streptavidin horseradish peroxidase  (Vector Laboratories)


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    Structured Review

    Vector Laboratories streptavidin horseradish peroxidase
    Prolectin acts as a cell adhesion molecule ( A ) Expression of fucosylated markers on normal CHO cells or CHO cells engineered to express fucosylated antigens tested by flow cytometry. Cells were introduced in the channels of a BioFlux microfluidic system coated with <t>prolectin/streptavidin</t> tetramers under a pressure of 0.1 Dyn/cm 2 . Unbound cells were washed away and pictures of the attached cells were taken at 10x magnification with a Leica DMI 6000B microscope. Bound cells were counted on three different segments along the channels and the mean cell densities and SEM are represented on the histogram. ( B ) DU-145 cells were introduced in the channels under a pressure of 0.1 Dyn/cm 2 and in presence or 25 mM D-galactose or L-fucose. Unbound cells were washed away and pictures of the attached cells were taken. Bound cells were counted in the whole channels. The results of five independent experiments are shown. Due to the high variation between experiments, the results are shown separately. Similar experiments were performed with the other tumor cell lines MCF-7, OVCAR-3 and HT-29. ( C ) MCF10A-LXSN and MCF10A-Kras(v12) cells were treated with dimethyl sulfoxide (DMSO, green), 5 μM kifunensin (orange), or 400 μM 2F-fucose (blue) during four days and stained with prolectin by flow cytometry as described above. Control cells and cells treated with the inhibitors were injected into prolectin-coated BioFlux channels. Unbound cells were washed away and pictures of the attached cells were taken. Bound cells were counted in the whole channels. ( D ) Prolectin expression was tested on Rat6-Neo and Rat6-CTLY11 fibroblasts by flow cytometry. Percentages of “positive” cells are indicated. Rat6-Neo and Rat6-CTLY11 or Rat6-CTLY11 in the presence of 25 mM galactose or fucose were injected in BioFlux channels covered with a layer of DU-145 cells under a pressure of 0.05 Dyn/cm 2 . Films of 1 minute (600 pictures) were acquired on a Leica DMI 6000B microscope using the Metamorph software. Chronophotographic images, corresponding to 10 pictures segments of the films, were constructed using the FiJi software and distances covered by individual cells were measured. Student tests were performed to assess statistical significance.
    Streptavidin Horseradish Peroxidase, supplied by Vector Laboratories, used in various techniques. Bioz Stars score: 97/100, based on 51 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Carcinoma-associated fucosylated antigens are markers of the epithelial state and can contribute to cell adhesion through CLEC17A (Prolectin)"

    Article Title: Carcinoma-associated fucosylated antigens are markers of the epithelial state and can contribute to cell adhesion through CLEC17A (Prolectin)

    Journal: Oncotarget

    doi: 10.18632/oncotarget.7476

    Prolectin acts as a cell adhesion molecule ( A ) Expression of fucosylated markers on normal CHO cells or CHO cells engineered to express fucosylated antigens tested by flow cytometry. Cells were introduced in the channels of a BioFlux microfluidic system coated with prolectin/streptavidin tetramers under a pressure of 0.1 Dyn/cm 2 . Unbound cells were washed away and pictures of the attached cells were taken at 10x magnification with a Leica DMI 6000B microscope. Bound cells were counted on three different segments along the channels and the mean cell densities and SEM are represented on the histogram. ( B ) DU-145 cells were introduced in the channels under a pressure of 0.1 Dyn/cm 2 and in presence or 25 mM D-galactose or L-fucose. Unbound cells were washed away and pictures of the attached cells were taken. Bound cells were counted in the whole channels. The results of five independent experiments are shown. Due to the high variation between experiments, the results are shown separately. Similar experiments were performed with the other tumor cell lines MCF-7, OVCAR-3 and HT-29. ( C ) MCF10A-LXSN and MCF10A-Kras(v12) cells were treated with dimethyl sulfoxide (DMSO, green), 5 μM kifunensin (orange), or 400 μM 2F-fucose (blue) during four days and stained with prolectin by flow cytometry as described above. Control cells and cells treated with the inhibitors were injected into prolectin-coated BioFlux channels. Unbound cells were washed away and pictures of the attached cells were taken. Bound cells were counted in the whole channels. ( D ) Prolectin expression was tested on Rat6-Neo and Rat6-CTLY11 fibroblasts by flow cytometry. Percentages of “positive” cells are indicated. Rat6-Neo and Rat6-CTLY11 or Rat6-CTLY11 in the presence of 25 mM galactose or fucose were injected in BioFlux channels covered with a layer of DU-145 cells under a pressure of 0.05 Dyn/cm 2 . Films of 1 minute (600 pictures) were acquired on a Leica DMI 6000B microscope using the Metamorph software. Chronophotographic images, corresponding to 10 pictures segments of the films, were constructed using the FiJi software and distances covered by individual cells were measured. Student tests were performed to assess statistical significance.
    Figure Legend Snippet: Prolectin acts as a cell adhesion molecule ( A ) Expression of fucosylated markers on normal CHO cells or CHO cells engineered to express fucosylated antigens tested by flow cytometry. Cells were introduced in the channels of a BioFlux microfluidic system coated with prolectin/streptavidin tetramers under a pressure of 0.1 Dyn/cm 2 . Unbound cells were washed away and pictures of the attached cells were taken at 10x magnification with a Leica DMI 6000B microscope. Bound cells were counted on three different segments along the channels and the mean cell densities and SEM are represented on the histogram. ( B ) DU-145 cells were introduced in the channels under a pressure of 0.1 Dyn/cm 2 and in presence or 25 mM D-galactose or L-fucose. Unbound cells were washed away and pictures of the attached cells were taken. Bound cells were counted in the whole channels. The results of five independent experiments are shown. Due to the high variation between experiments, the results are shown separately. Similar experiments were performed with the other tumor cell lines MCF-7, OVCAR-3 and HT-29. ( C ) MCF10A-LXSN and MCF10A-Kras(v12) cells were treated with dimethyl sulfoxide (DMSO, green), 5 μM kifunensin (orange), or 400 μM 2F-fucose (blue) during four days and stained with prolectin by flow cytometry as described above. Control cells and cells treated with the inhibitors were injected into prolectin-coated BioFlux channels. Unbound cells were washed away and pictures of the attached cells were taken. Bound cells were counted in the whole channels. ( D ) Prolectin expression was tested on Rat6-Neo and Rat6-CTLY11 fibroblasts by flow cytometry. Percentages of “positive” cells are indicated. Rat6-Neo and Rat6-CTLY11 or Rat6-CTLY11 in the presence of 25 mM galactose or fucose were injected in BioFlux channels covered with a layer of DU-145 cells under a pressure of 0.05 Dyn/cm 2 . Films of 1 minute (600 pictures) were acquired on a Leica DMI 6000B microscope using the Metamorph software. Chronophotographic images, corresponding to 10 pictures segments of the films, were constructed using the FiJi software and distances covered by individual cells were measured. Student tests were performed to assess statistical significance.

