells were infected with Bt-MetRS NLL bacteria at MOI 50 and grown for 18 hrs in DMEM supplemented with Anl. Cell lysates were biotinylated via cycloaddition and subjected to affinity purification using streptavidin beads. Western blotting with streptavidin-HRP was used to detect tagged proteins in input (I), unbound (U), and eluate (E) fractions. The eluate fraction is concentrated 10x relative to the input. Primary rabbit anti-GAPDH antibodies and secondary goat anti-rabbit antibodies conjugated to HRP were used to assess the relative amount of host protein in samples. (B) Venn diagram representing bacterial proteins identified by mass spectrometry in input and eluate fractions that were derived from infected host cells. “Eluate (culture)” corresponds to bacterial proteins identified in lysates of bacteria grown in culture.

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: Proteomic Profiling of Burkholderia thailandensis During Host Infection Using Bio-Orthogonal Noncanonical Amino Acid Tagging (BONCAT)

    doi: 10.3389/fcimb.2018.00370

    Figure Lengend Snippet: The enrichment of Anl-labeled Bt-MetRS NLL from infected host cells via affinity purification. (A) A549 cells were infected with Bt-MetRS NLL bacteria at MOI 50 and grown for 18 hrs in DMEM supplemented with Anl. Cell lysates were biotinylated via cycloaddition and subjected to affinity purification using streptavidin beads. Western blotting with streptavidin-HRP was used to detect tagged proteins in input (I), unbound (U), and eluate (E) fractions. The eluate fraction is concentrated 10x relative to the input. Primary rabbit anti-GAPDH antibodies and secondary goat anti-rabbit antibodies conjugated to HRP were used to assess the relative amount of host protein in samples. (B) Venn diagram representing bacterial proteins identified by mass spectrometry in input and eluate fractions that were derived from infected host cells. “Eluate (culture)” corresponds to bacterial proteins identified in lysates of bacteria grown in culture.

    Article Snippet: Blots were hybridized with 1:10,000 Pierce™ High Sensitivity Streptavidin-HRP (Thermo Fisher) in 5% milk TBS-T buffer for 1 h. After three 5 min TBS-T washes, membranes were developed using SuperSignalTM West Pico PLUS Chemiluminescent Substrate (Thermo Fisher).

    Techniques: Labeling, Infection, Affinity Purification, Western Blot, Mass Spectrometry, Derivative Assay

    Expression of MetRS NLL in B. thailandensis (Bt) leads to incorporation of Anl into Bt proteins. (A) Map of MiniTn7-kan-MetRS NLL plasmid used for integration of MetRS NLL into the genome of B. thailandensis strain E264. E. coli MetRS NLL gene, optimized for expression in Burkholderia spp., is constitutively expressed using the P S12 promoter. PC S12 promoter drives the constitutive expression of kanamycin resistance used for selection of transformed bacteria. (B) Scheme of Tn7 transposon attachment sites downstream of glucosamine-6-phosphate synthetase genes 1 and 2 (glmS1/2), each located on one of the two B. thailandensis chromosomes, allowing for site-specific directional transposition of genes into B. thailandensis genome. FRT, flippase recognition target sites for flippase-mediated excision of FRT flanked DNA; P S12 , B. thailandensis ribosomal protein S12 gene promoter; PC s12 , B. cenocepacia rpsL promoter; T 0 T 1 , transcriptional terminator; Tn7L and Tn7R, left and right transposase recognition sites of Tn7 transposon; R6K ⋎ ori , origin of replication; oriT, conjugal origin of transfer. Black arrows indicate genes and their transcriptional orientations (MetRS NLL , methionyl-tRNA synthetase; kan, kanamycin resistance; glmS, glucosamine-6 phosphate synthetase). (C) Growth curves of wild type Bt and Bt-MetRS NLL cultured in LB broth with or without azidonorleucine (Anl). Optical density (OD 600 ) measurements of cultures were taken during log phase. Each growth curve represents two biological replicates. (D) Survival of bacteria (wild-type Bt and Bt-MetRS NLL strains) assessed by infecting human epithelial cells (A549) at MOI 10. Cultures were grown in DMEM supplemented with or without Anl. At 2 h hpi, cells were washed with PBS to remove unassociated extracellular bacteria. Cell layers were washed and lysed with saponin at 6 and 24 hpi and bacterial counts determined by plating culture dilutions on LB agar. Colony forming units (CFUs) were normalized to the respective MOI for each strain. (E) Incorporation of Anl in Bt-MetRS NLL proteins was confirmed by subjecting lysates of Bt or Bt-MetRS NLL cultured in broth with or without Anl to click reaction using biotin-alkyne. Biotinylated proteins were detected by Western blot stained using streptavidin-HRP. Primary goat anti- Burkholderia antibodies and secondary donkey anti-goat antibodies conjugated to HRP were used to stain the blot as a loading control. (F) Relative protein quantity in lysates from E was assessed by loading the same amounts of lysates onto SDS-PAGE gel and detecting total protein using Sypro Ruby stain.

