pierce ecl western blotting substrate  (Thermo Fisher)


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    Name:
    Pierce ECL Plus Western Blotting Substrate
    Description:
    Thermo Scientific Pierce ECL Plus Substrate is an acridan based chemiluminescent and chemifluorescent HRP substrate for Western blot detection using X ray film or CCD or laser based imagers This product is sold as Pierce ECL 2 Substrate Cat PI80196 through Fisher Scientific and other channels Features of ECL Plus Substrate • Easy to use can be substituted for the discontinued GE ECL Plus Substrate without any re optimization • Higher sensitivity detect targets down to the low picogram level • Longer signal duration sustained light output for as long as 5 hours • More imaging options X ray CCD or laser based imagers • More affordable high quality and performance at a lower price than other suppliers similar substrates Pierce ECL Plus Substrate generates acridinium esters when it reacts with HRP As these ester intermediates react with peroxide they produce a strong and sustained chemiluminescent signal that can be captured by X ray film CCD imaging The substrate also produces a robust fluorescent signal that can be captured with fluorescence imagers Pierce ECL Plus Substrate enables the detection of low picogram amounts of target protein on nitrocellulose or PVDF membrane when probed with appropriate primary and secondary antibody concentrations Its broad dynamic range high sensitivity and long lasting signal make Pierce ECL Plus Substrate an excellent choice for Western blot analysis
    Catalog Number:
    32132
    Price:
    None
    Applications:
    Protein Assays and Analysis|Protein Biology|Western Blotting
    Category:
    Labeling Detection Products
    Buy from Supplier


    Structured Review

    Thermo Fisher pierce ecl western blotting substrate
    5f restored neural tube development and marker expression in chick embryos on EDD 5. Glucose concentration ( A ) and Pax3 protein expression ( B ) were determined in chick embryos. The plasma glucose concentration was measured using a glucose oxidase-coupled spectrophotometric assay kit. Proteins were detected using the monoclonal antibody anti-Pax3 diluted 1:1000 (DSHB, USA) and visualized using anti-mouse <t>IgG</t> conjugated with horseradish peroxidase (HRP) and Pierce <t>ECL</t> Western Blotting Substrate (Thermo Fisher Scientific, USA) as the substrate of HRP. The abbreviations “CON”, “GLU”, “CL”, “CM”, “CH”, “EPA”, “EDA” mean “controlled”, “glucose treated”, “low concentration of 5f treated”, “mild concentration of 5f treated”, “high concentration of 5f treated”, “epalrestat treated”, “edaravone treated” groups, respectively. Values were expressed as mean ± SD in each group (n = 10). * P
    Thermo Scientific Pierce ECL Plus Substrate is an acridan based chemiluminescent and chemifluorescent HRP substrate for Western blot detection using X ray film or CCD or laser based imagers This product is sold as Pierce ECL 2 Substrate Cat PI80196 through Fisher Scientific and other channels Features of ECL Plus Substrate • Easy to use can be substituted for the discontinued GE ECL Plus Substrate without any re optimization • Higher sensitivity detect targets down to the low picogram level • Longer signal duration sustained light output for as long as 5 hours • More imaging options X ray CCD or laser based imagers • More affordable high quality and performance at a lower price than other suppliers similar substrates Pierce ECL Plus Substrate generates acridinium esters when it reacts with HRP As these ester intermediates react with peroxide they produce a strong and sustained chemiluminescent signal that can be captured by X ray film CCD imaging The substrate also produces a robust fluorescent signal that can be captured with fluorescence imagers Pierce ECL Plus Substrate enables the detection of low picogram amounts of target protein on nitrocellulose or PVDF membrane when probed with appropriate primary and secondary antibody concentrations Its broad dynamic range high sensitivity and long lasting signal make Pierce ECL Plus Substrate an excellent choice for Western blot analysis
    https://www.bioz.com/result/pierce ecl western blotting substrate/product/Thermo Fisher
    Average 99 stars, based on 1641 article reviews
    Price from $9.99 to $1999.99
    pierce ecl western blotting substrate - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "Bioactivity Focus of α-Cyano-4-hydroxycinnamic acid (CHCA) Leads to Effective Multifunctional Aldose Reductase Inhibitors"