    Techniques Used: Expressing, Flow Cytometry, Cytometry, Microscopy, Staining, Injection, Software, Construct

    The fucose binding lectin BC2L-C-Nt stains epithelial cells rather than mesenchymal cells Breast cell lines were stained with biotinylated lectins BC2L-C-Nt followed by PE-conjugated streptavidin and analyzed by flow cytometry. Representative flow cytometry histograms as well as a histogram of the mean and SEM of Mean fluorescence intensities (MFI) for three independent experiments are presented.
    Figure Legend Snippet: The fucose binding lectin BC2L-C-Nt stains epithelial cells rather than mesenchymal cells Breast cell lines were stained with biotinylated lectins BC2L-C-Nt followed by PE-conjugated streptavidin and analyzed by flow cytometry. Representative flow cytometry histograms as well as a histogram of the mean and SEM of Mean fluorescence intensities (MFI) for three independent experiments are presented.

    Techniques Used: Binding Assay, Staining, Flow Cytometry, Cytometry, Fluorescence

    Prolectin is a human C-type lectin that recognizes similar fucosylated ligands as BC2L-C-Nt and efficiently binds to tumor cell lines and tumor tissues ( A ) Diagram of Prolectin structure (CRD: carbohydrate recognition domain; TM: transmembrane domain; IC: intra-cytoplasmic domain). ( B ) Glycan binding assay of prolectin and BC2L-C-Nt. Plates coated with a series of fucosylated HBGAs conjugated to HSA or PAA were probed with either biotinylated-BC2L-C-Nt followed by Avidin-HRP or tetramers of biotin-prolectin CRD bound to streptavidin-HRP. The results shown are the mean and SEM of three independent experiments. ( C ) Flow cytometry staining of tumor cell lines from various origins. Tetramers of biotinylated-prolectin CRD in complex with streptavidin-PE, pre-incubated in the absence of sugar or with L-fucose, were bound to the indicated cell lines, which were then analyzed by flow cytometry. The bar histogram shows the mean fluorescence intensities. Results are representative of at least two experiments. ( D ) Prolectin preferentially stains epithelial cell lines. The epithelial/mesenchymal cell lines pairs MCF10A-LXSN/MCF10A-Kras(v12) and MCF10A-Puro R /MCF10A-SNAIL were stained with prolectin as described above. Means and SEM of three independent experiments are shown ( E ) Prolectin binding to tumor tissues. Tetramers of biotinylated-prolectin CRD conjugated with streptavidin-HRP were incubated on fixed tissue sections from different tumor (T) or adjacent normal (NT) tissues. To control for the involvement of specific glycan binding in the prolectin staining, colon tumor sections were stained as above after prolectin tetramers were incubated with Ca 2+ , EDTA or Ca 2+ + L-fucose. Colon tissues had been fixed in ethanol whereas breast, lung and uterine tissues were from formalin-fixed TMAs. Magnifications of 20× are shown (the black bars represent 100 μm).
    Figure Legend Snippet: Prolectin is a human C-type lectin that recognizes similar fucosylated ligands as BC2L-C-Nt and efficiently binds to tumor cell lines and tumor tissues ( A ) Diagram of Prolectin structure (CRD: carbohydrate recognition domain; TM: transmembrane domain; IC: intra-cytoplasmic domain). ( B ) Glycan binding assay of prolectin and BC2L-C-Nt. Plates coated with a series of fucosylated HBGAs conjugated to HSA or PAA were probed with either biotinylated-BC2L-C-Nt followed by Avidin-HRP or tetramers of biotin-prolectin CRD bound to streptavidin-HRP. The results shown are the mean and SEM of three independent experiments. ( C ) Flow cytometry staining of tumor cell lines from various origins. Tetramers of biotinylated-prolectin CRD in complex with streptavidin-PE, pre-incubated in the absence of sugar or with L-fucose, were bound to the indicated cell lines, which were then analyzed by flow cytometry. The bar histogram shows the mean fluorescence intensities. Results are representative of at least two experiments. ( D ) Prolectin preferentially stains epithelial cell lines. The epithelial/mesenchymal cell lines pairs MCF10A-LXSN/MCF10A-Kras(v12) and MCF10A-Puro R /MCF10A-SNAIL were stained with prolectin as described above. Means and SEM of three independent experiments are shown ( E ) Prolectin binding to tumor tissues. Tetramers of biotinylated-prolectin CRD conjugated with streptavidin-HRP were incubated on fixed tissue sections from different tumor (T) or adjacent normal (NT) tissues. To control for the involvement of specific glycan binding in the prolectin staining, colon tumor sections were stained as above after prolectin tetramers were incubated with Ca 2+ , EDTA or Ca 2+ + L-fucose. Colon tissues had been fixed in ethanol whereas breast, lung and uterine tissues were from formalin-fixed TMAs. Magnifications of 20× are shown (the black bars represent 100 μm).