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: Proteomic Profiling of Burkholderia thailandensis During Host Infection Using Bio-Orthogonal Noncanonical Amino Acid Tagging (BONCAT)

    doi: 10.3389/fcimb.2018.00370

    Figure Lengend Snippet: Expression of MetRS NLL in B. thailandensis (Bt) leads to incorporation of Anl into Bt proteins. (A) Map of MiniTn7-kan-MetRS NLL plasmid used for integration of MetRS NLL into the genome of B. thailandensis strain E264. E. coli MetRS NLL gene, optimized for expression in Burkholderia spp., is constitutively expressed using the P S12 promoter. PC S12 promoter drives the constitutive expression of kanamycin resistance used for selection of transformed bacteria. (B) Scheme of Tn7 transposon attachment sites downstream of glucosamine-6-phosphate synthetase genes 1 and 2 (glmS1/2), each located on one of the two B. thailandensis chromosomes, allowing for site-specific directional transposition of genes into B. thailandensis genome. FRT, flippase recognition target sites for flippase-mediated excision of FRT flanked DNA; P S12 , B. thailandensis ribosomal protein S12 gene promoter; PC s12 , B. cenocepacia rpsL promoter; T 0 T 1 , transcriptional terminator; Tn7L and Tn7R, left and right transposase recognition sites of Tn7 transposon; R6K ⋎ ori , origin of replication; oriT, conjugal origin of transfer. Black arrows indicate genes and their transcriptional orientations (MetRS NLL , methionyl-tRNA synthetase; kan, kanamycin resistance; glmS, glucosamine-6 phosphate synthetase). (C) Growth curves of wild type Bt and Bt-MetRS NLL cultured in LB broth with or without azidonorleucine (Anl). Optical density (OD 600 ) measurements of cultures were taken during log phase. Each growth curve represents two biological replicates. (D) Survival of bacteria (wild-type Bt and Bt-MetRS NLL strains) assessed by infecting human epithelial cells (A549) at MOI 10. Cultures were grown in DMEM supplemented with or without Anl. At 2 h hpi, cells were washed with PBS to remove unassociated extracellular bacteria. Cell layers were washed and lysed with saponin at 6 and 24 hpi and bacterial counts determined by plating culture dilutions on LB agar. Colony forming units (CFUs) were normalized to the respective MOI for each strain. (E) Incorporation of Anl in Bt-MetRS NLL proteins was confirmed by subjecting lysates of Bt or Bt-MetRS NLL cultured in broth with or without Anl to click reaction using biotin-alkyne. Biotinylated proteins were detected by Western blot stained using streptavidin-HRP. Primary goat anti- Burkholderia antibodies and secondary donkey anti-goat antibodies conjugated to HRP were used to stain the blot as a loading control. (F) Relative protein quantity in lysates from E was assessed by loading the same amounts of lysates onto SDS-PAGE gel and detecting total protein using Sypro Ruby stain.

    Article Snippet: Blots were hybridized with 1:10,000 Pierce™ High Sensitivity Streptavidin-HRP (Thermo Fisher) in 5% milk TBS-T buffer for 1 h. After three 5 min TBS-T washes, membranes were developed using SuperSignalTM West Pico PLUS Chemiluminescent Substrate (Thermo Fisher).

    Techniques: Expressing, Plasmid Preparation, Selection, Transformation Assay, Cell Culture, Western Blot, Staining, SDS Page

    Anl-labeling of Bt-MetRS NLL during infection is bacteria-specific and allows for in-situ fluorescent detection of host-associated bacteria. (A) Human epithelial cells (A549) were infected at MOI 100 and cultured for 18 hrs in DMEM media supplemented with or without 1 mM Anl. Lysates from infected and uninfected monolayers were subjected to click chemistry using alkyne conjugated to biotin. Biotin-tagged proteins in cell lysates were detected by Western blotting with streptavidin-HRP. As a loading control, human GAPDH was detected using primary rabbit anti-GAPDH antibodies and secondary goat anti-rabbit antibodies conjugated to HRP. (B) A549 cells were infected at an MOI of 100 with Bt-MetRS NLL bacteria and grown in media supplemented with 1 mM Anl for 6 hrs. Infected cells were fixed and stained with Alexa Fluor 594-wheat germ agglutinin (WGA) conjugate to visualize host cell membranes (red). Cells were subjected to click chemistry using Alexa Fluor 488 conjugated to alkyne to tag Anl-labeled proteins (green). Host cell nuclei were stained using 6-diamidino-2-phenylindole (DAPI) (blue). White arrow indicates bacteria. Fluorescent signal was visualized using fluorescence microscopy; 100x magnification was used for all images. Scale bars indicate the distance of 10 μm.

