avidin biotin peroxidase complexes  (Millipore)


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    Name:
    Avidin Peroxidase
    Description:
    Avidin is homotetrameric protein 68kDa that is obtained from egg whites This egg protein binds strongly to biotin Thus avidin biotin association has been utilized in immunoassays to detect the localization of antigens in tissues The use of avidin biotin immunoassay enhances the sensitivity of the technique and facilitates the detection of antigens in low quantities
    Catalog Number:
    a3151
    Price:
    None
    Applications:
    To detect Ga1-deficient IgA1 antibodies in mouse serum, ELISA assays were performed using sera which was incubated with biotinylated HAA, which binds IgA1, in 96-well plates. HRP-conjugated avidin was then incubated with the sera for 40 minutes at 37 degrees.
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    Structured Review

    Millipore avidin biotin peroxidase complexes
    Avidin is homotetrameric protein 68kDa that is obtained from egg whites This egg protein binds strongly to biotin Thus avidin biotin association has been utilized in immunoassays to detect the localization of antigens in tissues The use of avidin biotin immunoassay enhances the sensitivity of the technique and facilitates the detection of antigens in low quantities
    https://www.bioz.com/result/avidin biotin peroxidase complexes/product/Millipore
    Average 99 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    avidin biotin peroxidase complexes - by Bioz Stars, 2020-09
    99/100 stars

    Images

    Related Articles

    Colorimetric Assay:

    Article Title: CD1d-restricted Human Natural Killer T Cells Are Highly Susceptible to Human Immunodeficiency Virus 1 Infection
    Article Snippet: .. Free biotin was removed by dialysis, and biotinylation efficiency was ascertained with a colorimetric assay using avidin peroxidase (Sigma-Aldrich). ..

    Incubation:

    Article Title: Signal Transducer and Activator of Transcription Factor 6 (Stat6)-deficient Mice Are Protected from Antigen-induced Airway Hyperresponsiveness and Mucus Production
    Article Snippet: .. Plates were washed with 0.05% Tween-20/PBS and incubated with biotin-conjugated anti– mouse IgG1 (1/2,000; γl chain specific) or IgG2a (1/2,000; γ2a chain specific; PharMingen , San Diego, CA; 100 μl/well) at room temperature for 1 h. After another wash procedure, plates were incubated for 1 h at room temperature with 100 μl/well of 0.0025 mg/ml avidin peroxidase ( Sigma Chemical Co. ) in 10% FBS/PBS. .. Plates were developed with ABTS (2.2′-azino-did[3-ethyl-benzthiazone sulfonate]; Kirkegaard & Perry Labs., Inc., Gaithersburg, MD; 100 μl/well).

    Article Title: Augmented reduction in colonic inflammatory markers of dextran sulfate sodium-induced colitis with a combination of 5-aminosalicylic acid and AD-lico™ from Glycyrrhiza inflata
    Article Snippet: .. This was followed by washing and incubation of the wells with avidin-peroxidase (Sigma-Aldrich) at 37°C for 30 min. .. The plates were washed once more and the color development substrate, (2,2′-azino-bis [3- ethylbenzothiazoline-6-sulfonic acid]-diammonium salt) (BD Bioscience), was added to each well.

    Article Title: CD4+ T helper responses in squamous cell carcinoma of the head and neck
    Article Snippet: .. After 2 h incubation, plates were washed with PBS/0.05% Tween 20, and developed with the avidin-peroxidase complex (Vectastain Elite kit; Vector) for 1 h. 3-Amino-9-ethyl-carbozole (Sigma, St. Louis, MO) was added and incubated for 5 min for the IFN-γ ELISPOT assay and the TMB substrate for peroxidase (3,3′,5,5′-Tetramethylbenzidine; Vector Laboratories) was added and incubated for 10 min for the IL-5 ELISPOT assay. ..

    Enzyme-linked Immunospot:

    Article Title: CD4+ T helper responses in squamous cell carcinoma of the head and neck
    Article Snippet: .. After 2 h incubation, plates were washed with PBS/0.05% Tween 20, and developed with the avidin-peroxidase complex (Vectastain Elite kit; Vector) for 1 h. 3-Amino-9-ethyl-carbozole (Sigma, St. Louis, MO) was added and incubated for 5 min for the IFN-γ ELISPOT assay and the TMB substrate for peroxidase (3,3′,5,5′-Tetramethylbenzidine; Vector Laboratories) was added and incubated for 10 min for the IL-5 ELISPOT assay. ..