    Techniques Used: Binding Assay, Avidin-Biotin Assay, Flow Cytometry, Cytometry, Staining, Incubation, Fluorescence

    2) Product Images from "Transcriptional and Proteomic Characterization of Telomere-Induced Senescence in a Human Alveolar Epithelial Cell Line"

    Article Title: Transcriptional and Proteomic Characterization of Telomere-Induced Senescence in a Human Alveolar Epithelial Cell Line

    Journal: Frontiers in Medicine

    doi: 10.3389/fmed.2021.600626

    ER-targeted BioID2 to label secreted proteins. (A) Schematic of lentivirus expressing ER-targeted BioID2 construct. (B) Photomicrographs of A549 and A549-BioID2 cells showing co-localization of the BioID2 (HA-tagged; green) and the ER marker calnexin (red). Nuclei are stained with DAPI (blue). Scale bar is 25 microns. (C) Proof of concept demonstrating that secreted proteins are biotinylated by ER-targeted BioID2. A549 or A549 cells stable expression ER-BioID2 were transfected with a plasmid encoding a V5-SFTPA2 construct. Eighteen hours after transfection, biotin was added to some samples and the media and cell lysates were examined the next day by western blotting for SFTPA2 (V5), BioID2 (HA), or biotinylated proteins (Strep-HRP). GAPDH was a load control for cell lysates. Biotinylated proteins were detected in the media only in cells that expressed ER-BioID2 and were cultured in excess biotin. Biotinylated SFTPA2 was detected in media only when ER-BioID2 was present. (D) Experimental procedure for unbiased proteomic analysis of secreted proteins. Cells were cultured for 5 days in the presence of Dox to induce senescence followed by incubation with excess biotin for 8 h. After biotin labeling, cells were washed and fresh media was added. Twenty-four hours later, media was collected and biotinylated proteins were isolated by incubation with streptavidin-coated beads and analyzed by stain-free SDS-page (E) .
    Figure Legend Snippet: ER-targeted BioID2 to label secreted proteins. (A) Schematic of lentivirus expressing ER-targeted BioID2 construct. (B) Photomicrographs of A549 and A549-BioID2 cells showing co-localization of the BioID2 (HA-tagged; green) and the ER marker calnexin (red). Nuclei are stained with DAPI (blue). Scale bar is 25 microns. (C) Proof of concept demonstrating that secreted proteins are biotinylated by ER-targeted BioID2. A549 or A549 cells stable expression ER-BioID2 were transfected with a plasmid encoding a V5-SFTPA2 construct. Eighteen hours after transfection, biotin was added to some samples and the media and cell lysates were examined the next day by western blotting for SFTPA2 (V5), BioID2 (HA), or biotinylated proteins (Strep-HRP). GAPDH was a load control for cell lysates. Biotinylated proteins were detected in the media only in cells that expressed ER-BioID2 and were cultured in excess biotin. Biotinylated SFTPA2 was detected in media only when ER-BioID2 was present. (D) Experimental procedure for unbiased proteomic analysis of secreted proteins. Cells were cultured for 5 days in the presence of Dox to induce senescence followed by incubation with excess biotin for 8 h. After biotin labeling, cells were washed and fresh media was added. Twenty-four hours later, media was collected and biotinylated proteins were isolated by incubation with streptavidin-coated beads and analyzed by stain-free SDS-page (E) .

    Techniques Used: Expressing, Construct, Marker, Staining, Transfection, Plasmid Preparation, Western Blot, Cell Culture, Incubation, Labeling, Isolation, SDS Page

    3) Product Images from "A developmentally regulated Na-H exchanger in Dictyostelium discoideum is necessary for cell polarity during chemotaxis"

    Article Title: A developmentally regulated Na-H exchanger in Dictyostelium discoideum is necessary for cell polarity during chemotaxis

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200412145

    Dd NHE1 groups with plasma membrane NHEs and localizes to the leading edge of chemotaxing cells. (A) Dictyostelium NHE1 ( Dd NHE1; AA052201 ), the nine known mammalian NHEs ( Hs NHE1–9; P19634 , Q9UBY0 , P48764 , XM_371544.2, Q14940 , Q92581 , Q96T83 , Q9Y2E8 , and Q8IVB4 , respectively), and three Drosophila NHEs ( Dm NHE1–3; Q8SZX8 , Q9VIF9, and Q81PJ4 , respectively) were aligned using ClustalW; the resulting tree diagram is shown. An asterisk (plasma membrane) or plus (intracellular membranes) denotes NHEs confirmed to have the indicated localization. (B) Localization of Dd NHE1-HA in chemotaxing cells, as determined by immunolabeling with anti-HA antibodies, was visualized by confocal microscopy. The three-dimensional projection of two representative cells is shown. White asterisks indicate direction of the point source of cAMP delivered by a micropipette. (C) Surface biotinylation followed by immunoprecipitating the indicated proteins and immunoblotting with HRP:Streptavidin-confirmed Dd NHE1-HA is at the plasma membrane. Immunoprecipitation of the plasma membrane cAMP receptor 1 (cAR1) in cells expressing Dd NHE1-HA and the GFP-tagged PH domain of CRAC (GFP-PH CRAC ) expressed in Ax2 cells were included as positive and negative controls, respectively. Immunoblot of total PH CRAC :GFP is shown separately (right).
    Figure Legend Snippet: Dd NHE1 groups with plasma membrane NHEs and localizes to the leading edge of chemotaxing cells. (A) Dictyostelium NHE1 ( Dd NHE1; AA052201 ), the nine known mammalian NHEs ( Hs NHE1–9; P19634 , Q9UBY0 , P48764 , XM_371544.2, Q14940 , Q92581 , Q96T83 , Q9Y2E8 , and Q8IVB4 , respectively), and three Drosophila NHEs ( Dm NHE1–3; Q8SZX8 , Q9VIF9, and Q81PJ4 , respectively) were aligned using ClustalW; the resulting tree diagram is shown. An asterisk (plasma membrane) or plus (intracellular membranes) denotes NHEs confirmed to have the indicated localization. (B) Localization of Dd NHE1-HA in chemotaxing cells, as determined by immunolabeling with anti-HA antibodies, was visualized by confocal microscopy. The three-dimensional projection of two representative cells is shown. White asterisks indicate direction of the point source of cAMP delivered by a micropipette. (C) Surface biotinylation followed by immunoprecipitating the indicated proteins and immunoblotting with HRP:Streptavidin-confirmed Dd NHE1-HA is at the plasma membrane. Immunoprecipitation of the plasma membrane cAMP receptor 1 (cAR1) in cells expressing Dd NHE1-HA and the GFP-tagged PH domain of CRAC (GFP-PH CRAC ) expressed in Ax2 cells were included as positive and negative controls, respectively. Immunoblot of total PH CRAC :GFP is shown separately (right).

