peroxidase conjugate secondary antibody  (Cell Signaling Technology Inc)

 
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    Name:
    Myeloperoxidase Antibody
    Description:
    Myeloperoxidase MPO is a peroxidase enzyme that is part of the host defense system of polymorphonuclear leukocytes reviewed in 1 The gene for MPO was cloned independently from several laboratories 2 5 A decrease in MPO expression was noticed upon differentiation of HL 60 cells 5 MPO catalyzes the reaction of hydrogen peroxide and chloride or other halides to produce hypochlorous acid and other potent antimicrobial oxidants Knockout mice of MPO are impaired in clearing select microbial infections 6 Processing of mature MPO from an initial 80 90 kDa translation product involves insertion of a heme moiety glycosylation and proteolytic cleavage The mature protein is a tetramer of two heavy chains 60 kDa and two light chains 12 kDa It is abundantly expressed in neutrophils and monocytes and secreted during their activation Heightened MPO levels have been associated with tissue damage and a number of pathological conditions 1
    Catalog Number:
    79623
    Price:
    None
    Applications:
    Western Blot
    Category:
    Primary Antibodies
    Source:
    Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of human myeloperoxidase protein. Antibodies are purified by protein A and peptide affinity chromatography.
    Reactivity:
    Human Mouse
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    Structured Review

    Cell Signaling Technology Inc peroxidase conjugate secondary antibody
    Myeloperoxidase MPO is a peroxidase enzyme that is part of the host defense system of polymorphonuclear leukocytes reviewed in 1 The gene for MPO was cloned independently from several laboratories 2 5 A decrease in MPO expression was noticed upon differentiation of HL 60 cells 5 MPO catalyzes the reaction of hydrogen peroxide and chloride or other halides to produce hypochlorous acid and other potent antimicrobial oxidants Knockout mice of MPO are impaired in clearing select microbial infections 6 Processing of mature MPO from an initial 80 90 kDa translation product involves insertion of a heme moiety glycosylation and proteolytic cleavage The mature protein is a tetramer of two heavy chains 60 kDa and two light chains 12 kDa It is abundantly expressed in neutrophils and monocytes and secreted during their activation Heightened MPO levels have been associated with tissue damage and a number of pathological conditions 1
    https://www.bioz.com/result/peroxidase conjugate secondary antibody/product/Cell Signaling Technology Inc
    Average 99 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    peroxidase conjugate secondary antibody - by Bioz Stars, 2020-08
    99/100 stars

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    Incubation:

    Article Title: Androgen Receptor Variants Occur Frequently in Castration Resistant Prostate Cancer Metastases
    Article Snippet: .. Membranes were incubated with a horseradish peroxidase-conjugated secondary antibody (Cell Signaling), and developed with ECL (Pharmacia Biotech). .. The membranes were stripped for 30 min in Stripping Buffer (Thermoscientific) and re-probed with anti-β-actin antibody as a loading control (Sigma-Aldrich).

    Article Title: The Traditional Chinese Medicine MLC901 inhibits inflammation processes after focal cerebral ischemia
    Article Snippet: .. After a PBS rinse, sections were incubated overnight with the following primary antibodies: rabbit polyclonal antibody against IL6 (Abcam, A6672, 1/500), GFAP (Dako, Z0334, 1/300) and MMP9 (Millipore, AB 19026, 1/200), mouse monoclonal antibody against Iba1 (Abcam 15690, 1/400), MPO (R & D, MAB3174) and rabbit monoclonal against phospho-NFκB p65 (Ser536) antibody (mAb#3033, diluted 1:1000; Cell Signal Technology), goat polyclonal antibody against TNFα (R & D, AF410—NA, 10μl/ml) and IL1β (R & D, AF401, 10μl/ml) and rabbit monoclonal antibody against Iba1 (Wako, 1/1000). .. After the primary incubation and three rinses in PBS, sections were then incubated in biotinylated horse anti-rabbit IgG (Jackson ImmunoResearch, diluted 1/15,000) for 2 h at room temperature.