    Avidin-Biotin Assay:

    Article Title: Anti-Lubricin Monoclonal Antibodies Created Using Lubricin-Knockout Mice Immunodetect Lubricin in Several Species and in Patients with Healthy and Diseased Joints
    Article Snippet: .. Detection of the avidin/biotin/peroxidase-conjugated secondary antibody was performed using DAB FastTabs substrate (Sigma-Aldrich) for 5 minutes, followed by counterstaining with hematoxylin or hematoxylin and eosin. ..

    Article Title: Signal Transducer and Activator of Transcription Factor 6 (Stat6)-deficient Mice Are Protected from Antigen-induced Airway Hyperresponsiveness and Mucus Production
    Article Snippet: .. Plates were washed with 0.05% Tween-20/PBS and incubated with biotin-conjugated anti– mouse IgG1 (1/2,000; γl chain specific) or IgG2a (1/2,000; γ2a chain specific; PharMingen , San Diego, CA; 100 μl/well) at room temperature for 1 h. After another wash procedure, plates were incubated for 1 h at room temperature with 100 μl/well of 0.0025 mg/ml avidin peroxidase ( Sigma Chemical Co. ) in 10% FBS/PBS. .. Plates were developed with ABTS (2.2′-azino-did[3-ethyl-benzthiazone sulfonate]; Kirkegaard & Perry Labs., Inc., Gaithersburg, MD; 100 μl/well).

    Article Title: CD1d-restricted Human Natural Killer T Cells Are Highly Susceptible to Human Immunodeficiency Virus 1 Infection
    Article Snippet: .. Free biotin was removed by dialysis, and biotinylation efficiency was ascertained with a colorimetric assay using avidin peroxidase (Sigma-Aldrich). ..

    Article Title: Augmented reduction in colonic inflammatory markers of dextran sulfate sodium-induced colitis with a combination of 5-aminosalicylic acid and AD-lico™ from Glycyrrhiza inflata
    Article Snippet: .. This was followed by washing and incubation of the wells with avidin-peroxidase (Sigma-Aldrich) at 37°C for 30 min. .. The plates were washed once more and the color development substrate, (2,2′-azino-bis [3- ethylbenzothiazoline-6-sulfonic acid]-diammonium salt) (BD Bioscience), was added to each well.

    Article Title: IgG1 antimycobacterial antibodies can reverse the inhibitory effect of pentoxifylline on tumour necrosis factor alpha (TNF-?) secreted by mycobacterial antigen-stimulated adherent cells
    Article Snippet: .. Avidin peroxidase (Sigma) was used as the revealing conjugate. .. Total cellular RNA was extracted from adherent cells using Trizol reagent ( Gibco BRL, Life Technologies, Frederick, MD, USA).

    Article Title: CD4+ T helper responses in squamous cell carcinoma of the head and neck
    Article Snippet: .. After 2 h incubation, plates were washed with PBS/0.05% Tween 20, and developed with the avidin-peroxidase complex (Vectastain Elite kit; Vector) for 1 h. 3-Amino-9-ethyl-carbozole (Sigma, St. Louis, MO) was added and incubated for 5 min for the IFN-γ ELISPOT assay and the TMB substrate for peroxidase (3,3′,5,5′-Tetramethylbenzidine; Vector Laboratories) was added and incubated for 10 min for the IL-5 ELISPOT assay. ..

    Article Title: Leukocyte, Rather than Tumor-produced SPARC, Determines Stroma and Collagen Type IV Deposition in Mammary Carcinoma
    Article Snippet: .. After washing, sections were sequentially overlaid with biotinylated goat anti–rabbit IgG for 30 min and with avidin–peroxidase complex for 30 min (Sigma-Aldrich). .. Avidin–peroxidase complex or peroxidase-conjugated rabbit anti-FITC antibody (DakoCytomation) was used as secondary antibody for sections incubated with biotinylated primary antibodies or FITC-conjugated primary antibody, respectively.

    Article Title: Transforming growth factor-β plays divergent roles in modulating vascular remodeling, inflammation, and pulmonary fibrosis in a murine model of scleroderma
    Article Snippet: .. Signals were visualized using avidin-biotin peroxidase (Vectastain Elite kit) with 3,3′-diaminobenzidine (Sigma). .. For immunofluorescence staining, Alexa 488- or Cy3-conjugated anti-α-SMA (clone 1A4, Sigma-Aldrich) was applied to sections for 1 h at room temperature.