    Techniques Used: Immunolabeling, Confocal Microscopy, Immunoprecipitation, Expressing

    4) Product Images from "Neutralizing and non-neutralizing monoclonal antibodies against dengue virus E protein derived from a naturally infected patient"

    Article Title: Neutralizing and non-neutralizing monoclonal antibodies against dengue virus E protein derived from a naturally infected patient

    Journal: Virology Journal

    doi: 10.1186/1743-422X-7-28

    Cross-competition between HMAbs . A cross-competition assay was performed to determine whether the three HMAbs and MMAb 4G2 recognized overlapping or non-overlapping sties on DENV-1 E protein. Purified MMAb 4G2 and HMAbs were incubated with biotinylated HMAbs, washed, and the presence of biotinylated antibodies was detected using streptavidin.
    Figure Legend Snippet: Cross-competition between HMAbs . A cross-competition assay was performed to determine whether the three HMAbs and MMAb 4G2 recognized overlapping or non-overlapping sties on DENV-1 E protein. Purified MMAb 4G2 and HMAbs were incubated with biotinylated HMAbs, washed, and the presence of biotinylated antibodies was detected using streptavidin.

    Techniques Used: Competitive Binding Assay, Purification, Incubation

    5) Product Images from "M Protein of the Group A Streptococcus Binds to the Seventh Short Consensus Repeat of Human Complement Factor H"

    Article Title: M Protein of the Group A Streptococcus Binds to the Seventh Short Consensus Repeat of Human Complement Factor H

    Journal: Infection and Immunity

    doi:

    Chemical cross-linking of fH, H15, and H5 to M6 protein. Biotinylated M6 protein and fH, H15, or H5 were incubated in 50 mM bicarbonate buffer (pH 7.4) with or without DSP cross-linker, as described in Materials and Methods. Proteins were separated by SDS–6% PAGE under nonreducing conditions, transferred to nitrocellulose, and then incubated in streptavidin-HRP. M6 protein and cross-linked M6 protein were detected by chemiluminescence. Cross-linking of M6 protein to fH and H15 is indicated by the arrows.
    Figure Legend Snippet: Chemical cross-linking of fH, H15, and H5 to M6 protein. Biotinylated M6 protein and fH, H15, or H5 were incubated in 50 mM bicarbonate buffer (pH 7.4) with or without DSP cross-linker, as described in Materials and Methods. Proteins were separated by SDS–6% PAGE under nonreducing conditions, transferred to nitrocellulose, and then incubated in streptavidin-HRP. M6 protein and cross-linked M6 protein were detected by chemiluminescence. Cross-linking of M6 protein to fH and H15 is indicated by the arrows.

    Techniques Used: Incubation, Polyacrylamide Gel Electrophoresis

    6) Product Images from "Pro-inflammatory State in Monoclonal Gammopathy of Undetermined Significance and in Multiple Myeloma Is Characterized by Low Sialylation of Pathogen-Specific and Other Monoclonal Immunoglobulins"

    Article Title: Pro-inflammatory State in Monoclonal Gammopathy of Undetermined Significance and in Multiple Myeloma Is Characterized by Low Sialylation of Pathogen-Specific and Other Monoclonal Immunoglobulins

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2017.01347

    Purification scheme of mc IgGs from the serum of monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM) patients. (A) Mc and pc IgGs from MGUS and MM patients were submitted to high-resolution agarose gel electrophoresis and then cut from the gel. (B) The purity of mc and pc IgGs was checked via isoelectro-focalization (IEF) and immunoblotting using a peroxydase coupled to an anti-human IgG-γ chain antibody (HRP anti-human IgG Ab). (C) The sialylation of mc IgG was assessed after incubation with biotinylated Sambucus nigra agglutinin (SNA) and streptavidin peroxidase. (B,C) are representative of purified basic or acid mc IgG, respectively.
    Figure Legend Snippet: Purification scheme of mc IgGs from the serum of monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM) patients. (A) Mc and pc IgGs from MGUS and MM patients were submitted to high-resolution agarose gel electrophoresis and then cut from the gel. (B) The purity of mc and pc IgGs was checked via isoelectro-focalization (IEF) and immunoblotting using a peroxydase coupled to an anti-human IgG-γ chain antibody (HRP anti-human IgG Ab). (C) The sialylation of mc IgG was assessed after incubation with biotinylated Sambucus nigra agglutinin (SNA) and streptavidin peroxidase. (B,C) are representative of purified basic or acid mc IgG, respectively.

