Structured Review

GE Healthcare hrp conjugated secondary antibody
Native EAG and <t>CMG</t> coimmunoprecipitate from Drosophila extracts. Protein extracts were prepared from the nervous system, imaginal discs, and body muscle fibers of third-instar larvae. CMG antisera were used to immunopreciptate CMG and associating proteins from extracts prepared from the indicated genotypes. Precipitated proteins were resolved by SDS-PAGE, transferred to nitrocellulose, and then probed with either CMG ( A ) or EAG (NT) antisera ( B , right), followed by <t>HRP-conjugated</t> anti-guinea pig or anti-rabbit IgG, respectively. For comparison, input extract probed with EAG (NT) antisera is shown in the left panel of B . Respective loads were 5, 10, and 20 μl for extracts, immunoprecipitated proteins, and coimmunoprecipitated proteins, respectively (see Materials and Methods). Similar results were obtained in five experiments. Bands were visualized using ECL. WT, Wild type; IP, immunoprecipitation.
Hrp Conjugated Secondary Antibody, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 94/100, based on 732 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/hrp conjugated secondary antibody/product/GE Healthcare
Average 94 stars, based on 732 article reviews
Price from $9.99 to $1999.99
hrp conjugated secondary antibody - by Bioz Stars, 2020-07
94/100 stars

Images

1) Product Images from "Camguk/CASK Enhances Ether-Á-Go-Go Potassium Current by a Phosphorylation-Dependent Mechanism"

Article Title: Camguk/CASK Enhances Ether-Á-Go-Go Potassium Current by a Phosphorylation-Dependent Mechanism

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.4566-04.2005

Native EAG and CMG coimmunoprecipitate from Drosophila extracts. Protein extracts were prepared from the nervous system, imaginal discs, and body muscle fibers of third-instar larvae. CMG antisera were used to immunopreciptate CMG and associating proteins from extracts prepared from the indicated genotypes. Precipitated proteins were resolved by SDS-PAGE, transferred to nitrocellulose, and then probed with either CMG ( A ) or EAG (NT) antisera ( B , right), followed by HRP-conjugated anti-guinea pig or anti-rabbit IgG, respectively. For comparison, input extract probed with EAG (NT) antisera is shown in the left panel of B . Respective loads were 5, 10, and 20 μl for extracts, immunoprecipitated proteins, and coimmunoprecipitated proteins, respectively (see Materials and Methods). Similar results were obtained in five experiments. Bands were visualized using ECL. WT, Wild type; IP, immunoprecipitation.
Figure Legend Snippet: Native EAG and CMG coimmunoprecipitate from Drosophila extracts. Protein extracts were prepared from the nervous system, imaginal discs, and body muscle fibers of third-instar larvae. CMG antisera were used to immunopreciptate CMG and associating proteins from extracts prepared from the indicated genotypes. Precipitated proteins were resolved by SDS-PAGE, transferred to nitrocellulose, and then probed with either CMG ( A ) or EAG (NT) antisera ( B , right), followed by HRP-conjugated anti-guinea pig or anti-rabbit IgG, respectively. For comparison, input extract probed with EAG (NT) antisera is shown in the left panel of B . Respective loads were 5, 10, and 20 μl for extracts, immunoprecipitated proteins, and coimmunoprecipitated proteins, respectively (see Materials and Methods). Similar results were obtained in five experiments. Bands were visualized using ECL. WT, Wild type; IP, immunoprecipitation.