    Labeling:

    Article Title: MicroRNA-1826 directly targets beta-catenin (CTNNB1) and MEK1 (MAP2K1) in VHL-inactivated renal cancer
    Article Snippet: .. Blots were washed in Tris-buffered saline containing 0.1% Tween 20 and labeled with horseradish peroxidase-conjugated secondary anti-mouse or anti-rabbit antibody (Cell signaling Technology). .. Proteins were enhanced by chemiluminescence (Amersham ECL plus Western Blotting detection system; Amersham Biosciences) for visualization.

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  • 88
    Cell Signaling Technology Inc anti rabbit secondary antibody horseradish peroxidase conjugate
    (A) Schematic illustration of the opsonization assays, in which K. pneumoniae was incubated with PCCs conjugated with either biotin or DNP, opsonized by corresponding <t>antibody,</t> and then detected. (B) Cell-binding ELISA results shows that biotinylated cy(LLFFF) at 50 μM binds two strains of MrkA-expressing K. pneumoniae cells with excellent selectivity versus E. coli and S. typhimurium . By comparison a biotinylated dummy ligand cy(HNGPT) shows no or minimal binding above baseline. Asterisks (*) indicate ELISA data for target K. pneumoniae strains, and error bars reflect standard deviations. (C) Flow cytometry results for K. pneumoniae cells (strain BAA 2146) opsonized by DNP-conjugated PCCs and fluorescently-tagged <t>anti-DNP</t> antibodies. Cells exposed to cy(LLFFF)-DNP, at either 1 or 5 μM concentrations, plus <t>secondary</t> antibody (light and dark green) showed much higher fluorescence than cells incubated with 50 μM of cy(HGNPT)-DNP <t>conjugate</t> plus secondary antibody (orange) and secondary antibody only (teal). Untreated cells showed the lowest fluorescence (red). Cytometry measurements were gated for single cells, and each distribution comprises > 18,000 cells.
    Anti Rabbit Secondary Antibody Horseradish Peroxidase Conjugate, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti rabbit secondary antibody horseradish peroxidase conjugate/product/Cell Signaling Technology Inc
    Average 88 stars, based on 1 article reviews
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    94
    Cell Signaling Technology Inc horseradish peroxidase hrp conjugated secondary antibody
    Oncogenic GNAQ Q209P -mediated activation of ERK and YAP signalling in choroidal melanocytes at the junction between RPE and choroid A-D. Transverse sections of formalin-fixed and paraffin-embedded eye tissues of F 1 generation, 5-month-old Tg ( mitfa :GNAQ Q209P ) zebrafish. (A) H E staining demonstrating choroidal hyperplasia (black arrowheads). White dashed box indicates the region of the choroid magnified in B-D. (B-D) Transverse sections of formalin-fixed and paraffin-embedded eye tissues were stained by IHC, visualized by ImmPact NovaRed peroxidase <t>(HRP)</t> substrate then counterstained with hematoxylin (blue). (B) Negative control: section incubated with <t>1x</t> PBS instead of primary antibody. (C) Immunoreactivity to pERK1/2 (read-out of ERK activation; black arrowheads) in melanocytes at the interface between the RPE and choroid. (D) YAP-positive nuclei (read-out of YAP activation; black arrowheads) in the same cells. Scale bars, 20 μm.
    Horseradish Peroxidase Hrp Conjugated Secondary Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 116 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/horseradish peroxidase hrp conjugated secondary antibody/product/Cell Signaling Technology Inc
    Average 94 stars, based on 116 article reviews
    Price from $9.99 to $1999.99
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    90
    Cell Signaling Technology Inc horse anti mouse horseradish peroxidase hrp conjugated igg secondary antibody