    Techniques Used: Purification, Agarose Gel Electrophoresis, Electrofocusing, Incubation

    7) Product Images from "Passive Immunization with Tau Oligomer Monoclonal Antibody Reverses Tauopathy Phenotypes without Affecting Hyperphosphorylated Neurofibrillary Tangles"

    Article Title: Passive Immunization with Tau Oligomer Monoclonal Antibody Reverses Tauopathy Phenotypes without Affecting Hyperphosphorylated Neurofibrillary Tangles

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.3192-13.2014

    Extracellular and peripheral clearance of tau oligomers by TOMA. A – D , In vivo imaging demonstrates that a fraction of TOMA injected into the tail vein crosses the BBB and binds to tau oligomers in the 8-month-old P301L brain and spinal cord. A , TOMA at the injection site in 8-month-old wild-type-C57 and P301L mice at time 0; animals were imaged immediately after the injection. B , Animals imaged 4 h after injection showed retention of TOMA only in the brains of P301L animals, whereas TOMA was cleared from the wild-type animals. C – D , To confirm that TOMA entered the CNS, we imaged brain and spinal cord sections derived from cryotome cross-sectioning of P301L and wild-type brains and spinal cords extracted from animals killed 4 h after TOMA injection. E – J , Biotinylated TOMA antibody (30 μg/animal) was injected into the tail veins of 8-month-old P301L mice. E – H , Representative images of brain sections stained with streptavidin-peroxidase demonstrate that biotinylated TOMA antibody crosses the BBB and remains in hippocampus 2, 6, and 24 h after injection. Scale bar, 30 μm. I , Dot blot analyses of PBS soluble fractions from brain homogenates of mice injected with biotinylated TOMA confirmed the presence of the biotinylated antibody in the brain compared with the control (brain homogenate from P301L mouse injected with unlabeled TOMA). A 0.5 μl aliquot of biotinylated-TOMA was used as a positive control. J , Quantification of the dot intensities. ★★★ p
    Figure Legend Snippet: Extracellular and peripheral clearance of tau oligomers by TOMA. A – D , In vivo imaging demonstrates that a fraction of TOMA injected into the tail vein crosses the BBB and binds to tau oligomers in the 8-month-old P301L brain and spinal cord. A , TOMA at the injection site in 8-month-old wild-type-C57 and P301L mice at time 0; animals were imaged immediately after the injection. B , Animals imaged 4 h after injection showed retention of TOMA only in the brains of P301L animals, whereas TOMA was cleared from the wild-type animals. C – D , To confirm that TOMA entered the CNS, we imaged brain and spinal cord sections derived from cryotome cross-sectioning of P301L and wild-type brains and spinal cords extracted from animals killed 4 h after TOMA injection. E – J , Biotinylated TOMA antibody (30 μg/animal) was injected into the tail veins of 8-month-old P301L mice. E – H , Representative images of brain sections stained with streptavidin-peroxidase demonstrate that biotinylated TOMA antibody crosses the BBB and remains in hippocampus 2, 6, and 24 h after injection. Scale bar, 30 μm. I , Dot blot analyses of PBS soluble fractions from brain homogenates of mice injected with biotinylated TOMA confirmed the presence of the biotinylated antibody in the brain compared with the control (brain homogenate from P301L mouse injected with unlabeled TOMA). A 0.5 μl aliquot of biotinylated-TOMA was used as a positive control. J , Quantification of the dot intensities. ★★★ p

    Techniques Used: In Vivo Imaging, Injection, Mouse Assay, Derivative Assay, Staining, Dot Blot, Positive Control

    8) Product Images from "Novel Adhesin from Pasteurella multocida That Binds to the Integrin-Binding Fibronectin FnIII9-10 Repeats ▿"

    Article Title: Novel Adhesin from Pasteurella multocida That Binds to the Integrin-Binding Fibronectin FnIII9-10 Repeats ▿

    Journal: Infection and Immunity

    doi: 10.1128/IAI.01349-07

    Direct binding ELISAs to determine binding of rGST-PM1665 to fibronectin or recombinant fibronectin fragments. Wells were coated with whole fibronectin (control) or FnIII fragments 1 and 2, 4 to 7, or 9 and 10. Biotinylated rGST-PM1665 (A and C) or rGST-PM1665 (B) was added to these coated wells, and the amounts of bound rGST-PM1665 were detected with streptavidin-HRP (A and C) or with anti-GST antibody, followed by an HRP-conjugated anti-goat antibody (B). The error bars indicate standard errors of the mean.
    Figure Legend Snippet: Direct binding ELISAs to determine binding of rGST-PM1665 to fibronectin or recombinant fibronectin fragments. Wells were coated with whole fibronectin (control) or FnIII fragments 1 and 2, 4 to 7, or 9 and 10. Biotinylated rGST-PM1665 (A and C) or rGST-PM1665 (B) was added to these coated wells, and the amounts of bound rGST-PM1665 were detected with streptavidin-HRP (A and C) or with anti-GST antibody, followed by an HRP-conjugated anti-goat antibody (B). The error bars indicate standard errors of the mean.

    Techniques Used: Binding Assay, Recombinant

    Ligand blotting showing binding of biotinylated rGST-PM1665 to fibronectin or fragments of fibronectin. (A) SDS-PAGE gel stained with colloidal blue. Lanes: 1, whole fibronectin; 2, Fn III 1-2 ; 3, Fn III 4-7 ; 4, GST-Fn III 8-10; , 5, Fn III 9-10 ; 6, GST-Fn III 11 . (B) Ligand blot of a gel identical to that shown in panel A. The nitrocellulose membrane was incubated with biotinylated rGST-PM1665, followed by incubation with streptavidin-HRP. Recombinant GST-PM1665 bound preferentially to the GST-FnIII 8-10 and FnIII 9-10 fragments.
    Figure Legend Snippet: Ligand blotting showing binding of biotinylated rGST-PM1665 to fibronectin or fragments of fibronectin. (A) SDS-PAGE gel stained with colloidal blue. Lanes: 1, whole fibronectin; 2, Fn III 1-2 ; 3, Fn III 4-7 ; 4, GST-Fn III 8-10; , 5, Fn III 9-10 ; 6, GST-Fn III 11 . (B) Ligand blot of a gel identical to that shown in panel A. The nitrocellulose membrane was incubated with biotinylated rGST-PM1665, followed by incubation with streptavidin-HRP. Recombinant GST-PM1665 bound preferentially to the GST-FnIII 8-10 and FnIII 9-10 fragments.