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: Proteomic Profiling of Burkholderia thailandensis During Host Infection Using Bio-Orthogonal Noncanonical Amino Acid Tagging (BONCAT)

    doi: 10.3389/fcimb.2018.00370

    Figure Lengend Snippet: Anl-labeling of Bt-MetRS NLL during infection is bacteria-specific and allows for in-situ fluorescent detection of host-associated bacteria. (A) Human epithelial cells (A549) were infected at MOI 100 and cultured for 18 hrs in DMEM media supplemented with or without 1 mM Anl. Lysates from infected and uninfected monolayers were subjected to click chemistry using alkyne conjugated to biotin. Biotin-tagged proteins in cell lysates were detected by Western blotting with streptavidin-HRP. As a loading control, human GAPDH was detected using primary rabbit anti-GAPDH antibodies and secondary goat anti-rabbit antibodies conjugated to HRP. (B) A549 cells were infected at an MOI of 100 with Bt-MetRS NLL bacteria and grown in media supplemented with 1 mM Anl for 6 hrs. Infected cells were fixed and stained with Alexa Fluor 594-wheat germ agglutinin (WGA) conjugate to visualize host cell membranes (red). Cells were subjected to click chemistry using Alexa Fluor 488 conjugated to alkyne to tag Anl-labeled proteins (green). Host cell nuclei were stained using 6-diamidino-2-phenylindole (DAPI) (blue). White arrow indicates bacteria. Fluorescent signal was visualized using fluorescence microscopy; 100x magnification was used for all images. Scale bars indicate the distance of 10 μm.

    Article Snippet: Blots were hybridized with 1:10,000 Pierce™ High Sensitivity Streptavidin-HRP (Thermo Fisher) in 5% milk TBS-T buffer for 1 h. After three 5 min TBS-T washes, membranes were developed using SuperSignalTM West Pico PLUS Chemiluminescent Substrate (Thermo Fisher).

    Techniques: Labeling, Infection, In Situ, Cell Culture, Western Blot, Staining, Whole Genome Amplification, Fluorescence, Microscopy

    Anl-labeled proteins expressed by Bt-MetRS NLL can be purified from bacterial lysates. (A) Bt-MetRS NLL bacteria were grown for 4 hrs in DMEM media with or without Anl. Bacterial lysates were subjected to cycloaddition reaction using biotin-conjugated alkyne and biotin-tagged proteins were purified using magnetic streptavidin beads. Input (I), unbound (U), and eluate (E) samples obtained by affinity purification were analyzed by Western blotting using streptavidin-HRP to visualize biotin-tagged proteins. Primary goat anti- Burkholderia antibodies and secondary donkey anti-goat antibodies conjugated to HRP were used to determine relative bacterial protein abundance in samples. (B) Venn diagram representing the number of proteins identified by mass spectrometry in input and eluate fractions from Anl-labeled bacterial lysates and an eluate derived from unlabeled bacterial lysate.

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: Proteomic Profiling of Burkholderia thailandensis During Host Infection Using Bio-Orthogonal Noncanonical Amino Acid Tagging (BONCAT)

    doi: 10.3389/fcimb.2018.00370

    Figure Lengend Snippet: Anl-labeled proteins expressed by Bt-MetRS NLL can be purified from bacterial lysates. (A) Bt-MetRS NLL bacteria were grown for 4 hrs in DMEM media with or without Anl. Bacterial lysates were subjected to cycloaddition reaction using biotin-conjugated alkyne and biotin-tagged proteins were purified using magnetic streptavidin beads. Input (I), unbound (U), and eluate (E) samples obtained by affinity purification were analyzed by Western blotting using streptavidin-HRP to visualize biotin-tagged proteins. Primary goat anti- Burkholderia antibodies and secondary donkey anti-goat antibodies conjugated to HRP were used to determine relative bacterial protein abundance in samples. (B) Venn diagram representing the number of proteins identified by mass spectrometry in input and eluate fractions from Anl-labeled bacterial lysates and an eluate derived from unlabeled bacterial lysate.