    Article Title: Bioactivity Focus of α-Cyano-4-hydroxycinnamic acid (CHCA) Leads to Effective Multifunctional Aldose Reductase Inhibitors

    Journal: Scientific Reports

    doi: 10.1038/srep24942

    5f restored neural tube development and marker expression in chick embryos on EDD 5. Glucose concentration ( A ) and Pax3 protein expression ( B ) were determined in chick embryos. The plasma glucose concentration was measured using a glucose oxidase-coupled spectrophotometric assay kit. Proteins were detected using the monoclonal antibody anti-Pax3 diluted 1:1000 (DSHB, USA) and visualized using anti-mouse IgG conjugated with horseradish peroxidase (HRP) and Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, USA) as the substrate of HRP. The abbreviations “CON”, “GLU”, “CL”, “CM”, “CH”, “EPA”, “EDA” mean “controlled”, “glucose treated”, “low concentration of 5f treated”, “mild concentration of 5f treated”, “high concentration of 5f treated”, “epalrestat treated”, “edaravone treated” groups, respectively. Values were expressed as mean ± SD in each group (n = 10). * P
    Figure Legend Snippet: 5f restored neural tube development and marker expression in chick embryos on EDD 5. Glucose concentration ( A ) and Pax3 protein expression ( B ) were determined in chick embryos. The plasma glucose concentration was measured using a glucose oxidase-coupled spectrophotometric assay kit. Proteins were detected using the monoclonal antibody anti-Pax3 diluted 1:1000 (DSHB, USA) and visualized using anti-mouse IgG conjugated with horseradish peroxidase (HRP) and Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, USA) as the substrate of HRP. The abbreviations “CON”, “GLU”, “CL”, “CM”, “CH”, “EPA”, “EDA” mean “controlled”, “glucose treated”, “low concentration of 5f treated”, “mild concentration of 5f treated”, “high concentration of 5f treated”, “epalrestat treated”, “edaravone treated” groups, respectively. Values were expressed as mean ± SD in each group (n = 10). * P

    Techniques Used: Marker, Expressing, Concentration Assay, Spectrophotometric Assay, Western Blot

    2) Product Images from "Chemotactic Chemokines Are Important in the Pathogenesis of Irritable Bowel Syndrome"

    Article Title: Chemotactic Chemokines Are Important in the Pathogenesis of Irritable Bowel Syndrome

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0093144

    A representative image of the various biomarkers detected in the sera of IBS patients and healthy volunteers. Each pair of horizontal blots (bands) represent a different biomarker present in the serum, whereas the intensities of the blots characterize the amount of the respective biomarker. Sera (250 μL) from 60 IBS patients (44 idiopathic IBS and 16 post-infectious IBS) and 40 healthy volunteers were evaluated for the presence of 36 different biomarkers using the Proteome Profiler Human Cytokine Array Panel A Kit (R D Systems, Minneapolis, MN). Samples were incubated with a membrane embedded with biotinylated antibodies that are specific for each of the 36 biomarkers analyzed. For detection, the membranes were probed with the Pierce ECL Western Blotting Substrate (Thermo Scientific, Rockford, IL) and exposed to an X-ray film, following incubation with a secondary antibody labelled with streptavidin-horse radish peroxidase. The exposed film was processed using SRX-101A Medical Film Processor (Konica Minolta). Intensities of the blots were determined and expressed as pixel densities using ImageJ (National Institutes of Health, Bethesda, MD).
    Figure Legend Snippet: A representative image of the various biomarkers detected in the sera of IBS patients and healthy volunteers. Each pair of horizontal blots (bands) represent a different biomarker present in the serum, whereas the intensities of the blots characterize the amount of the respective biomarker. Sera (250 μL) from 60 IBS patients (44 idiopathic IBS and 16 post-infectious IBS) and 40 healthy volunteers were evaluated for the presence of 36 different biomarkers using the Proteome Profiler Human Cytokine Array Panel A Kit (R D Systems, Minneapolis, MN). Samples were incubated with a membrane embedded with biotinylated antibodies that are specific for each of the 36 biomarkers analyzed. For detection, the membranes were probed with the Pierce ECL Western Blotting Substrate (Thermo Scientific, Rockford, IL) and exposed to an X-ray film, following incubation with a secondary antibody labelled with streptavidin-horse radish peroxidase. The exposed film was processed using SRX-101A Medical Film Processor (Konica Minolta). Intensities of the blots were determined and expressed as pixel densities using ImageJ (National Institutes of Health, Bethesda, MD).