Techniques Used: SDS Page, Immunoprecipitation

2) Product Images from "Mitochondrial targeting of the electrophilic lipid 15-deoxy-?12,14-Prostaglandin J2 increases apoptotic efficacy via redox cell signaling mechanisms"

Article Title: Mitochondrial targeting of the electrophilic lipid 15-deoxy-?12,14-Prostaglandin J2 increases apoptotic efficacy via redox cell signaling mechanisms

Journal: The Biochemical journal

doi: 10.1042/BJ20091293

Visualization of bt-15d-PGJ 2 and mito-15d-PGJ 2 modified proteins by 2D- IEF-SDS-PAGE Cell lysates from MDA-MB231 cells were separated by 2D-IEF. Ethanol vehicle control (1 h) treated cell lysates were probed using Streptavidin-HRP ( A ) and TPP+ antibody with HRP-conjugated secondary antibody ( B ) to determine background reactivity. Cells were treated (10 μM, 1 h) with bt-15d-PGJ 2 ( C ) or mito-15d-PGJ 2 ( D ) and protein adducts were detected using Streptavidin-HRP and TPP+ antibody respectively. A merged image of bt-15d-PGJ 2 (red) and mito-15d-PGJ 2 (green) adduct blots is shown ( E ). Analysis of spot quantity was determined and common and unique spots from bt-15d-PGJ 2 and mito-15d-PGJ 2 membranes are shown schematically ( F ).
Figure Legend Snippet: Visualization of bt-15d-PGJ 2 and mito-15d-PGJ 2 modified proteins by 2D- IEF-SDS-PAGE Cell lysates from MDA-MB231 cells were separated by 2D-IEF. Ethanol vehicle control (1 h) treated cell lysates were probed using Streptavidin-HRP ( A ) and TPP+ antibody with HRP-conjugated secondary antibody ( B ) to determine background reactivity. Cells were treated (10 μM, 1 h) with bt-15d-PGJ 2 ( C ) or mito-15d-PGJ 2 ( D ) and protein adducts were detected using Streptavidin-HRP and TPP+ antibody respectively. A merged image of bt-15d-PGJ 2 (red) and mito-15d-PGJ 2 (green) adduct blots is shown ( E ). Analysis of spot quantity was determined and common and unique spots from bt-15d-PGJ 2 and mito-15d-PGJ 2 membranes are shown schematically ( F ).

Techniques Used: Modification, Electrofocusing, SDS Page, Multiple Displacement Amplification

Localization of 15d-PGJ 2 and mito-15d-PGJ 2 to the mitochondrion and effects on mitochondrial membrane potential (Δ ψ) MDA-MB231 cells were treated with 10 μM bt-15d-PGJ 2 (bt-15d) or mito-15d-PGJ 2 (Mito-15d) for 1 h then mitochondrial and cytosolic fractions were prepared and resolved using 10% SDS-PAGE. Adduct formation was detected using an anti-TPP antibody (for mito-15d-PGJ 2 ) or streptavidin-HRP (for bt-15d-PGJ 2 ). ECL+ coupled to horseradish peroxidase was used to detect chemifluorescence protein adducts ( A ). VDAC protein levels were also determined using Western blot analysis to confirm mitochondrial preparation purity. Protein adducts were quantified and expressed as a percent of homogenate ( B ). MDA-MB231 cells were exposed to increasing concentrations (1–10 μM) of 15d-PGJ 2 , mito-15d-PGJ 2 , or TPMP for 4 h then Δ ψ was assessed using JC-1 ( C ). Data are represented as the ratio of red/green fluorescence. Results represent means ± s.e.m., n = 3–6. * p
Figure Legend Snippet: Localization of 15d-PGJ 2 and mito-15d-PGJ 2 to the mitochondrion and effects on mitochondrial membrane potential (Δ ψ) MDA-MB231 cells were treated with 10 μM bt-15d-PGJ 2 (bt-15d) or mito-15d-PGJ 2 (Mito-15d) for 1 h then mitochondrial and cytosolic fractions were prepared and resolved using 10% SDS-PAGE. Adduct formation was detected using an anti-TPP antibody (for mito-15d-PGJ 2 ) or streptavidin-HRP (for bt-15d-PGJ 2 ). ECL+ coupled to horseradish peroxidase was used to detect chemifluorescence protein adducts ( A ). VDAC protein levels were also determined using Western blot analysis to confirm mitochondrial preparation purity. Protein adducts were quantified and expressed as a percent of homogenate ( B ). MDA-MB231 cells were exposed to increasing concentrations (1–10 μM) of 15d-PGJ 2 , mito-15d-PGJ 2 , or TPMP for 4 h then Δ ψ was assessed using JC-1 ( C ). Data are represented as the ratio of red/green fluorescence. Results represent means ± s.e.m., n = 3–6. * p