    Aberrant localisation of proteinases in subcellular fractions of the pg1058 mutant. P . gingivalis W50, pg1058 mutant and pg1058 + complement strain cultures were fractionated, separated via SDS-PAGE and immunoblotted on nitrocellulose membranes. The immunoblots have material derived from whole-cells (WC; 2 x 10 8 cells), total membrane (3 x 10 8 cells) and soluble (1 x 10 8 cells) fractions; WC, periplasm and osmotically-shocked cell (OSC) fractions (5 x 10 8 cells each); WC (2 x 10 8 cells), culture fluid (CF; from culture containing 2 x 10 9 cells) and vesicle-free cleared culture fluid (CCF; from culture containing 2 x 10 9 cells). Immunoblots were probed with either anti-rKgp cat ( A , 1/200,000 dilution), pre-adsorbed anti-rRgpA cat ( B , 1/10,000 dilution), anti-rKgp A1 ( C , 1/10,000 dilution) or anti-rRgpB-CTD 422 ( D , 1/200,000 dilution) followed by goat anti-rabbit ( A ) or horse anti-mouse ( B , C and D ) <t>IgG-conjugated</t> <t>HRP</t> secondary antibodies (1/3,000 dilution).
    Horse Anti Mouse Horseradish Peroxidase Hrp Conjugated Igg Secondary Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/horse anti mouse horseradish peroxidase hrp conjugated igg secondary antibody/product/Cell Signaling Technology Inc
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    93
    Cell Signaling Technology Inc peroxidase conjugated goat anti rabbit secondary antibody
    The hybrids 7, 9, 5, 17, 18, 19, 13 and the etoposide positive control induced double-strand DNA breaks in K562 cells as indicated by formation of γH2AX. K562 cells were treated with 20 µM of the drugs indicated for 4 h in growth medium, lysed and subjected to SDS-PAGE electrophoresis and Western blotting. The blots were probed with <t>antibodies</t> to γH2AX and with GAPDH (glyceraldehyde 3-phosphate dehydrogenase) as a loading control and a chemiluminescent-inducing horseradish <t>peroxidase-conjugated</t> <t>secondary</t> <t>antibody.</t> The results were typical of experiments carried out on 2 different days.
    Peroxidase Conjugated Goat Anti Rabbit Secondary Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 45 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/peroxidase conjugated goat anti rabbit secondary antibody/product/Cell Signaling Technology Inc
    Average 93 stars, based on 45 article reviews
    Price from $9.99 to $1999.99
    peroxidase conjugated goat anti rabbit secondary antibody - by Bioz Stars, 2020-08
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    (A) Schematic illustration of the opsonization assays, in which K. pneumoniae was incubated with PCCs conjugated with either biotin or DNP, opsonized by corresponding antibody, and then detected. (B) Cell-binding ELISA results shows that biotinylated cy(LLFFF) at 50 μM binds two strains of MrkA-expressing K. pneumoniae cells with excellent selectivity versus E. coli and S. typhimurium . By comparison a biotinylated dummy ligand cy(HNGPT) shows no or minimal binding above baseline. Asterisks (*) indicate ELISA data for target K. pneumoniae strains, and error bars reflect standard deviations. (C) Flow cytometry results for K. pneumoniae cells (strain BAA 2146) opsonized by DNP-conjugated PCCs and fluorescently-tagged anti-DNP antibodies. Cells exposed to cy(LLFFF)-DNP, at either 1 or 5 μM concentrations, plus secondary antibody (light and dark green) showed much higher fluorescence than cells incubated with 50 μM of cy(HGNPT)-DNP conjugate plus secondary antibody (orange) and secondary antibody only (teal). Untreated cells showed the lowest fluorescence (red). Cytometry measurements were gated for single cells, and each distribution comprises > 18,000 cells.