    Techniques Used: Biomarker Assay, Incubation, Western Blot

    3) Product Images from "Benefits and Pitfalls of Secondary Antibodies: Why Choosing the Right Secondary Is of Primary Importance"

    Article Title: Benefits and Pitfalls of Secondary Antibodies: Why Choosing the Right Secondary Is of Primary Importance

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0038313

    HL detection bias is seen in 2°Ab preparations from different suppliers, with different fluorophores, and with enzyme conjugates. (A) Kv1.2-transfected COS-1 cells were labeled with 1°Ab as in Figure 4C , and HL 2°Ab and the respective SCS 2°Abs, and the ratios of fluorescence intensities from three fields each of three independent samples normalized to the HL/IgG1 ratio. Letters are supplier (L = Life Technologies, R = Rockland), numbers are Alex or DyLight fluorophore conjugates; high: highly adsorbed; fab: F(ab′) 2 fragment of HL ( e.g. , L488 SCS is Life Technologies Alexa 488 conjugated SCS). 4/09 and 7/11 refer to two lots of Life Technologies HL. (B) FLISAs showing detection bias of 2°Abs is present at all 2°Ab concentrations. Upper left: Life Technologies HL. Upper right: Life Technologies SCS. Lower left: Jackson ImmunoResearch HL. Lower right: Jackson ImmunoResearch HL (highly cross-adsorbed). (C) HRP conjugated HL secondaries show detection bias by immunoblot. Purified mAb IgG preparations were analyzed by reducing SDS-PAGE and coomassie blue staining (CB), or immunoblotting and detection with two different HRP-conjugated H+L 2°Abs and ECL. HL: Kirkegaard Perry Laboratories. HL*: Antibodies Incorporated. Note subclass-specific differences in detection of heavy chain (HC) but not light chain (LC) bands in IgG preparations.
    Figure Legend Snippet: HL detection bias is seen in 2°Ab preparations from different suppliers, with different fluorophores, and with enzyme conjugates. (A) Kv1.2-transfected COS-1 cells were labeled with 1°Ab as in Figure 4C , and HL 2°Ab and the respective SCS 2°Abs, and the ratios of fluorescence intensities from three fields each of three independent samples normalized to the HL/IgG1 ratio. Letters are supplier (L = Life Technologies, R = Rockland), numbers are Alex or DyLight fluorophore conjugates; high: highly adsorbed; fab: F(ab′) 2 fragment of HL ( e.g. , L488 SCS is Life Technologies Alexa 488 conjugated SCS). 4/09 and 7/11 refer to two lots of Life Technologies HL. (B) FLISAs showing detection bias of 2°Abs is present at all 2°Ab concentrations. Upper left: Life Technologies HL. Upper right: Life Technologies SCS. Lower left: Jackson ImmunoResearch HL. Lower right: Jackson ImmunoResearch HL (highly cross-adsorbed). (C) HRP conjugated HL secondaries show detection bias by immunoblot. Purified mAb IgG preparations were analyzed by reducing SDS-PAGE and coomassie blue staining (CB), or immunoblotting and detection with two different HRP-conjugated H+L 2°Abs and ECL. HL: Kirkegaard Perry Laboratories. HL*: Antibodies Incorporated. Note subclass-specific differences in detection of heavy chain (HC) but not light chain (LC) bands in IgG preparations.

    Techniques Used: Transfection, Labeling, Fluorescence, Purification, SDS Page, Staining

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    Western Blot:

    Article Title: Swimming Exercise Alleviated Insulin Resistance by Regulating Tripartite Motif Family Protein 72 Expression and AKT Signal Pathway in Sprague-Dawley Rats Fed with High-Fat Diet
    Article Snippet: .. Immunoreactive protein bands were visualized with the Pierce ECL Plus Western Blot Substrate (Thermo Fisher Scientific, Rockford, IL, USA). .. GAPDH was performed as an internal loading control, and quantification of the band intensity was performed using ImageJ software (NIH) and defined as the fold-change of the CON after being normalized against GAPDH.