Techniques Used: Multiple Displacement Amplification, SDS Page, Western Blot, Fluorescence

3) Product Images from "Fibulin-1 Binds to Fibroblast Growth Factor 8 with High Affinity"

Article Title: Fibulin-1 Binds to Fibroblast Growth Factor 8 with High Affinity

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M115.702761

Placental fibulin-1 protects FGF8b from enzymatic degradation. FGF8b (150 ng) was incubated with and without ADAM17 (0.2 μg) in the presence or absence of FBLN1 (1.4 μg). After 18 h of incubation, 10 μl of 4× sample buffer was added to the 30-μl reaction mixture and then loaded on 4–12% NUPAGE gel. Proteins were then transferred to a PVDF membrane. FGF8 was detected using monoclonal primary antibodies and anti-mouse horseradish peroxidase (HRP)-conjugated IgG. Visualization was done using ECL chemiluminescent reagent ( A ). Reblotting with ADAM17/TACE antibodies detected the enzyme lanes 3 and 4 ( B ). Data presented are representative of three independent experiments.
Figure Legend Snippet: Placental fibulin-1 protects FGF8b from enzymatic degradation. FGF8b (150 ng) was incubated with and without ADAM17 (0.2 μg) in the presence or absence of FBLN1 (1.4 μg). After 18 h of incubation, 10 μl of 4× sample buffer was added to the 30-μl reaction mixture and then loaded on 4–12% NUPAGE gel. Proteins were then transferred to a PVDF membrane. FGF8 was detected using monoclonal primary antibodies and anti-mouse horseradish peroxidase (HRP)-conjugated IgG. Visualization was done using ECL chemiluminescent reagent ( A ). Reblotting with ADAM17/TACE antibodies detected the enzyme lanes 3 and 4 ( B ). Data presented are representative of three independent experiments.

Techniques Used: Incubation

4) Product Images from "MMP-2 Downregulation Mediates Differential Regulation of Cell Death via ErbB-2 in Glioma Xenografts"

Article Title: MMP-2 Downregulation Mediates Differential Regulation of Cell Death via ErbB-2 in Glioma Xenografts

Journal: International journal of oncology

doi:

Downregulation of MMP-2 causes the up regulation of APAF-1 and ErbB-2 in 5310 but not in 4910 human glioma xenograft cells in vivo Nude mice with pre-established intracranial human glioma tumors (4910 or 5310) were treated with Ad-MMP-2 by intracranial injections (1 or 10×10 6 pfu). 10 days following Ad-MMP-2 administration the brains were harvested sectioned and immunoprobed for ErbB-2 and APAF-1 using appropriate HRP conjugated (for ErbB2) or TxRed conjugated (for APAF-1) secondary antibody.
Figure Legend Snippet: Downregulation of MMP-2 causes the up regulation of APAF-1 and ErbB-2 in 5310 but not in 4910 human glioma xenograft cells in vivo Nude mice with pre-established intracranial human glioma tumors (4910 or 5310) were treated with Ad-MMP-2 by intracranial injections (1 or 10×10 6 pfu). 10 days following Ad-MMP-2 administration the brains were harvested sectioned and immunoprobed for ErbB-2 and APAF-1 using appropriate HRP conjugated (for ErbB2) or TxRed conjugated (for APAF-1) secondary antibody.

Techniques Used: In Vivo, Mouse Assay

Related Articles

Western Blot:

Article Title: Bradykinin 1 receptor blockade subdues systemic autoimmunity, renal inflammation, and blood pressure in murine lupus nephritis
Article Snippet: .. HRP-conjugated secondary antibodies and the ECL-plus detection kit (Amersham, Little Chalfont, UK) were used for western blot. ..