    Plasmid Preparation:

    Article Title: CD4+ T helper responses in squamous cell carcinoma of the head and neck
    Article Snippet: .. After 2 h incubation, plates were washed with PBS/0.05% Tween 20, and developed with the avidin-peroxidase complex (Vectastain Elite kit; Vector) for 1 h. 3-Amino-9-ethyl-carbozole (Sigma, St. Louis, MO) was added and incubated for 5 min for the IFN-γ ELISPOT assay and the TMB substrate for peroxidase (3,3′,5,5′-Tetramethylbenzidine; Vector Laboratories) was added and incubated for 10 min for the IL-5 ELISPOT assay. ..

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  • 99
    Millipore avidin biotin peroxidase complex technique
    Avidin Biotin Peroxidase Complex Technique, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/avidin biotin peroxidase complex technique/product/Millipore
    Average 99 stars, based on 13 article reviews
    Price from $9.99 to $1999.99
    avidin biotin peroxidase complex technique - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    91
    Millipore streptavidin biotinylated hrp complex
    Swarm sensing with dropicles. (A) Histograms of fluorescence intensities for populations of dropicles formed from O-shaped particle design 2 after the QuantaRed assay readout at 45 min. Concentration of <t>streptavidin-HRP</t> ranges from 0 (control) to 1 nM. Results correlate with the mean intensity results reported in Figure 6C. (B) Normalized standard error of the mean vs number of dropicles in the sample for the amplified assay showing that a larger number of dropicles leads to a more accurate representation of the concentration of analyte.
    Streptavidin Biotinylated Hrp Complex, supplied by Millipore, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/streptavidin biotinylated hrp complex/product/Millipore
    Average 91 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    streptavidin biotinylated hrp complex - by Bioz Stars, 2020-09
    91/100 stars
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    90
    Millipore avidin biotin immunoperoxidase technique
    Swarm sensing with dropicles. (A) Histograms of fluorescence intensities for populations of dropicles formed from O-shaped particle design 2 after the QuantaRed assay readout at 45 min. Concentration of <t>streptavidin-HRP</t> ranges from 0 (control) to 1 nM. Results correlate with the mean intensity results reported in Figure 6C. (B) Normalized standard error of the mean vs number of dropicles in the sample for the amplified assay showing that a larger number of dropicles leads to a more accurate representation of the concentration of analyte.
    Avidin Biotin Immunoperoxidase Technique, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/avidin biotin immunoperoxidase technique/product/Millipore
    Average 90 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    avidin biotin immunoperoxidase technique - by Bioz Stars, 2020-09
    90/100 stars
      Buy from Supplier

    Image Search Results


    Swarm sensing with dropicles. (A) Histograms of fluorescence intensities for populations of dropicles formed from O-shaped particle design 2 after the QuantaRed assay readout at 45 min. Concentration of streptavidin-HRP ranges from 0 (control) to 1 nM. Results correlate with the mean intensity results reported in Figure 6C. (B) Normalized standard error of the mean vs number of dropicles in the sample for the amplified assay showing that a larger number of dropicles leads to a more accurate representation of the concentration of analyte.

    Journal: bioRxiv

    Article Title: Formation of uniform reaction volumes using concentric amphiphilic microparticles

    doi: 10.1101/2020.03.15.992321

    Figure Lengend Snippet: Swarm sensing with dropicles. (A) Histograms of fluorescence intensities for populations of dropicles formed from O-shaped particle design 2 after the QuantaRed assay readout at 45 min. Concentration of streptavidin-HRP ranges from 0 (control) to 1 nM. Results correlate with the mean intensity results reported in Figure 6C. (B) Normalized standard error of the mean vs number of dropicles in the sample for the amplified assay showing that a larger number of dropicles leads to a more accurate representation of the concentration of analyte.

    Article Snippet: For the biotin to streptavidin-HRP binding assays reported, the inner PEGDA layer is also biotinylated to enable binding of streptavidin.