    Article Snippet: Blots were hybridized with 1:10,000 Pierce™ High Sensitivity Streptavidin-HRP (Thermo Fisher) in 5% milk TBS-T buffer for 1 h. After three 5 min TBS-T washes, membranes were developed using SuperSignalTM West Pico PLUS Chemiluminescent Substrate (Thermo Fisher).

    Techniques: Labeling, Purification, Affinity Purification, Western Blot, Mass Spectrometry, Derivative Assay

    ECs have higher levels of dimeric rsFcγRIIIa-binding antibodies than viremic subjects, (A) Dimeric rsFcγRIIIa-binding assay setup. HIV antigen (50 ng/well) was applied to a 96-well MaxiSorp plate, followed by addition of 1:10 diluted serum (containing IgGs). Subsequently, biotinylated dimeric rsFcγRIIIa was added, binding between antibodies and dimeric rsFcγRIIIa was measured using HRP-conjugated streptavidin and TMB, and reaction stopped with HCl. Absorbance was measured at 450 nm. (B) Dimeric rsFcγRIIIa V158-binding antibodies detected in ECs ( n = 22) compared to those of viremic subjects ( n = 44) against HIV Env (left panel), Vpu protein (middle panel), and Vpu19 peptide (right panel) using a 1:10 serum dilution; for Env, the dotted line at OD = 0.455 represent a cutoff of dimeric rsFcγRIIIa-binding activity calculated using ROC analyses. All data were normalized to 5 μg/ml HIV IgG immunoglobulin. Bars represent medians ( P values from Mann-Whitney U test). (C) Comparison of dimeric rsFcγRIIIa V158-binding antibody responses in ECs carrying protective versus nonprotective HLA I to those in viremic subjects. Bars represent medians ( P values, Kruskal-Wallis test). For both panels B and C, the dashed lines represent three times the mean OD obtained using five different HIV-negative serum samples against each antigen tested. (D) Endpoint titration curves of dimeric rsFcγRIIIa V158 binding to half-log dilutions of sera against HIV Env for 10 ECs (left panel) and case-matched 20 viremic subjects (right panel); the dotted lines at OD = 0.455 represent a cutoff for dimeric rsFcγRIIIa binding activity calculated using ROC analyses of the data shown in panel B. (E) Dimeric rsFcγRIIIa V158-binding antibody endpoint titer comparison of ECs with viremic subjects. Bars represent medians ( P values, Mann-Whitney U test).

    Journal: Journal of Virology

    Article Title: HIV-1 Env- and Vpu-Specific Antibody-Dependent Cellular Cytotoxicity Responses Associated with Elite Control of HIV

    doi: 10.1128/JVI.00700-17

    Figure Lengend Snippet: ECs have higher levels of dimeric rsFcγRIIIa-binding antibodies than viremic subjects, (A) Dimeric rsFcγRIIIa-binding assay setup. HIV antigen (50 ng/well) was applied to a 96-well MaxiSorp plate, followed by addition of 1:10 diluted serum (containing IgGs). Subsequently, biotinylated dimeric rsFcγRIIIa was added, binding between antibodies and dimeric rsFcγRIIIa was measured using HRP-conjugated streptavidin and TMB, and reaction stopped with HCl. Absorbance was measured at 450 nm. (B) Dimeric rsFcγRIIIa V158-binding antibodies detected in ECs ( n = 22) compared to those of viremic subjects ( n = 44) against HIV Env (left panel), Vpu protein (middle panel), and Vpu19 peptide (right panel) using a 1:10 serum dilution; for Env, the dotted line at OD = 0.455 represent a cutoff of dimeric rsFcγRIIIa-binding activity calculated using ROC analyses. All data were normalized to 5 μg/ml HIV IgG immunoglobulin. Bars represent medians ( P values from Mann-Whitney U test). (C) Comparison of dimeric rsFcγRIIIa V158-binding antibody responses in ECs carrying protective versus nonprotective HLA I to those in viremic subjects. Bars represent medians ( P values, Kruskal-Wallis test). For both panels B and C, the dashed lines represent three times the mean OD obtained using five different HIV-negative serum samples against each antigen tested. (D) Endpoint titration curves of dimeric rsFcγRIIIa V158 binding to half-log dilutions of sera against HIV Env for 10 ECs (left panel) and case-matched 20 viremic subjects (right panel); the dotted lines at OD = 0.455 represent a cutoff for dimeric rsFcγRIIIa binding activity calculated using ROC analyses of the data shown in panel B. (E) Dimeric rsFcγRIIIa V158-binding antibody endpoint titer comparison of ECs with viremic subjects. Bars represent medians ( P values, Mann-Whitney U test).