Incubation:

Article Title: The Myosin Va Head Domain Binds to the Neurofilament-L Rod and Modulates Endoplasmic Reticulum (ER) Content and Distribution within Axons
Article Snippet: .. After washing, the membranes were incubated with the corresponding HRP-conjugated secondary antibodies and immunoreactive bands were detected with ECL reagent (GE Healthcare, NJ). .. Images were scanned, and bands were quantified with a multigauge program (Fuji film, Japan).

Binding Assay:

Article Title: Specific Binding of the Karyopherin Kap121p to a Subunit of the Nuclear Pore Complex Containing Nup53p, Nup59p, and Nup170p
Article Snippet: .. Antibody binding was visualized with HRP-conjugated secondary antibodies and ECL using procedures outlined by the manufacturer ( Amersham Corp. , Oakville, Ontario, Canada). .. In Vitro Binding Assays In vitro binding assays were performed using the recombinant proteins GST-Nup53p, GST-Nup59p, GST-Kap121p, and GST purified as described above.

Blocking Assay:

Article Title: Mitochondrial targeting of the electrophilic lipid 15-deoxy-?12,14-Prostaglandin J2 increases apoptotic efficacy via redox cell signaling mechanisms
Article Snippet: .. After blocking for 1 h, the membranes were probed using an anti-TPP+ antibody (1:10,000; received from Dr. Michael P. Murphy) with a HRP-conjugated secondary antibody and streptavidin conjugated to HRP (GE Healthcare) for detection of bt-15d-PGJ2 and mito-15d-PGJ2 adducts, respectively. .. Membranes were developed as described above, and total protein was detected using Deep Purple stain (GE Healthcare).

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  • 88
    GE Healthcare anti mouse secondary antibody conjugated to hrp
    LGG induces ROS that oxidizes cysteines. (A) Intestinal epithelial cells (SKCO15) were contacted with 10 8 CFU/mL of E. coli or LGG for 15 min, and then with 15 µM HydroCy3 for 30 min before confocal microscopic analysis at 555 nm, (scale bars 20 µm). (B) SKCO15 were contacted with 10 8 CFU/mL of E. coli or LGG for 15 min, labeled for 30 min with a thiol-reactive, Thiol Tracker™ fluorescence probe, and then analyzed by fluorescence microscopy at 405 nm (scale bars 200 µm). Mean image intensity is shown at bottom left for A and B. (C) Biotinylated-iodoacetamide (BIAM) labeling of cysteine residues in lysates of LGG or E. coli contacted SKCO15 cells, followed by pull-down of labeled residues with <t>streptavidin</t> conjugated agarose and detected by Western blot using <t>HRP</t> conjugated streptavidin as a probe. The relative intensity of each lane of the blot is shown in arbitrary units to the left. Each value was normalized to calnexin that served as a loading control to give the relative oxidation amounts. (D) Dual labeling of LGG or E. coli contacted (15 mins) SKCO15 cultured cells with HydroCy3 (red) and Lysotracker (green). Note co-localization of lysotracker and hydro-Cy3 in LGG contacted cells (bars 10 µm).
    Anti Mouse Secondary Antibody Conjugated To Hrp, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 88/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti mouse secondary antibody conjugated to hrp/product/GE Healthcare
    Average 88 stars, based on 20 article reviews
    Price from $9.99 to $1999.99
    anti mouse secondary antibody conjugated to hrp - by Bioz Stars, 2020-07
    88/100 stars
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    85
    GE Healthcare horse raddish peroxidase hrp conjugated secondary antibodies sheep anti mouse igg
    Degradation of deposited C3b by B. anthracis spores-bound PLG. (A) Spores (2×10 7 /well) were immobilized onto a microplate and incubated with 10% NHS (100 µl) for 30 min at room temperature. Washed spores were incubated with 2 µg/well PLG for 1 h in the presence or absence of protease cocktail. Bound PLG was activated by uPA (20 units/well) for 3 h at 37°C. Deposited C3b was detected by anti-C3c <t>IgG</t> and <t>HRP-conjugated</t> secondary antibody followed by TMB reaction. C3b deposition is expressed as the mean absorbance at 450 nm of quadruplicates. Error bars indicate ± standard deviation. *P
    Horse Raddish Peroxidase Hrp Conjugated Secondary Antibodies Sheep Anti Mouse Igg, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/horse raddish peroxidase hrp conjugated secondary antibodies sheep anti mouse igg/product/GE Healthcare
    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    horse raddish peroxidase hrp conjugated secondary antibodies sheep anti mouse igg - by Bioz Stars, 2020-07
    85/100 stars
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    94
    GE Healthcare hrp conjugated secondary antibody
    Visualization of bt-15d-PGJ 2 and mito-15d-PGJ 2 modified proteins by 2D- IEF-SDS-PAGE Cell lysates from MDA-MB231 cells were separated by 2D-IEF. Ethanol vehicle control (1 h) treated cell lysates were probed using <t>Streptavidin-HRP</t> ( A ) and <t>TPP+</t> antibody with HRP-conjugated secondary antibody ( B ) to determine background reactivity. Cells were treated (10 μM, 1 h) with bt-15d-PGJ 2 ( C ) or mito-15d-PGJ 2 ( D ) and protein adducts were detected using Streptavidin-HRP and TPP+ antibody respectively. A merged image of bt-15d-PGJ 2 (red) and mito-15d-PGJ 2 (green) adduct blots is shown ( E ). Analysis of spot quantity was determined and common and unique spots from bt-15d-PGJ 2 and mito-15d-PGJ 2 membranes are shown schematically ( F ).
    Hrp Conjugated Secondary Antibody, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 94/100, based on 732 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hrp conjugated secondary antibody/product/GE Healthcare
    Average 94 stars, based on 732 article reviews
    Price from $9.99 to $1999.99
    hrp conjugated secondary antibody - by Bioz Stars, 2020-07
    94/100 stars
      Buy from Supplier