    Techniques: Fluorescence, Concentration Assay, Amplification

    Minimal cross talk is observed between reactions in separate dropicles over time. Microscopic images showing the same well at 15 min, 35 min, 60 min, and 48 hrs after initiating the QuantaRed reaction. Two types of particles (with and without biotin) are introduced and incubated with 0.1 nM of streptavidin-HRP.

    Journal: bioRxiv

    Article Title: Formation of uniform reaction volumes using concentric amphiphilic microparticles

    doi: 10.1101/2020.03.15.992321

    Figure Lengend Snippet: Minimal cross talk is observed between reactions in separate dropicles over time. Microscopic images showing the same well at 15 min, 35 min, 60 min, and 48 hrs after initiating the QuantaRed reaction. Two types of particles (with and without biotin) are introduced and incubated with 0.1 nM of streptavidin-HRP.

    Article Snippet: For the biotin to streptavidin-HRP binding assays reported, the inner PEGDA layer is also biotinylated to enable binding of streptavidin.

    Techniques: Incubation

    Amplified bioassay in dropicles. (A) Schematic of the assay workflow for biotin streptavidin affinity and the mechanism of amplification using horse radish peroxidase (HRP) turnover of the fluorogenic substrate ADHP to generate resorufin. (B) Microscopic images of a single well at different steps of the assay workflow. Images are captured when particles are in ethanol (step 1), PBS (step 2), and PSDS oil (step 4) where dropicles are formed. Zoomed in inset images of the same field-of-view highlight the particle morphology changes as the inner PEG and outer PPG layers swell or shrink in different solutions. Scale bar for the whole well is 1 mm, and the scale bar for the insets is 100 μm.

    Journal: bioRxiv

    Article Title: Formation of uniform reaction volumes using concentric amphiphilic microparticles

    doi: 10.1101/2020.03.15.992321

    Figure Lengend Snippet: Amplified bioassay in dropicles. (A) Schematic of the assay workflow for biotin streptavidin affinity and the mechanism of amplification using horse radish peroxidase (HRP) turnover of the fluorogenic substrate ADHP to generate resorufin. (B) Microscopic images of a single well at different steps of the assay workflow. Images are captured when particles are in ethanol (step 1), PBS (step 2), and PSDS oil (step 4) where dropicles are formed. Zoomed in inset images of the same field-of-view highlight the particle morphology changes as the inner PEG and outer PPG layers swell or shrink in different solutions. Scale bar for the whole well is 1 mm, and the scale bar for the insets is 100 μm.

    Article Snippet: For the biotin to streptavidin-HRP binding assays reported, the inner PEGDA layer is also biotinylated to enable binding of streptavidin.

    Techniques: Amplification

    Duplex assay using shape-coded particles demonstrating minimal cross talk. (A) Schematic of the duplex assay showing the two particle populations: plus-shaped particles without biotin in the PEGDA layer are used as negative control population; H-shaped particles with biotin in the PEGDA layer are used as a positive population. (B) A merged image of bright field and fluorescent channels, after mixing plus- and H-shaped particles, incubating with 0.1 nM streptavidin-HRP solution, and initiating the HRP amplification reaction. There is contrast in the red fluorescent signal between plus-shaped particles and H-shaped particles. (C) Fluorescence images of the same field of view as in (B) at 15, 35 and 60 min after initiating the reaction. Red dotted lines in the images outline the PPG boundary of H-particles (positive) while white dotted lines outline the PPG boundary of plus-particle (negative). Scale bars in (B-C) represent 100μm. (D) Histograms showing the intensity distribution for a population of plus-shaped and H-shaped dropicles at 15, 35 and 60 min. (E) The mean of fluorescent intensity at these three timepoints 15, 35, and 60 min for H- and plus-shaped particles, showing the negative volume does not appear to have an appreciable increase in intensity over time even as the positive volume shows high levels of fluorescence intensity increase. The error bars represent standard deviation.