    Techniques Used: Binding Assay, SDS Page, Staining, Incubation, Recombinant

    9) Product Images from "High Expression Level of α2-3-Linked Sialic Acids on Salivary Glycoproteins of Breastfeeding Women May Help to Protect Them from Avian Influenza Virus Infection"

    Article Title: High Expression Level of α2-3-Linked Sialic Acids on Salivary Glycoproteins of Breastfeeding Women May Help to Protect Them from Avian Influenza Virus Infection

    Journal: Molecules

    doi: 10.3390/molecules27134285

    Comparison of theterminal α2-3/6-linked Sia expression level on MUC5B and IgA between saliva groups of women with and without breastfeeding. Salivary MUC5B or IgA was captured by the anti-human MUC5B or anti-human IgA antibody on 96-well plate, and the expression level of terminal α2-3/6-linked Sia, which was recognized by the biotinylated SNA/MAL-II and finally detected by the Avidin-HPR system. The absorbance at 450 nm was read for the analysis of SNA/MAL-II binding to IgA ( A ) and MUC5B ( B ) from different groups. Data shown are mean ± SD, statistical notations and group abbreviations are same as shown in Figure 1 .
    Figure Legend Snippet: Comparison of theterminal α2-3/6-linked Sia expression level on MUC5B and IgA between saliva groups of women with and without breastfeeding. Salivary MUC5B or IgA was captured by the anti-human MUC5B or anti-human IgA antibody on 96-well plate, and the expression level of terminal α2-3/6-linked Sia, which was recognized by the biotinylated SNA/MAL-II and finally detected by the Avidin-HPR system. The absorbance at 450 nm was read for the analysis of SNA/MAL-II binding to IgA ( A ) and MUC5B ( B ) from different groups. Data shown are mean ± SD, statistical notations and group abbreviations are same as shown in Figure 1 .

    Techniques Used: Expressing, Avidin-Biotin Assay, Binding Assay

    10) Product Images from "Altered β1,6-GlcNAc branched N-glycans impair TGF-β-mediated Epithelial-to-Mesenchymal Transition through Smad signalling pathway in human lung cancer"

    Article Title: Altered β1,6-GlcNAc branched N-glycans impair TGF-β-mediated Epithelial-to-Mesenchymal Transition through Smad signalling pathway in human lung cancer

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.12331

    Expression of β1,6-GlcNAc branched N -glycans and GnT-V is down-regulated after TGF-β1 treatment in human lung adenocarcinoma A549 cells. A549 cells were cultured to confluency in six-well plates, and treated with TGF-β1 (2 or 10 ng/ml) for indicated 48 hrs. ( A ) Cell morphological changes were observed under microscopy (top) and the protein levels of EMT markers including E-cadherin, N -cadherin and Vimentin were examined by western blot (bottom). ( B ) The cell surface N -glycans were quantified by flow cytometry with biotinylated L-PHA, DSA, WGA, E-PHA, ConA and GNA lectins, followed by incubation with FITC-conjugated streptavidin. The binding specificity of the lectins used was as indicated: L-PHA, complex-type N -glycans containing β1,6-GlcNAc branched structure; DSA, tri-and tetra-antennary complex type of N -glycans; WGA, tri-and tetra-antennary complex type of N -glycans; E-PHA, bisected type of N -glycans; ConA, hybrid and bi-antennary type of N -glycans; GNA, high-mannose type of N -glycans. ( C ) The N -glycans in cell total lysates were detected by L-PHA, DSA, WGA, E-PHA, ConA and GNA lectin blot. GAPDH was used as a load control. ( D ) TGF-β1 reduces GnT-V expression in A549 cells. mRNA (left) and protein (right) expression of GnT-V was determined by qRT-PCR and western blot respectively.
    Figure Legend Snippet: Expression of β1,6-GlcNAc branched N -glycans and GnT-V is down-regulated after TGF-β1 treatment in human lung adenocarcinoma A549 cells. A549 cells were cultured to confluency in six-well plates, and treated with TGF-β1 (2 or 10 ng/ml) for indicated 48 hrs. ( A ) Cell morphological changes were observed under microscopy (top) and the protein levels of EMT markers including E-cadherin, N -cadherin and Vimentin were examined by western blot (bottom). ( B ) The cell surface N -glycans were quantified by flow cytometry with biotinylated L-PHA, DSA, WGA, E-PHA, ConA and GNA lectins, followed by incubation with FITC-conjugated streptavidin. The binding specificity of the lectins used was as indicated: L-PHA, complex-type N -glycans containing β1,6-GlcNAc branched structure; DSA, tri-and tetra-antennary complex type of N -glycans; WGA, tri-and tetra-antennary complex type of N -glycans; E-PHA, bisected type of N -glycans; ConA, hybrid and bi-antennary type of N -glycans; GNA, high-mannose type of N -glycans. ( C ) The N -glycans in cell total lysates were detected by L-PHA, DSA, WGA, E-PHA, ConA and GNA lectin blot. GAPDH was used as a load control. ( D ) TGF-β1 reduces GnT-V expression in A549 cells. mRNA (left) and protein (right) expression of GnT-V was determined by qRT-PCR and western blot respectively.