    Article Snippet: Subsequently, 100 ng/ml of Pierce high-sensitivity streptavidin-HRP (Thermo Scientific, Pittsburgh, PA) diluted in PBS with 1 mM EDTA and 1% BSA was added and left for 1 h at 37°C.

    Techniques: Binding Assay, Activity Assay, MANN-WHITNEY, Titration

    ABP labelling of endogenous UBE3C in cell lysate. Lysates from HEK293T and HeLa cells were incubated for 3 h with 5 μ m probe and analysed by western blotting (WB specifies antibody). A shift in the molecular weight of the ligase indicates labelling (arrows). An asterisk indicates a contaminating band detected by the antibody. Weak yet specific labelling of endogenous UBE3C with His‐UBE2D2_WT‐ABP (but not the F62A mutant) was detected under basal/unstimulated conditions in A) HEK293T and B) HeLa cells lysed in buffer with a 0.5 % Triton X‐100. An HRP‐conjugated His antibody was used as a loading control for His‐UBE2D2‐ABPs. In contrast to UBE3C, labelling of other HECT ligases was not detected. C) Western blot shows improved labelling of endogenous UBE3C under basal/unstimulated condition after sonication (no detergent). Addition of 2 m m DTT during labelling resulted in a slight increase in labelling of endogenous UBE3C. Similarly to A) and B), no labelling is detected for other HECT ligases. D) Western blot analysis of HeLa cell lysates obtained by sonication and incubation with E2–Ub‐ABPs (His‐UBE2D2_WT‐ABP and the F62A mutant) or Ub‐ABPs (Ub‐VME and Ub‐PA). As in C), UBE3C was efficiently labelled by His‐UBE2D2_WT‐ABP. We also observed some weak labelling with biotin‐Ub‐PA but not with Ub‐VME. HRP‐conjugated His and streptavidin‐HRP antibodies were used as loading control for the probes. Note that Ub‐VME did not have a tag. E) Cell lysate from HEK293T overexpressing HA‐tagged full‐length‐mouse HECTD1 was incubated with ABPs. UBE3C was used as a positive control for probe functionality. However, labelling of HA‐FL‐mHECTD1 was not detected in this assay.

    Journal: Chembiochem

    Article Title: Activity‐Based Probes for HECT E3 Ubiquitin Ligases

    doi: 10.1002/cbic.201700006

    Figure Lengend Snippet: ABP labelling of endogenous UBE3C in cell lysate. Lysates from HEK293T and HeLa cells were incubated for 3 h with 5 μ m probe and analysed by western blotting (WB specifies antibody). A shift in the molecular weight of the ligase indicates labelling (arrows). An asterisk indicates a contaminating band detected by the antibody. Weak yet specific labelling of endogenous UBE3C with His‐UBE2D2_WT‐ABP (but not the F62A mutant) was detected under basal/unstimulated conditions in A) HEK293T and B) HeLa cells lysed in buffer with a 0.5 % Triton X‐100. An HRP‐conjugated His antibody was used as a loading control for His‐UBE2D2‐ABPs. In contrast to UBE3C, labelling of other HECT ligases was not detected. C) Western blot shows improved labelling of endogenous UBE3C under basal/unstimulated condition after sonication (no detergent). Addition of 2 m m DTT during labelling resulted in a slight increase in labelling of endogenous UBE3C. Similarly to A) and B), no labelling is detected for other HECT ligases. D) Western blot analysis of HeLa cell lysates obtained by sonication and incubation with E2–Ub‐ABPs (His‐UBE2D2_WT‐ABP and the F62A mutant) or Ub‐ABPs (Ub‐VME and Ub‐PA). As in C), UBE3C was efficiently labelled by His‐UBE2D2_WT‐ABP. We also observed some weak labelling with biotin‐Ub‐PA but not with Ub‐VME. HRP‐conjugated His and streptavidin‐HRP antibodies were used as loading control for the probes. Note that Ub‐VME did not have a tag. E) Cell lysate from HEK293T overexpressing HA‐tagged full‐length‐mouse HECTD1 was incubated with ABPs. UBE3C was used as a positive control for probe functionality. However, labelling of HA‐FL‐mHECTD1 was not detected in this assay.

    Article Snippet: PIERCE high‐sensitivity streptavidin‐HRP (Thermo Fisher Scientific) was used to detect biotin‐Ub‐PA (also referred to as Ub‐PA in the main text).

    Techniques: Incubation, Western Blot, Molecular Weight, Mutagenesis, Sonication, Positive Control