    Image Search Results


    LGG induces ROS that oxidizes cysteines. (A) Intestinal epithelial cells (SKCO15) were contacted with 10 8 CFU/mL of E. coli or LGG for 15 min, and then with 15 µM HydroCy3 for 30 min before confocal microscopic analysis at 555 nm, (scale bars 20 µm). (B) SKCO15 were contacted with 10 8 CFU/mL of E. coli or LGG for 15 min, labeled for 30 min with a thiol-reactive, Thiol Tracker™ fluorescence probe, and then analyzed by fluorescence microscopy at 405 nm (scale bars 200 µm). Mean image intensity is shown at bottom left for A and B. (C) Biotinylated-iodoacetamide (BIAM) labeling of cysteine residues in lysates of LGG or E. coli contacted SKCO15 cells, followed by pull-down of labeled residues with streptavidin conjugated agarose and detected by Western blot using HRP conjugated streptavidin as a probe. The relative intensity of each lane of the blot is shown in arbitrary units to the left. Each value was normalized to calnexin that served as a loading control to give the relative oxidation amounts. (D) Dual labeling of LGG or E. coli contacted (15 mins) SKCO15 cultured cells with HydroCy3 (red) and Lysotracker (green). Note co-localization of lysotracker and hydro-Cy3 in LGG contacted cells (bars 10 µm).