    Journal: bioRxiv

    Article Title: Antibody-Recruiting Protein-Catalyzed Capture Agents to Combat Antibiotic-Resistant Bacteria

    doi: 10.1101/822346

    Figure Lengend Snippet: (A) Schematic illustration of the opsonization assays, in which K. pneumoniae was incubated with PCCs conjugated with either biotin or DNP, opsonized by corresponding antibody, and then detected. (B) Cell-binding ELISA results shows that biotinylated cy(LLFFF) at 50 μM binds two strains of MrkA-expressing K. pneumoniae cells with excellent selectivity versus E. coli and S. typhimurium . By comparison a biotinylated dummy ligand cy(HNGPT) shows no or minimal binding above baseline. Asterisks (*) indicate ELISA data for target K. pneumoniae strains, and error bars reflect standard deviations. (C) Flow cytometry results for K. pneumoniae cells (strain BAA 2146) opsonized by DNP-conjugated PCCs and fluorescently-tagged anti-DNP antibodies. Cells exposed to cy(LLFFF)-DNP, at either 1 or 5 μM concentrations, plus secondary antibody (light and dark green) showed much higher fluorescence than cells incubated with 50 μM of cy(HGNPT)-DNP conjugate plus secondary antibody (orange) and secondary antibody only (teal). Untreated cells showed the lowest fluorescence (red). Cytometry measurements were gated for single cells, and each distribution comprises > 18,000 cells.

    Article Snippet: The general procedure for an ELISA assay was: wash 3×200 μL/well, conjugate with 2 μM of biotinylated compound for 2 h at room temperature (100 μL/well), wash 3×200 μL/well, blocked overnight in 5 wt% milk at 4°C, wash 3×200 μL/well with wash buffer, incubate with the titrated compound for 1 h, 3×200 μL/well, incubate with primary antibody (either or) for 1 h (100 μL/well), wash 3×200 μL/well, incubate with 1:2,000 anti-rabbit secondary antibody-horseradish peroxidase conjugate (Cell signaling Technologies, 7074S) for 1 h, 3×200 μL/well, develop with the Microwell Peroxidase Substrate System (2-C) (SeraCare, 5120-0047) using 100 μL/well for 1-40 minutes, and quench using 1 M H2 SO4 (aq.) at 100 μL/well.

    Techniques: Incubation, Binding Assay, Enzyme-linked Immunosorbent Assay, Expressing, Flow Cytometry, Fluorescence, Cytometry

    Oncogenic GNAQ Q209P -mediated activation of ERK and YAP signalling in choroidal melanocytes at the junction between RPE and choroid A-D. Transverse sections of formalin-fixed and paraffin-embedded eye tissues of F 1 generation, 5-month-old Tg ( mitfa :GNAQ Q209P ) zebrafish. (A) H E staining demonstrating choroidal hyperplasia (black arrowheads). White dashed box indicates the region of the choroid magnified in B-D. (B-D) Transverse sections of formalin-fixed and paraffin-embedded eye tissues were stained by IHC, visualized by ImmPact NovaRed peroxidase (HRP) substrate then counterstained with hematoxylin (blue). (B) Negative control: section incubated with 1x PBS instead of primary antibody. (C) Immunoreactivity to pERK1/2 (read-out of ERK activation; black arrowheads) in melanocytes at the interface between the RPE and choroid. (D) YAP-positive nuclei (read-out of YAP activation; black arrowheads) in the same cells. Scale bars, 20 μm.

    Journal: Oncotarget

    Article Title: Minimal contribution of ERK1/2-MAPK signalling towards the maintenance of oncogenic GNAQQ209P-driven uveal melanomas in zebrafish

    doi: 10.18632/oncotarget.9207

    Figure Lengend Snippet: Oncogenic GNAQ Q209P -mediated activation of ERK and YAP signalling in choroidal melanocytes at the junction between RPE and choroid A-D. Transverse sections of formalin-fixed and paraffin-embedded eye tissues of F 1 generation, 5-month-old Tg ( mitfa :GNAQ Q209P ) zebrafish. (A) H E staining demonstrating choroidal hyperplasia (black arrowheads). White dashed box indicates the region of the choroid magnified in B-D. (B-D) Transverse sections of formalin-fixed and paraffin-embedded eye tissues were stained by IHC, visualized by ImmPact NovaRed peroxidase (HRP) substrate then counterstained with hematoxylin (blue). (B) Negative control: section incubated with 1x PBS instead of primary antibody. (C) Immunoreactivity to pERK1/2 (read-out of ERK activation; black arrowheads) in melanocytes at the interface between the RPE and choroid. (D) YAP-positive nuclei (read-out of YAP activation; black arrowheads) in the same cells. Scale bars, 20 μm.

    Article Snippet: To remove excess unbound primary antibody, membranes were washed three times with 1x TBST buffer then incubated with an appropriate horseradish peroxidase (HRP)-conjugated secondary antibody (Cell Signalling Technology, 1:1000 dilution) for 1 hour at room temperature.