    Journal: bioRxiv

    Article Title: Formation of uniform reaction volumes using concentric amphiphilic microparticles

    doi: 10.1101/2020.03.15.992321

    Figure Lengend Snippet: Duplex assay using shape-coded particles demonstrating minimal cross talk. (A) Schematic of the duplex assay showing the two particle populations: plus-shaped particles without biotin in the PEGDA layer are used as negative control population; H-shaped particles with biotin in the PEGDA layer are used as a positive population. (B) A merged image of bright field and fluorescent channels, after mixing plus- and H-shaped particles, incubating with 0.1 nM streptavidin-HRP solution, and initiating the HRP amplification reaction. There is contrast in the red fluorescent signal between plus-shaped particles and H-shaped particles. (C) Fluorescence images of the same field of view as in (B) at 15, 35 and 60 min after initiating the reaction. Red dotted lines in the images outline the PPG boundary of H-particles (positive) while white dotted lines outline the PPG boundary of plus-particle (negative). Scale bars in (B-C) represent 100μm. (D) Histograms showing the intensity distribution for a population of plus-shaped and H-shaped dropicles at 15, 35 and 60 min. (E) The mean of fluorescent intensity at these three timepoints 15, 35, and 60 min for H- and plus-shaped particles, showing the negative volume does not appear to have an appreciable increase in intensity over time even as the positive volume shows high levels of fluorescence intensity increase. The error bars represent standard deviation.

    Article Snippet: For the biotin to streptavidin-HRP binding assays reported, the inner PEGDA layer is also biotinylated to enable binding of streptavidin.

    Techniques: Negative Control, Amplification, Fluorescence, Standard Deviation

    Amplified assay performance in dropicles. (A) Microscopic images of QuantaRed assay results using two O-shaped particle designs for two concentrations of streptavidin-HRP, i.e., negative control and 1 nM. The look-up table was adjusted to be the same for each condition for visibility. The scale bar represents 100 μm. (B-C) Mean amplified intensity across a population of dropicles as a function of concentration of streptavidin-HRP using (B) particle design 1 (N=11-38) and (C) particle design 2 (N=307-382), each tuned by varying the thickness ( t ) and width ( w ) of the internal PEG layer. (D) Mean amplified fluorescent intensity across a population of dropicles as a function of concentration of NT-proBNP. Data reported in (A-D) were obtained after 45 min of reaction. Error bars in (B-D) represent standard error. *** represents p

    Journal: bioRxiv

    Article Title: Formation of uniform reaction volumes using concentric amphiphilic microparticles

    doi: 10.1101/2020.03.15.992321

    Figure Lengend Snippet: Amplified assay performance in dropicles. (A) Microscopic images of QuantaRed assay results using two O-shaped particle designs for two concentrations of streptavidin-HRP, i.e., negative control and 1 nM. The look-up table was adjusted to be the same for each condition for visibility. The scale bar represents 100 μm. (B-C) Mean amplified intensity across a population of dropicles as a function of concentration of streptavidin-HRP using (B) particle design 1 (N=11-38) and (C) particle design 2 (N=307-382), each tuned by varying the thickness ( t ) and width ( w ) of the internal PEG layer. (D) Mean amplified fluorescent intensity across a population of dropicles as a function of concentration of NT-proBNP. Data reported in (A-D) were obtained after 45 min of reaction. Error bars in (B-D) represent standard error. *** represents p

    Article Snippet: For the biotin to streptavidin-HRP binding assays reported, the inner PEGDA layer is also biotinylated to enable binding of streptavidin.

    Techniques: Amplification, Negative Control, Concentration Assay

    Time dependence of intensity change in dropicles using a QuantaRed assay. (A) Fluorescence signal increase over time from 10 to 45 min for binding of biotinylated particles with streptavidin-HRP at 10 pM and 100 fM concentrations. The control condition is the same particles and reagents without streptavidin-HRP. Data represents mean of intensity for dropicles with error bars representing standard error of the mean. Intensity drop at early time points is likely due to changes in shape/swelling upon introduction of the oil phase. (B-C) Fluorescence intensity from individual particles are shown over time for (B) 100 fM and (C) 10 pM concentrations of streptavidin-HRP respectively.

    Journal: bioRxiv

    Article Title: Formation of uniform reaction volumes using concentric amphiphilic microparticles

    doi: 10.1101/2020.03.15.992321

    Figure Lengend Snippet: Time dependence of intensity change in dropicles using a QuantaRed assay. (A) Fluorescence signal increase over time from 10 to 45 min for binding of biotinylated particles with streptavidin-HRP at 10 pM and 100 fM concentrations. The control condition is the same particles and reagents without streptavidin-HRP. Data represents mean of intensity for dropicles with error bars representing standard error of the mean. Intensity drop at early time points is likely due to changes in shape/swelling upon introduction of the oil phase. (B-C) Fluorescence intensity from individual particles are shown over time for (B) 100 fM and (C) 10 pM concentrations of streptavidin-HRP respectively.