    Journal: Redox Biology

    Article Title: Proteomic analysis of microbial induced redox-dependent intestinal signaling

    doi: 10.1016/j.redox.2018.11.011

    Figure Lengend Snippet: LGG induces ROS that oxidizes cysteines. (A) Intestinal epithelial cells (SKCO15) were contacted with 10 8 CFU/mL of E. coli or LGG for 15 min, and then with 15 µM HydroCy3 for 30 min before confocal microscopic analysis at 555 nm, (scale bars 20 µm). (B) SKCO15 were contacted with 10 8 CFU/mL of E. coli or LGG for 15 min, labeled for 30 min with a thiol-reactive, Thiol Tracker™ fluorescence probe, and then analyzed by fluorescence microscopy at 405 nm (scale bars 200 µm). Mean image intensity is shown at bottom left for A and B. (C) Biotinylated-iodoacetamide (BIAM) labeling of cysteine residues in lysates of LGG or E. coli contacted SKCO15 cells, followed by pull-down of labeled residues with streptavidin conjugated agarose and detected by Western blot using HRP conjugated streptavidin as a probe. The relative intensity of each lane of the blot is shown in arbitrary units to the left. Each value was normalized to calnexin that served as a loading control to give the relative oxidation amounts. (D) Dual labeling of LGG or E. coli contacted (15 mins) SKCO15 cultured cells with HydroCy3 (red) and Lysotracker (green). Note co-localization of lysotracker and hydro-Cy3 in LGG contacted cells (bars 10 µm).

    Article Snippet: Anti-mouse secondary antibody conjugated to HRP was obtained from GE, while the streptavidin conjugated HRP was obtained from Abcam.

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

    mAb binding specificities. A , mAb binding to ZnT8-His immobilized to Ni-NTA plate. Bound mAb20 ( red ) or mAb39 ( magenta ) was detected by an HRP-conjugated secondary antibody. Solid lines , least-squares fits of binding data points to a one-component binding process. Error bars , S.E. ( n = 8). B , mouse ZnT8-His ( M ) and human ZnT8-His ( H ) were SDS-denatured on immunoblots and detected by mAb20 or mAb39, as indicated. A replicated immunoblot was probed by an anti-His tag antibody to show that approximately the same amounts of HEK293 crude lysates were loaded to each lane. All immunoblots were recorded using an identical exposure time on Amersham Biosciences Imager 600. C , detection of endogenous human ZnT8 in EndoC-βH cells by mAb20 or mAb39 immunoblotting. An equal amount of EndoC-βH lysate (∼50 μg of total protein) was loaded to each lane. D , immunoblotting of HEK lysates with overexpression of a ZnT homolog as indicated. An approximately equal amount of total lysate protein (5 μg) was located to each lane , probed by either mAb20, mAb39, or anti-FLAG antibody, as indicated. Note that the mAb39 immunoblot was recorded with an over 100-fold longer exposure time as compared with that of the mAb20 immunoblot. a.u. , arbitrary units.

    Journal: The Journal of Biological Chemistry

    Article Title: Highly specific monoclonal antibodies for allosteric inhibition and immunodetection of the human pancreatic zinc transporter ZnT8

    doi: 10.1074/jbc.RA118.005136

    Figure Lengend Snippet: mAb binding specificities. A , mAb binding to ZnT8-His immobilized to Ni-NTA plate. Bound mAb20 ( red ) or mAb39 ( magenta ) was detected by an HRP-conjugated secondary antibody. Solid lines , least-squares fits of binding data points to a one-component binding process. Error bars , S.E. ( n = 8). B , mouse ZnT8-His ( M ) and human ZnT8-His ( H ) were SDS-denatured on immunoblots and detected by mAb20 or mAb39, as indicated. A replicated immunoblot was probed by an anti-His tag antibody to show that approximately the same amounts of HEK293 crude lysates were loaded to each lane. All immunoblots were recorded using an identical exposure time on Amersham Biosciences Imager 600. C , detection of endogenous human ZnT8 in EndoC-βH cells by mAb20 or mAb39 immunoblotting. An equal amount of EndoC-βH lysate (∼50 μg of total protein) was loaded to each lane. D , immunoblotting of HEK lysates with overexpression of a ZnT homolog as indicated. An approximately equal amount of total lysate protein (5 μg) was located to each lane , probed by either mAb20, mAb39, or anti-FLAG antibody, as indicated. Note that the mAb39 immunoblot was recorded with an over 100-fold longer exposure time as compared with that of the mAb20 immunoblot. a.u. , arbitrary units.