    Techniques: Activation Assay, Staining, Immunohistochemistry, Negative Control, Incubation

    Aberrant localisation of proteinases in subcellular fractions of the pg1058 mutant. P . gingivalis W50, pg1058 mutant and pg1058 + complement strain cultures were fractionated, separated via SDS-PAGE and immunoblotted on nitrocellulose membranes. The immunoblots have material derived from whole-cells (WC; 2 x 10 8 cells), total membrane (3 x 10 8 cells) and soluble (1 x 10 8 cells) fractions; WC, periplasm and osmotically-shocked cell (OSC) fractions (5 x 10 8 cells each); WC (2 x 10 8 cells), culture fluid (CF; from culture containing 2 x 10 9 cells) and vesicle-free cleared culture fluid (CCF; from culture containing 2 x 10 9 cells). Immunoblots were probed with either anti-rKgp cat ( A , 1/200,000 dilution), pre-adsorbed anti-rRgpA cat ( B , 1/10,000 dilution), anti-rKgp A1 ( C , 1/10,000 dilution) or anti-rRgpB-CTD 422 ( D , 1/200,000 dilution) followed by goat anti-rabbit ( A ) or horse anti-mouse ( B , C and D ) IgG-conjugated HRP secondary antibodies (1/3,000 dilution).

    Journal: PLoS ONE

    Article Title: PG1058 Is a Novel Multidomain Protein Component of the Bacterial Type IX Secretion System

    doi: 10.1371/journal.pone.0164313

    Figure Lengend Snippet: Aberrant localisation of proteinases in subcellular fractions of the pg1058 mutant. P . gingivalis W50, pg1058 mutant and pg1058 + complement strain cultures were fractionated, separated via SDS-PAGE and immunoblotted on nitrocellulose membranes. The immunoblots have material derived from whole-cells (WC; 2 x 10 8 cells), total membrane (3 x 10 8 cells) and soluble (1 x 10 8 cells) fractions; WC, periplasm and osmotically-shocked cell (OSC) fractions (5 x 10 8 cells each); WC (2 x 10 8 cells), culture fluid (CF; from culture containing 2 x 10 9 cells) and vesicle-free cleared culture fluid (CCF; from culture containing 2 x 10 9 cells). Immunoblots were probed with either anti-rKgp cat ( A , 1/200,000 dilution), pre-adsorbed anti-rRgpA cat ( B , 1/10,000 dilution), anti-rKgp A1 ( C , 1/10,000 dilution) or anti-rRgpB-CTD 422 ( D , 1/200,000 dilution) followed by goat anti-rabbit ( A ) or horse anti-mouse ( B , C and D ) IgG-conjugated HRP secondary antibodies (1/3,000 dilution).

    Article Snippet: The membrane was blocked with 5% non-fat skim milk in PBST0.1 (0.1% v/v Tween-20 in PBS) and probed with primary antibody followed by a goat anti-rabbit or horse anti-mouse horseradish peroxidase (HRP)-conjugated IgG secondary antibody (Cell Signaling Technology Inc.).

    Techniques: Mutagenesis, SDS Page, Western Blot, Derivative Assay

    Absence of surface-associated Kgp in the pg1058 mutant determined by whole-cell ELISA analysis. FKWC (10 9 per well) prepared from P . gingivalis W50 (positive control), pg1058 mutant and kgp mutant (negative control) were probed with two-fold dilutions of anti-rKgp cat antisera (1/5,000–1/80,000) followed by goat anti-rabbit HRP-conjugated IgG (1/3,000) and developed with ABTS chromogenic substrate. The chromogenic reaction was stopped and detected at 405 nm. Controls included no primary antibody control (No 1°Ab), no secondary antibody control (No 2°Ab) and no cell control (No Bacteria). Mean ± SEM (standard error of the mean), N = 3 for each strain.