    Techniques Used: Expressing, Cell Culture, Microscopy, Western Blot, Flow Cytometry, Cytometry, Whole Genome Amplification, Incubation, Binding Assay, Quantitative RT-PCR

    11) Product Images from "Sialidase Fusion Protein as a Novel Broad-Spectrum Inhibitor of Influenza Virus Infection"

    Article Title: Sialidase Fusion Protein as a Novel Broad-Spectrum Inhibitor of Influenza Virus Infection

    Journal:

    doi: 10.1128/AAC.50.4.1470-1479.2006

    IFV binding to MDCK and fetuin-coated plates. Biotinylated A/PR/8/34 was allowed to bind to the DAS181-treated fetuin or MDCK monolayers for 30 min at 4°C. The bound virus was detected by using streptavidin-HRP and developed by using TMB. Virus
    Figure Legend Snippet: IFV binding to MDCK and fetuin-coated plates. Biotinylated A/PR/8/34 was allowed to bind to the DAS181-treated fetuin or MDCK monolayers for 30 min at 4°C. The bound virus was detected by using streptavidin-HRP and developed by using TMB. Virus

    Techniques Used: Binding Assay

    12) Product Images from "Identification of candidate biomarkers with cancer-specific glycosylation in the tissue and serum of endometrioid ovarian cancer patients by glycoproteomic analysis"

    Article Title: Identification of candidate biomarkers with cancer-specific glycosylation in the tissue and serum of endometrioid ovarian cancer patients by glycoproteomic analysis

    Journal: Proteomics

    doi: 10.1002/pmic.200900537

    Tissue validation. (A) Lectin blot analysis of POSTN immunoprecipitations. POSTN was immunoprecitated from 500 μg of total cell lysate using a polyclonal antibody (Abcam, Cambridge, MA) prior to separation on 4–12% polyacrylamide gel and transfer to PVDF membrane. Blots were probed with biotinylated lectins (1:5000) and detected using streptavidin-coupled HRP (1:5000) and chemiluminescent development. (B) Lectin blot analysis of LAMP-1 immunoprecipitation reactions. LAMP-1 was immunoprecipitated from 500 μg of total cell lysates (normal-NL and tumor-TU) using a monoclonal antibody (E-Biosciences, San Diego, CA) detected by lectin blot as described above.
    Figure Legend Snippet: Tissue validation. (A) Lectin blot analysis of POSTN immunoprecipitations. POSTN was immunoprecitated from 500 μg of total cell lysate using a polyclonal antibody (Abcam, Cambridge, MA) prior to separation on 4–12% polyacrylamide gel and transfer to PVDF membrane. Blots were probed with biotinylated lectins (1:5000) and detected using streptavidin-coupled HRP (1:5000) and chemiluminescent development. (B) Lectin blot analysis of LAMP-1 immunoprecipitation reactions. LAMP-1 was immunoprecipitated from 500 μg of total cell lysates (normal-NL and tumor-TU) using a monoclonal antibody (E-Biosciences, San Diego, CA) detected by lectin blot as described above.

    Techniques Used: Immunoprecipitation

    13) Product Images from "Extrasynaptic α7-Nicotinic Acetylcholine Receptor Expression in Developing Neurons Is Regulated by Inputs, Targets, and Activity"

    Article Title: Extrasynaptic α7-Nicotinic Acetylcholine Receptor Expression in Developing Neurons Is Regulated by Inputs, Targets, and Activity

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.22-18-08101.2002

    α7-nAChR staining is specifically reduced in CG neurons deprived of synaptic partners compared with control neurons. Cryostat sections of CGs from operated and control embryos at E12–E14 were incubated with biotinylated α-Bgt followed by streptavidin–HRP. The sections were then reacted for peroxidase activity and examined by bright-field microscopy. A , Sham-operated E14 CG; B , input-deprived E14 CG; C , contralateral control E12 CG; D , target tissue-deprived E12 CG; E , staining control E14 CG; F , both input- and target-deprived E12 CG. Most of the neuronal somata are intensely stained in the unoperated control ganglion sections ( A , C ). The interiors of the somata are filled with HRP reaction product deposits, with the exception of the nuclei, which, when visible, are not stained above background levels ( E ). In contrast, the majority of the neuronal somata are moderately stained in the input-deprived ganglion section ( B ), only lightly stained in the target-deprived ganglion section ( D ), and just slightly stained above background levels in the input- and target-deprived ganglion section ( F ). Specific α-Bgt staining is demonstrated by the absence of HRP reaction product in the sham-operated E14 ganglion section that was incubated with PBS in place of biotinylated α-Bgt ( C ). In contrast to the declines in α7-nAChRs, similar relative levels of MAP2 immunolabeling are present in the input- and target-deprived E8 ganglion section ( H ) and the sham-operated E8 ganglion section ( G ). Intense MAP2 immunoperoxidase labeling fills the soma and dendrites of the developing CG neurons in G and H . The decreases in α7-nAChR levels are specific. Scale bar: A–F , 30 μm; G , H , 35 μm.
    Figure Legend Snippet: α7-nAChR staining is specifically reduced in CG neurons deprived of synaptic partners compared with control neurons. Cryostat sections of CGs from operated and control embryos at E12–E14 were incubated with biotinylated α-Bgt followed by streptavidin–HRP. The sections were then reacted for peroxidase activity and examined by bright-field microscopy. A , Sham-operated E14 CG; B , input-deprived E14 CG; C , contralateral control E12 CG; D , target tissue-deprived E12 CG; E , staining control E14 CG; F , both input- and target-deprived E12 CG. Most of the neuronal somata are intensely stained in the unoperated control ganglion sections ( A , C ). The interiors of the somata are filled with HRP reaction product deposits, with the exception of the nuclei, which, when visible, are not stained above background levels ( E ). In contrast, the majority of the neuronal somata are moderately stained in the input-deprived ganglion section ( B ), only lightly stained in the target-deprived ganglion section ( D ), and just slightly stained above background levels in the input- and target-deprived ganglion section ( F ). Specific α-Bgt staining is demonstrated by the absence of HRP reaction product in the sham-operated E14 ganglion section that was incubated with PBS in place of biotinylated α-Bgt ( C ). In contrast to the declines in α7-nAChRs, similar relative levels of MAP2 immunolabeling are present in the input- and target-deprived E8 ganglion section ( H ) and the sham-operated E8 ganglion section ( G ). Intense MAP2 immunoperoxidase labeling fills the soma and dendrites of the developing CG neurons in G and H . The decreases in α7-nAChR levels are specific. Scale bar: A–F , 30 μm; G , H , 35 μm.