    Article Title: Trimethylation of Elongation Factor-Tu by the Dual Thermoregulated Methyltransferase EftM Does Not Impact Its Canonical Function in Translation
    Article Snippet: .. Next membranes were incubated with appropriate secondary antibody for one hour at room temperature, exposed with Pierce ECL Western Blotting Substrate (ThermoFisher), and imaged on a ChemiDoc MP Imaging System. .. Depletion of EF-Tu from P . aeruginosa lysates and in vitro methylation PAO1ΔeftM was grown to mid-exponential at 25 °C or 37 °C.

    Article Title: Platelet-rich plasma protects rat chondrocytes from interleukin-1β-induced apoptosis
    Article Snippet: .. The membranes were washed three times (15 min each wash) between antibody incubations with TBS containing 0.1% Tween 20, and the blots were developed using ECL Plus Western Blotting Substrate (Pierce Biotechnology; Thermo Fisher Scientific, Inc.). .. Blots were semi-quantified using ImageJ 2x software (Rawak Software, Inc., Stuttgart, Germany).

    Article Title: Benefits and Pitfalls of Secondary Antibodies: Why Choosing the Right Secondary Is of Primary Importance
    Article Snippet: .. After 3×10 min washes with TBS, the immunoblots were visualized directly (for fluorescent 2°Abs) or after visualization with Pierce ECL Western Blotting Substrate (Thermo Scientific; for HRP 2°Abs) in a FluorChem Q imager (Cell Biosciences). .. Pure mAb preparations were boiled in RSB, and size-fractionated by 12% SDS–PAGE.

    Article Title: GPI-anchor signal sequence influences PrPC sorting, shedding and signalling, and impacts on different pathomechanistic aspects of prion disease in mice
    Article Snippet: .. Membranes were incubated with appropriate secondary antibodies and then developed either with Pierce ECL Western Blotting Substrate or Pierce Femto (Thermo Scientific) in a CD camera imaging system (BioRad) or with Odyssey Image system (Licor) and quantified with Image Studio software (Licor). .. Immunohistochemistry Processing of samples, including cutting and staining of paraffin sections with HE, was performed as published [ ].

    Article Title: Chemotactic Chemokines Are Important in the Pathogenesis of Irritable Bowel Syndrome
    Article Snippet: .. Following a washing step, 3 ml of a 1∶1000 dilution of secondary antibody conjugated with streptavidin-HRP was added to each membrane and incubated at room temperature for 45 min. For detection, the membranes were probed with the Pierce ECL Western Blotting Substrate (Thermo Scientific, Rockford, IL) and exposed to an X-ray film. .. The exposed film was processed using SRX-101A Medical Film Processor (Konica Minolta).

    Article Title: Bioactivity Focus of α-Cyano-4-hydroxycinnamic acid (CHCA) Leads to Effective Multifunctional Aldose Reductase Inhibitors
    Article Snippet: .. Proteins were detected using the monoclonal antibody anti-Pax3 diluted 1:1000 (DSHB, USA) and visualized using anti-mouse IgG conjugated with horseradish peroxidase (HRP) and Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, USA) as the substrate of HRP. ..

    Incubation:

    Article Title: Trimethylation of Elongation Factor-Tu by the Dual Thermoregulated Methyltransferase EftM Does Not Impact Its Canonical Function in Translation
    Article Snippet: .. Next membranes were incubated with appropriate secondary antibody for one hour at room temperature, exposed with Pierce ECL Western Blotting Substrate (ThermoFisher), and imaged on a ChemiDoc MP Imaging System. .. Depletion of EF-Tu from P . aeruginosa lysates and in vitro methylation PAO1ΔeftM was grown to mid-exponential at 25 °C or 37 °C.

    Article Title: GPI-anchor signal sequence influences PrPC sorting, shedding and signalling, and impacts on different pathomechanistic aspects of prion disease in mice
    Article Snippet: .. Membranes were incubated with appropriate secondary antibodies and then developed either with Pierce ECL Western Blotting Substrate or Pierce Femto (Thermo Scientific) in a CD camera imaging system (BioRad) or with Odyssey Image system (Licor) and quantified with Image Studio software (Licor). .. Immunohistochemistry Processing of samples, including cutting and staining of paraffin sections with HE, was performed as published [ ].