    Journal: PLoS ONE

    Article Title: PG1058 Is a Novel Multidomain Protein Component of the Bacterial Type IX Secretion System

    doi: 10.1371/journal.pone.0164313

    Figure Lengend Snippet: Absence of surface-associated Kgp in the pg1058 mutant determined by whole-cell ELISA analysis. FKWC (10 9 per well) prepared from P . gingivalis W50 (positive control), pg1058 mutant and kgp mutant (negative control) were probed with two-fold dilutions of anti-rKgp cat antisera (1/5,000–1/80,000) followed by goat anti-rabbit HRP-conjugated IgG (1/3,000) and developed with ABTS chromogenic substrate. The chromogenic reaction was stopped and detected at 405 nm. Controls included no primary antibody control (No 1°Ab), no secondary antibody control (No 2°Ab) and no cell control (No Bacteria). Mean ± SEM (standard error of the mean), N = 3 for each strain.

    Article Snippet: The membrane was blocked with 5% non-fat skim milk in PBST0.1 (0.1% v/v Tween-20 in PBS) and probed with primary antibody followed by a goat anti-rabbit or horse anti-mouse horseradish peroxidase (HRP)-conjugated IgG secondary antibody (Cell Signaling Technology Inc.).

    Techniques: Mutagenesis, Enzyme-linked Immunosorbent Assay, Positive Control, Negative Control

    PG1058 is an OM-associated periplasmic protein. A. P . gingivalis W50, pg1058 mutant and pg1058 + complement strain cultures were fractionated, separated via SDS-PAGE and immunoblotted on nitrocellulose membranes. The immunoblots have material derived from vesicles (5 x 10 9 cells); whole-cells (WC; 2 x 10 8 cells), culture fluid (CF; from culture containing 2 x 10 9 cells) and vesicle-free cleared culture fluid (CCF; from culture containing 2 x 10 9 cells); WC (2 x 10 8 cells), soluble (5 x 10 9 cells), total membrane (Membrane; 1.6 x 10 10 cells), sarkosyl-insoluble membrane (SI; 1.6 x 10 10 cells) and sarkosyl-soluble membrane (SS; 1.6 x 10 10 cells) fractions; WC (5 x 10 8 cells), periplasm (5 x 10 8 cells) and osmotically-shocked cell (OSC; 5 x 10 8 cells) fractions. Immunoblots were probed with anti-rPG1058 (1/10,000 dilution) and horse anti-mouse IgG-conjugated HRP secondary antibody (1/3,000 dilution). B. Whole-cell ELISA. P . gingivalis W50, pg1058 mutant, pg1058 + complement FKWC or lysed P . gingivalis W50 FKWC as a positive control (2 x 10 9 cells per well) were probed with anti-rPG1058 antiserum (1/100, 1/200, 1/400, 1/800 and 1/1600 dilutions) followed by horse anti-mouse HRP-conjugated IgG secondary antibody (1/3,000) and developed with ABTS chromogenic substrate. The chromogenic reaction was suspended and detected at 405 nm. Other controls included no primary antibody control (No 1°Ab), no secondary antibody control (No 2°Ab) and no cell control (No Bacteria). Mean ± SEM, N = 3 for each strain except where only one replicate ( N = 1) of the positive control (Positive) was assessed. C. P . gingivalis W50 total membrane was separated via sucrose density gradient centrifugation then fractions were separated by SDS-PAGE and immunoblotted on nitrocellulose membrane. Fraction numbers are indicated above the lanes and P refers to the pellet (unequal loading). Immunoblots were probed with anti-rPG1058 (1/10,000 dilution) or anti-Omp41 (1/10,000 dilution) followed by horse anti-mouse IgG-conjugated HRP secondary antibody (1/3,000 dilution).