    Article Snippet: SDS-solubilized cell lysate of EndoC-βH cells or HEK293 cells overexpressing ZnT8 was subjected to SDS-PAGE and probed with a 1:3000 mAb20 or mAb39, followed by 1:3000 anti-mouse HRP-conjugated secondary antibody (GE Healthcare, catalog no. NA931V).

    Techniques: Binding Assay, Western Blot, Over Expression

    Degradation of deposited C3b by B. anthracis spores-bound PLG. (A) Spores (2×10 7 /well) were immobilized onto a microplate and incubated with 10% NHS (100 µl) for 30 min at room temperature. Washed spores were incubated with 2 µg/well PLG for 1 h in the presence or absence of protease cocktail. Bound PLG was activated by uPA (20 units/well) for 3 h at 37°C. Deposited C3b was detected by anti-C3c IgG and HRP-conjugated secondary antibody followed by TMB reaction. C3b deposition is expressed as the mean absorbance at 450 nm of quadruplicates. Error bars indicate ± standard deviation. *P

    Journal: PLoS ONE

    Article Title: Bacillus anthracis Interacts with Plasmin(ogen) to Evade C3b-Dependent Innate Immunity

    doi: 10.1371/journal.pone.0018119

    Figure Lengend Snippet: Degradation of deposited C3b by B. anthracis spores-bound PLG. (A) Spores (2×10 7 /well) were immobilized onto a microplate and incubated with 10% NHS (100 µl) for 30 min at room temperature. Washed spores were incubated with 2 µg/well PLG for 1 h in the presence or absence of protease cocktail. Bound PLG was activated by uPA (20 units/well) for 3 h at 37°C. Deposited C3b was detected by anti-C3c IgG and HRP-conjugated secondary antibody followed by TMB reaction. C3b deposition is expressed as the mean absorbance at 450 nm of quadruplicates. Error bars indicate ± standard deviation. *P

    Article Snippet: Horse raddish peroxidase (HRP)-conjugated secondary antibodies sheep anti-mouse IgG and donkey anti-rabbit IgG antibody were purchased from GE Healthcare and rabbit anti-goat IgG antibody from Jackson Immuno Research.

    Techniques: Incubation, Standard Deviation

    Visualization of bt-15d-PGJ 2 and mito-15d-PGJ 2 modified proteins by 2D- IEF-SDS-PAGE Cell lysates from MDA-MB231 cells were separated by 2D-IEF. Ethanol vehicle control (1 h) treated cell lysates were probed using Streptavidin-HRP ( A ) and TPP+ antibody with HRP-conjugated secondary antibody ( B ) to determine background reactivity. Cells were treated (10 μM, 1 h) with bt-15d-PGJ 2 ( C ) or mito-15d-PGJ 2 ( D ) and protein adducts were detected using Streptavidin-HRP and TPP+ antibody respectively. A merged image of bt-15d-PGJ 2 (red) and mito-15d-PGJ 2 (green) adduct blots is shown ( E ). Analysis of spot quantity was determined and common and unique spots from bt-15d-PGJ 2 and mito-15d-PGJ 2 membranes are shown schematically ( F ).

    Journal: The Biochemical journal

    Article Title: Mitochondrial targeting of the electrophilic lipid 15-deoxy-?12,14-Prostaglandin J2 increases apoptotic efficacy via redox cell signaling mechanisms

    doi: 10.1042/BJ20091293

    Figure Lengend Snippet: Visualization of bt-15d-PGJ 2 and mito-15d-PGJ 2 modified proteins by 2D- IEF-SDS-PAGE Cell lysates from MDA-MB231 cells were separated by 2D-IEF. Ethanol vehicle control (1 h) treated cell lysates were probed using Streptavidin-HRP ( A ) and TPP+ antibody with HRP-conjugated secondary antibody ( B ) to determine background reactivity. Cells were treated (10 μM, 1 h) with bt-15d-PGJ 2 ( C ) or mito-15d-PGJ 2 ( D ) and protein adducts were detected using Streptavidin-HRP and TPP+ antibody respectively. A merged image of bt-15d-PGJ 2 (red) and mito-15d-PGJ 2 (green) adduct blots is shown ( E ). Analysis of spot quantity was determined and common and unique spots from bt-15d-PGJ 2 and mito-15d-PGJ 2 membranes are shown schematically ( F ).