    Article Snippet: For the biotin to streptavidin-HRP binding assays reported, the inner PEGDA layer is also biotinylated to enable binding of streptavidin.

    Techniques: Fluorescence, Binding Assay

    Conception and applicability of autotransporter (AT)—complement receptor ligand (CRL) fusions. ( a ) Graphical abstract. ( b ) The modification of YadA to yield YFC (YadA-FLAG-C3d) and YFP (YadA-FLAG-p28); SS: signal sequence. F: FLAG tag. ( c ) Heterologous expression of plasmid-borne YadA by Ec and Ft detected by western blot with α-YadA sera followed by horseradish peroxidase (HRP)-conjugated secondary Ab. Strains bearing empty vector are denoted by “-”. Arrows indicate the trimers and monomers of unmodified YadA. ( d ) Collagen-coated ELISA plates were incubated with serial dilutions of intact Ft :-, Ft :YadA, and Ft :YFC; bound bacteria were detected by α- Ft LPS Ab. ( e , f ) Whole cell lysates of Ec , Kp , and Ft containing empty vector (-) or the YFP or YFC expression vectors were probed by western blot with primary Ab specific for C3d and the FLAG epitope followed sequentially by biotinylated secondary Abs and streptavidin-HRP (SA-HRP). YFC and YFP trimers and monomers are designated with black and grey arrows. The ~20 kDa bands evident in all lanes are endogenously biotinylated bacterial proteins (annotated AccB in Ec and Ft [ 40 ]) detected by SA-HRP. ( g ) Intact bacteria as indicated were incubated in solution with α-FLAG Ab. ( h ) Intact Ft as indicated were incubated in solution with the indicated Ab. IglC is primarily a cytoplasmic Ft protein. Washed bacteria in ( g , h ) were probed for IgG heavy chain (HC), followed by biotinylated secondary Abs and SA-HRP.

    Journal: Pathogens

    Article Title: Autotransporter-Mediated Display of Complement Receptor Ligands by Gram-Negative Bacteria Increases Antibody Responses and Limits Disease Severity

    doi: 10.3390/pathogens9050375

    Figure Lengend Snippet: Conception and applicability of autotransporter (AT)—complement receptor ligand (CRL) fusions. ( a ) Graphical abstract. ( b ) The modification of YadA to yield YFC (YadA-FLAG-C3d) and YFP (YadA-FLAG-p28); SS: signal sequence. F: FLAG tag. ( c ) Heterologous expression of plasmid-borne YadA by Ec and Ft detected by western blot with α-YadA sera followed by horseradish peroxidase (HRP)-conjugated secondary Ab. Strains bearing empty vector are denoted by “-”. Arrows indicate the trimers and monomers of unmodified YadA. ( d ) Collagen-coated ELISA plates were incubated with serial dilutions of intact Ft :-, Ft :YadA, and Ft :YFC; bound bacteria were detected by α- Ft LPS Ab. ( e , f ) Whole cell lysates of Ec , Kp , and Ft containing empty vector (-) or the YFP or YFC expression vectors were probed by western blot with primary Ab specific for C3d and the FLAG epitope followed sequentially by biotinylated secondary Abs and streptavidin-HRP (SA-HRP). YFC and YFP trimers and monomers are designated with black and grey arrows. The ~20 kDa bands evident in all lanes are endogenously biotinylated bacterial proteins (annotated AccB in Ec and Ft [ 40 ]) detected by SA-HRP. ( g ) Intact bacteria as indicated were incubated in solution with α-FLAG Ab. ( h ) Intact Ft as indicated were incubated in solution with the indicated Ab. IglC is primarily a cytoplasmic Ft protein. Washed bacteria in ( g , h ) were probed for IgG heavy chain (HC), followed by biotinylated secondary Abs and SA-HRP.

    Article Snippet: Secondary horseradish peroxidase (HRP)-conjugated antibodies were diluted to the indicated concentrations in PBST and incubated at 4 °C for 1 h. Where indicated, a biotinylated secondary antibody was used, followed by streptavidin-linked HRP, each for 1 h incubations at 4 °C.

    Techniques: Modification, Sequencing, FLAG-tag, Expressing, Plasmid Preparation, Western Blot, Enzyme-linked Immunosorbent Assay, Incubation