    Journal: PLoS ONE

    Article Title: PG1058 Is a Novel Multidomain Protein Component of the Bacterial Type IX Secretion System

    doi: 10.1371/journal.pone.0164313

    Figure Lengend Snippet: PG1058 is an OM-associated periplasmic protein. A. P . gingivalis W50, pg1058 mutant and pg1058 + complement strain cultures were fractionated, separated via SDS-PAGE and immunoblotted on nitrocellulose membranes. The immunoblots have material derived from vesicles (5 x 10 9 cells); whole-cells (WC; 2 x 10 8 cells), culture fluid (CF; from culture containing 2 x 10 9 cells) and vesicle-free cleared culture fluid (CCF; from culture containing 2 x 10 9 cells); WC (2 x 10 8 cells), soluble (5 x 10 9 cells), total membrane (Membrane; 1.6 x 10 10 cells), sarkosyl-insoluble membrane (SI; 1.6 x 10 10 cells) and sarkosyl-soluble membrane (SS; 1.6 x 10 10 cells) fractions; WC (5 x 10 8 cells), periplasm (5 x 10 8 cells) and osmotically-shocked cell (OSC; 5 x 10 8 cells) fractions. Immunoblots were probed with anti-rPG1058 (1/10,000 dilution) and horse anti-mouse IgG-conjugated HRP secondary antibody (1/3,000 dilution). B. Whole-cell ELISA. P . gingivalis W50, pg1058 mutant, pg1058 + complement FKWC or lysed P . gingivalis W50 FKWC as a positive control (2 x 10 9 cells per well) were probed with anti-rPG1058 antiserum (1/100, 1/200, 1/400, 1/800 and 1/1600 dilutions) followed by horse anti-mouse HRP-conjugated IgG secondary antibody (1/3,000) and developed with ABTS chromogenic substrate. The chromogenic reaction was suspended and detected at 405 nm. Other controls included no primary antibody control (No 1°Ab), no secondary antibody control (No 2°Ab) and no cell control (No Bacteria). Mean ± SEM, N = 3 for each strain except where only one replicate ( N = 1) of the positive control (Positive) was assessed. C. P . gingivalis W50 total membrane was separated via sucrose density gradient centrifugation then fractions were separated by SDS-PAGE and immunoblotted on nitrocellulose membrane. Fraction numbers are indicated above the lanes and P refers to the pellet (unequal loading). Immunoblots were probed with anti-rPG1058 (1/10,000 dilution) or anti-Omp41 (1/10,000 dilution) followed by horse anti-mouse IgG-conjugated HRP secondary antibody (1/3,000 dilution).

    Article Snippet: The membrane was blocked with 5% non-fat skim milk in PBST0.1 (0.1% v/v Tween-20 in PBS) and probed with primary antibody followed by a goat anti-rabbit or horse anti-mouse horseradish peroxidase (HRP)-conjugated IgG secondary antibody (Cell Signaling Technology Inc.).

    Techniques: Mutagenesis, SDS Page, Western Blot, Derivative Assay, Enzyme-linked Immunosorbent Assay, Positive Control, Gradient Centrifugation

    Presence of A-LPS in the pg1058 mutant. A. P . gingivalis W50, pg105 8 mutant and pg1058 + complement strain cultures were fractionated, separated via SDS-PAGE and immunoblotted on nitrocellulose membranes (as per Fig 5 ). Immunoblots were probed with MAb 1B5 (1/10,000 dilution) followed by horse anti-mouse IgG-conjugated HRP secondary antibody (1/3,000 dilution). B. Whole-cell ELISA of P . gingivalis W50 and pg1058 mutant FKWC (10 9 per well) probed with two-fold dilutions of MAb 1B5 from 1/100–1/1,600 followed by horse anti-mouse HRP-conjugated IgG secondary antibody (1/3,000) and developed with ABTS chromogenic substrate. The chromogenic reaction was stopped and detected at 405 nm. Controls included no primary antibody control (No 1°Ab), no secondary antibody control (No 2°Ab) and no cell control (No Bacteria). Mean ± SEM, N = 3 for each strain. C. Immunogold TEM micrographs representative of the P . gingivalis W50 ( N = 19) and pg1058 mutant ( N = 29). Probing with MAb 1B5 (1/10,000 dilution) was followed by detection with anti-mouse IgG conjugated to 18 nm colloidal gold particles (1/40 dilution). D. The immunogold particle distribution. Distance between IM and centre of the gold particle measured in nm, distances less than 0 nm correspond to a cytoplasmic and IM localisation, 0–20 nm corresponds to the periplasmic space, 21–120 corresponds to the OM and extracellular environment. Mean ± SEM.