    Article Title: Chemotactic Chemokines Are Important in the Pathogenesis of Irritable Bowel Syndrome
    Article Snippet: .. Following a washing step, 3 ml of a 1∶1000 dilution of secondary antibody conjugated with streptavidin-HRP was added to each membrane and incubated at room temperature for 45 min. For detection, the membranes were probed with the Pierce ECL Western Blotting Substrate (Thermo Scientific, Rockford, IL) and exposed to an X-ray film. .. The exposed film was processed using SRX-101A Medical Film Processor (Konica Minolta).

    Imaging:

    Article Title: Trimethylation of Elongation Factor-Tu by the Dual Thermoregulated Methyltransferase EftM Does Not Impact Its Canonical Function in Translation
    Article Snippet: .. Next membranes were incubated with appropriate secondary antibody for one hour at room temperature, exposed with Pierce ECL Western Blotting Substrate (ThermoFisher), and imaged on a ChemiDoc MP Imaging System. .. Depletion of EF-Tu from P . aeruginosa lysates and in vitro methylation PAO1ΔeftM was grown to mid-exponential at 25 °C or 37 °C.

    Article Title: GPI-anchor signal sequence influences PrPC sorting, shedding and signalling, and impacts on different pathomechanistic aspects of prion disease in mice
    Article Snippet: .. Membranes were incubated with appropriate secondary antibodies and then developed either with Pierce ECL Western Blotting Substrate or Pierce Femto (Thermo Scientific) in a CD camera imaging system (BioRad) or with Odyssey Image system (Licor) and quantified with Image Studio software (Licor). .. Immunohistochemistry Processing of samples, including cutting and staining of paraffin sections with HE, was performed as published [ ].

    Software:

    Article Title: GPI-anchor signal sequence influences PrPC sorting, shedding and signalling, and impacts on different pathomechanistic aspects of prion disease in mice
    Article Snippet: .. Membranes were incubated with appropriate secondary antibodies and then developed either with Pierce ECL Western Blotting Substrate or Pierce Femto (Thermo Scientific) in a CD camera imaging system (BioRad) or with Odyssey Image system (Licor) and quantified with Image Studio software (Licor). .. Immunohistochemistry Processing of samples, including cutting and staining of paraffin sections with HE, was performed as published [ ].

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  • 99
    Thermo Fisher pierce ecl western blotting substrate
    5f restored neural tube development and marker expression in chick embryos on EDD 5. Glucose concentration ( A ) and Pax3 protein expression ( B ) were determined in chick embryos. The plasma glucose concentration was measured using a glucose oxidase-coupled spectrophotometric assay kit. Proteins were detected using the monoclonal antibody anti-Pax3 diluted 1:1000 (DSHB, USA) and visualized using anti-mouse <t>IgG</t> conjugated with horseradish peroxidase (HRP) and Pierce <t>ECL</t> Western Blotting Substrate (Thermo Fisher Scientific, USA) as the substrate of HRP. The abbreviations “CON”, “GLU”, “CL”, “CM”, “CH”, “EPA”, “EDA” mean “controlled”, “glucose treated”, “low concentration of 5f treated”, “mild concentration of 5f treated”, “high concentration of 5f treated”, “epalrestat treated”, “edaravone treated” groups, respectively. Values were expressed as mean ± SD in each group (n = 10). * P
    Pierce Ecl Western Blotting Substrate, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1837 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pierce ecl western blotting substrate/product/Thermo Fisher
    Average 99 stars, based on 1837 article reviews
    Price from $9.99 to $1999.99
    pierce ecl western blotting substrate - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    Image Search Results