    Article Snippet: After blocking for 1 h, the membranes were probed using an anti-TPP+ antibody (1:10,000; received from Dr. Michael P. Murphy) with a HRP-conjugated secondary antibody and streptavidin conjugated to HRP (GE Healthcare) for detection of bt-15d-PGJ2 and mito-15d-PGJ2 adducts, respectively.

    Techniques: Modification, Electrofocusing, SDS Page, Multiple Displacement Amplification

    Localization of 15d-PGJ 2 and mito-15d-PGJ 2 to the mitochondrion and effects on mitochondrial membrane potential (Δ ψ) MDA-MB231 cells were treated with 10 μM bt-15d-PGJ 2 (bt-15d) or mito-15d-PGJ 2 (Mito-15d) for 1 h then mitochondrial and cytosolic fractions were prepared and resolved using 10% SDS-PAGE. Adduct formation was detected using an anti-TPP antibody (for mito-15d-PGJ 2 ) or streptavidin-HRP (for bt-15d-PGJ 2 ). ECL+ coupled to horseradish peroxidase was used to detect chemifluorescence protein adducts ( A ). VDAC protein levels were also determined using Western blot analysis to confirm mitochondrial preparation purity. Protein adducts were quantified and expressed as a percent of homogenate ( B ). MDA-MB231 cells were exposed to increasing concentrations (1–10 μM) of 15d-PGJ 2 , mito-15d-PGJ 2 , or TPMP for 4 h then Δ ψ was assessed using JC-1 ( C ). Data are represented as the ratio of red/green fluorescence. Results represent means ± s.e.m., n = 3–6. * p

    Journal: The Biochemical journal

    Article Title: Mitochondrial targeting of the electrophilic lipid 15-deoxy-?12,14-Prostaglandin J2 increases apoptotic efficacy via redox cell signaling mechanisms

    doi: 10.1042/BJ20091293

    Figure Lengend Snippet: Localization of 15d-PGJ 2 and mito-15d-PGJ 2 to the mitochondrion and effects on mitochondrial membrane potential (Δ ψ) MDA-MB231 cells were treated with 10 μM bt-15d-PGJ 2 (bt-15d) or mito-15d-PGJ 2 (Mito-15d) for 1 h then mitochondrial and cytosolic fractions were prepared and resolved using 10% SDS-PAGE. Adduct formation was detected using an anti-TPP antibody (for mito-15d-PGJ 2 ) or streptavidin-HRP (for bt-15d-PGJ 2 ). ECL+ coupled to horseradish peroxidase was used to detect chemifluorescence protein adducts ( A ). VDAC protein levels were also determined using Western blot analysis to confirm mitochondrial preparation purity. Protein adducts were quantified and expressed as a percent of homogenate ( B ). MDA-MB231 cells were exposed to increasing concentrations (1–10 μM) of 15d-PGJ 2 , mito-15d-PGJ 2 , or TPMP for 4 h then Δ ψ was assessed using JC-1 ( C ). Data are represented as the ratio of red/green fluorescence. Results represent means ± s.e.m., n = 3–6. * p

    Article Snippet: After blocking for 1 h, the membranes were probed using an anti-TPP+ antibody (1:10,000; received from Dr. Michael P. Murphy) with a HRP-conjugated secondary antibody and streptavidin conjugated to HRP (GE Healthcare) for detection of bt-15d-PGJ2 and mito-15d-PGJ2 adducts, respectively.

    Techniques: Multiple Displacement Amplification, SDS Page, Western Blot, Fluorescence