    Journal: PLoS ONE

    Article Title: PG1058 Is a Novel Multidomain Protein Component of the Bacterial Type IX Secretion System

    doi: 10.1371/journal.pone.0164313

    Figure Lengend Snippet: Presence of A-LPS in the pg1058 mutant. A. P . gingivalis W50, pg105 8 mutant and pg1058 + complement strain cultures were fractionated, separated via SDS-PAGE and immunoblotted on nitrocellulose membranes (as per Fig 5 ). Immunoblots were probed with MAb 1B5 (1/10,000 dilution) followed by horse anti-mouse IgG-conjugated HRP secondary antibody (1/3,000 dilution). B. Whole-cell ELISA of P . gingivalis W50 and pg1058 mutant FKWC (10 9 per well) probed with two-fold dilutions of MAb 1B5 from 1/100–1/1,600 followed by horse anti-mouse HRP-conjugated IgG secondary antibody (1/3,000) and developed with ABTS chromogenic substrate. The chromogenic reaction was stopped and detected at 405 nm. Controls included no primary antibody control (No 1°Ab), no secondary antibody control (No 2°Ab) and no cell control (No Bacteria). Mean ± SEM, N = 3 for each strain. C. Immunogold TEM micrographs representative of the P . gingivalis W50 ( N = 19) and pg1058 mutant ( N = 29). Probing with MAb 1B5 (1/10,000 dilution) was followed by detection with anti-mouse IgG conjugated to 18 nm colloidal gold particles (1/40 dilution). D. The immunogold particle distribution. Distance between IM and centre of the gold particle measured in nm, distances less than 0 nm correspond to a cytoplasmic and IM localisation, 0–20 nm corresponds to the periplasmic space, 21–120 corresponds to the OM and extracellular environment. Mean ± SEM.

    Article Snippet: The membrane was blocked with 5% non-fat skim milk in PBST0.1 (0.1% v/v Tween-20 in PBS) and probed with primary antibody followed by a goat anti-rabbit or horse anti-mouse horseradish peroxidase (HRP)-conjugated IgG secondary antibody (Cell Signaling Technology Inc.).

    Techniques: Mutagenesis, SDS Page, Western Blot, Enzyme-linked Immunosorbent Assay, Transmission Electron Microscopy

    The hybrids 7, 9, 5, 17, 18, 19, 13 and the etoposide positive control induced double-strand DNA breaks in K562 cells as indicated by formation of γH2AX. K562 cells were treated with 20 µM of the drugs indicated for 4 h in growth medium, lysed and subjected to SDS-PAGE electrophoresis and Western blotting. The blots were probed with antibodies to γH2AX and with GAPDH (glyceraldehyde 3-phosphate dehydrogenase) as a loading control and a chemiluminescent-inducing horseradish peroxidase-conjugated secondary antibody. The results were typical of experiments carried out on 2 different days.

    Journal: Bioorganic & medicinal chemistry

    Article Title: Structure-based design, synthesis and biological testing of etoposide analog epipodophyllotoxin-N-mustard hybrid compounds designed to covalently bind to topoisomerase II and DNA

    doi: 10.1016/j.bmc.2014.09.014

    Figure Lengend Snippet: The hybrids 7, 9, 5, 17, 18, 19, 13 and the etoposide positive control induced double-strand DNA breaks in K562 cells as indicated by formation of γH2AX. K562 cells were treated with 20 µM of the drugs indicated for 4 h in growth medium, lysed and subjected to SDS-PAGE electrophoresis and Western blotting. The blots were probed with antibodies to γH2AX and with GAPDH (glyceraldehyde 3-phosphate dehydrogenase) as a loading control and a chemiluminescent-inducing horseradish peroxidase-conjugated secondary antibody. The results were typical of experiments carried out on 2 different days.

    Article Snippet: This was followed by incubation for 1 h with peroxidase-conjugated goat-anti-rabbit secondary antibody (Cell Signaling Technology) diluted 1:2000.

    Techniques: Positive Control, SDS Page, Electrophoresis, Western Blot