    5f restored neural tube development and marker expression in chick embryos on EDD 5. Glucose concentration ( A ) and Pax3 protein expression ( B ) were determined in chick embryos. The plasma glucose concentration was measured using a glucose oxidase-coupled spectrophotometric assay kit. Proteins were detected using the monoclonal antibody anti-Pax3 diluted 1:1000 (DSHB, USA) and visualized using anti-mouse IgG conjugated with horseradish peroxidase (HRP) and Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, USA) as the substrate of HRP. The abbreviations “CON”, “GLU”, “CL”, “CM”, “CH”, “EPA”, “EDA” mean “controlled”, “glucose treated”, “low concentration of 5f treated”, “mild concentration of 5f treated”, “high concentration of 5f treated”, “epalrestat treated”, “edaravone treated” groups, respectively. Values were expressed as mean ± SD in each group (n = 10). * P

    Journal: Scientific Reports

    Article Title: Bioactivity Focus of α-Cyano-4-hydroxycinnamic acid (CHCA) Leads to Effective Multifunctional Aldose Reductase Inhibitors

    doi: 10.1038/srep24942

    Figure Lengend Snippet: 5f restored neural tube development and marker expression in chick embryos on EDD 5. Glucose concentration ( A ) and Pax3 protein expression ( B ) were determined in chick embryos. The plasma glucose concentration was measured using a glucose oxidase-coupled spectrophotometric assay kit. Proteins were detected using the monoclonal antibody anti-Pax3 diluted 1:1000 (DSHB, USA) and visualized using anti-mouse IgG conjugated with horseradish peroxidase (HRP) and Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, USA) as the substrate of HRP. The abbreviations “CON”, “GLU”, “CL”, “CM”, “CH”, “EPA”, “EDA” mean “controlled”, “glucose treated”, “low concentration of 5f treated”, “mild concentration of 5f treated”, “high concentration of 5f treated”, “epalrestat treated”, “edaravone treated” groups, respectively. Values were expressed as mean ± SD in each group (n = 10). * P

    Article Snippet: Proteins were detected using the monoclonal antibody anti-Pax3 diluted 1:1000 (DSHB, USA) and visualized using anti-mouse IgG conjugated with horseradish peroxidase (HRP) and Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, USA) as the substrate of HRP.

    Techniques: Marker, Expressing, Concentration Assay, Spectrophotometric Assay, Western Blot

    A representative image of the various biomarkers detected in the sera of IBS patients and healthy volunteers. Each pair of horizontal blots (bands) represent a different biomarker present in the serum, whereas the intensities of the blots characterize the amount of the respective biomarker. Sera (250 μL) from 60 IBS patients (44 idiopathic IBS and 16 post-infectious IBS) and 40 healthy volunteers were evaluated for the presence of 36 different biomarkers using the Proteome Profiler Human Cytokine Array Panel A Kit (R D Systems, Minneapolis, MN). Samples were incubated with a membrane embedded with biotinylated antibodies that are specific for each of the 36 biomarkers analyzed. For detection, the membranes were probed with the Pierce ECL Western Blotting Substrate (Thermo Scientific, Rockford, IL) and exposed to an X-ray film, following incubation with a secondary antibody labelled with streptavidin-horse radish peroxidase. The exposed film was processed using SRX-101A Medical Film Processor (Konica Minolta). Intensities of the blots were determined and expressed as pixel densities using ImageJ (National Institutes of Health, Bethesda, MD).

    Journal: PLoS ONE

    Article Title: Chemotactic Chemokines Are Important in the Pathogenesis of Irritable Bowel Syndrome

    doi: 10.1371/journal.pone.0093144

    Figure Lengend Snippet: A representative image of the various biomarkers detected in the sera of IBS patients and healthy volunteers. Each pair of horizontal blots (bands) represent a different biomarker present in the serum, whereas the intensities of the blots characterize the amount of the respective biomarker. Sera (250 μL) from 60 IBS patients (44 idiopathic IBS and 16 post-infectious IBS) and 40 healthy volunteers were evaluated for the presence of 36 different biomarkers using the Proteome Profiler Human Cytokine Array Panel A Kit (R D Systems, Minneapolis, MN). Samples were incubated with a membrane embedded with biotinylated antibodies that are specific for each of the 36 biomarkers analyzed. For detection, the membranes were probed with the Pierce ECL Western Blotting Substrate (Thermo Scientific, Rockford, IL) and exposed to an X-ray film, following incubation with a secondary antibody labelled with streptavidin-horse radish peroxidase. The exposed film was processed using SRX-101A Medical Film Processor (Konica Minolta). Intensities of the blots were determined and expressed as pixel densities using ImageJ (National Institutes of Health, Bethesda, MD).

    Article Snippet: Following a washing step, 3 ml of a 1∶1000 dilution of secondary antibody conjugated with streptavidin-HRP was added to each membrane and incubated at room temperature for 45 min. For detection, the membranes were probed with the Pierce ECL Western Blotting Substrate (Thermo Scientific, Rockford, IL) and exposed to an X-ray film.

    Techniques: Biomarker Assay, Incubation, Western Blot

    HL detection bias is seen in 2°Ab preparations from different suppliers, with different fluorophores, and with enzyme conjugates. (A) Kv1.2-transfected COS-1 cells were labeled with 1°Ab as in Figure 4C , and HL 2°Ab and the respective SCS 2°Abs, and the ratios of fluorescence intensities from three fields each of three independent samples normalized to the HL/IgG1 ratio. Letters are supplier (L = Life Technologies, R = Rockland), numbers are Alex or DyLight fluorophore conjugates; high: highly adsorbed; fab: F(ab′) 2 fragment of HL ( e.g. , L488 SCS is Life Technologies Alexa 488 conjugated SCS). 4/09 and 7/11 refer to two lots of Life Technologies HL. (B) FLISAs showing detection bias of 2°Abs is present at all 2°Ab concentrations. Upper left: Life Technologies HL. Upper right: Life Technologies SCS. Lower left: Jackson ImmunoResearch HL. Lower right: Jackson ImmunoResearch HL (highly cross-adsorbed). (C) HRP conjugated HL secondaries show detection bias by immunoblot. Purified mAb IgG preparations were analyzed by reducing SDS-PAGE and coomassie blue staining (CB), or immunoblotting and detection with two different HRP-conjugated H+L 2°Abs and ECL. HL: Kirkegaard Perry Laboratories. HL*: Antibodies Incorporated. Note subclass-specific differences in detection of heavy chain (HC) but not light chain (LC) bands in IgG preparations.

    Journal: PLoS ONE

    Article Title: Benefits and Pitfalls of Secondary Antibodies: Why Choosing the Right Secondary Is of Primary Importance

    doi: 10.1371/journal.pone.0038313

    Figure Lengend Snippet: HL detection bias is seen in 2°Ab preparations from different suppliers, with different fluorophores, and with enzyme conjugates. (A) Kv1.2-transfected COS-1 cells were labeled with 1°Ab as in Figure 4C , and HL 2°Ab and the respective SCS 2°Abs, and the ratios of fluorescence intensities from three fields each of three independent samples normalized to the HL/IgG1 ratio. Letters are supplier (L = Life Technologies, R = Rockland), numbers are Alex or DyLight fluorophore conjugates; high: highly adsorbed; fab: F(ab′) 2 fragment of HL ( e.g. , L488 SCS is Life Technologies Alexa 488 conjugated SCS). 4/09 and 7/11 refer to two lots of Life Technologies HL. (B) FLISAs showing detection bias of 2°Abs is present at all 2°Ab concentrations. Upper left: Life Technologies HL. Upper right: Life Technologies SCS. Lower left: Jackson ImmunoResearch HL. Lower right: Jackson ImmunoResearch HL (highly cross-adsorbed). (C) HRP conjugated HL secondaries show detection bias by immunoblot. Purified mAb IgG preparations were analyzed by reducing SDS-PAGE and coomassie blue staining (CB), or immunoblotting and detection with two different HRP-conjugated H+L 2°Abs and ECL. HL: Kirkegaard Perry Laboratories. HL*: Antibodies Incorporated. Note subclass-specific differences in detection of heavy chain (HC) but not light chain (LC) bands in IgG preparations.

    Article Snippet: After 3×10 min washes with TBS, the immunoblots were visualized directly (for fluorescent 2°Abs) or after visualization with Pierce ECL Western Blotting Substrate (Thermo Scientific; for HRP 2°Abs) in a FluorChem Q imager (Cell Biosciences).

    Techniques: Transfection, Labeling, Fluorescence, Purification, SDS Page, Staining