    Techniques Used: Staining, Incubation, Activity Assay, Microscopy, Immunolabeling, Labeling

    14) Product Images from "Treatment of murine lupus with cDNA encoding IFN-?R/Fc"

    Article Title: Treatment of murine lupus with cDNA encoding IFN-?R/Fc

    Journal: Journal of Clinical Investigation

    doi:

    CD3 + and F4/80 + cells in kidney tissue from MRL-Fas lpr mice treated with blank (VR1255) or active (VR1255-IFN-γR/Fc) plasmid with electroporation from 1 month of age. The numbers of T cells (CD3 + ), and especially macrophages (F4/80 + ), are markedly reduced in the active plasmid–treated groups. Similar reductions were observed in mice treated from 4 months of age with active plasmid (data not shown). Tissue sections were stained with either biotinylated anti-CD3 or anti-F4/80, incubated with streptavidin-horseradish peroxidase, and developed with AEC (see Methods). Photomicrographs are representative sections from four mice in each group. ×25.
    Figure Legend Snippet: CD3 + and F4/80 + cells in kidney tissue from MRL-Fas lpr mice treated with blank (VR1255) or active (VR1255-IFN-γR/Fc) plasmid with electroporation from 1 month of age. The numbers of T cells (CD3 + ), and especially macrophages (F4/80 + ), are markedly reduced in the active plasmid–treated groups. Similar reductions were observed in mice treated from 4 months of age with active plasmid (data not shown). Tissue sections were stained with either biotinylated anti-CD3 or anti-F4/80, incubated with streptavidin-horseradish peroxidase, and developed with AEC (see Methods). Photomicrographs are representative sections from four mice in each group. ×25.

    Techniques Used: Mouse Assay, Plasmid Preparation, Electroporation, Staining, Incubation

    15) Product Images from "Interleukin-1β-induced Reduction of CD44 Ser-325 Phosphorylation in Human Epidermal Keratinocytes Promotes CD44 Homomeric Complexes, Binding to Ezrin, and Extended, Monocyte-adhesive Hyaluronan Coats *"

    Article Title: Interleukin-1β-induced Reduction of CD44 Ser-325 Phosphorylation in Human Epidermal Keratinocytes Promotes CD44 Homomeric Complexes, Binding to Ezrin, and Extended, Monocyte-adhesive Hyaluronan Coats *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M114.620864

    IL-1β and inhibition of CAMKII induce the formation of extended hyaluronan-coats. HaCaT cells were treated with either IL-1β or KN93 for 20 h. A , B , E , F , and G , staining of live cultures for hyaluronan in situ with 5 μg/ml Alexa Fluor 568-labeled HABC in the culture medium. C and D , fixed cultures stained with biotinylated HABC and the anti-CD44 antibody Hermes 3, visualized by FITC-streptavidin and TR-labeled secondary antibody, respectively. Red in A , B , E , F , and G and green in C and D , hyaluronan. Red in C and D , CD44; blue (DRAQ5 TM dye), nuclei. Cell protrusions and hyaluronan cables are indicated by arrows and arrowheads , respectively. Scale bars , 10 μm ( A–D ) and 20 μm ( E ). F and G , specificity of HABC label was checked with hyaluronidase treatment prior to staining. H and J , hyaluronan content in the culture medium. I and K , pericellular hyaluronan (released by trypsin). The treatment time was 20 h for IL-1β and 6 h for KN-93. The values represent means ± S.E. ( error bars ) from five (IL-1β) and three (KN93) independent experiments. **, p
    Figure Legend Snippet: IL-1β and inhibition of CAMKII induce the formation of extended hyaluronan-coats. HaCaT cells were treated with either IL-1β or KN93 for 20 h. A , B , E , F , and G , staining of live cultures for hyaluronan in situ with 5 μg/ml Alexa Fluor 568-labeled HABC in the culture medium. C and D , fixed cultures stained with biotinylated HABC and the anti-CD44 antibody Hermes 3, visualized by FITC-streptavidin and TR-labeled secondary antibody, respectively. Red in A , B , E , F , and G and green in C and D , hyaluronan. Red in C and D , CD44; blue (DRAQ5 TM dye), nuclei. Cell protrusions and hyaluronan cables are indicated by arrows and arrowheads , respectively. Scale bars , 10 μm ( A–D ) and 20 μm ( E ). F and G , specificity of HABC label was checked with hyaluronidase treatment prior to staining. H and J , hyaluronan content in the culture medium. I and K , pericellular hyaluronan (released by trypsin). The treatment time was 20 h for IL-1β and 6 h for KN-93. The values represent means ± S.E. ( error bars ) from five (IL-1β) and three (KN93) independent experiments. **, p

    Techniques Used: Inhibition, Staining, In Situ, Labeling

    16) Product Images from "A Selective Irreversible Inhibitor of Furin Does Not Prevent Pseudomonas Aeruginosa Exotoxin A-Induced Airway Epithelial Cytotoxicity"

    Article Title: A Selective Irreversible Inhibitor of Furin Does Not Prevent Pseudomonas Aeruginosa Exotoxin A-Induced Airway Epithelial Cytotoxicity

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0159868

    Irreversible inhibition and visualization of recombinant human furin by QUB-F1. Western blot analysis was performed under reducing and denaturing conditions demonstrating QUB-F1 (100 μM) is irreversibly bound to furin (0.4 μg). The inhibitor-proteases complex can be visualized using streptavidin-HRP, which detects the biotin reporter group on the compound.
    Figure Legend Snippet: Irreversible inhibition and visualization of recombinant human furin by QUB-F1. Western blot analysis was performed under reducing and denaturing conditions demonstrating QUB-F1 (100 μM) is irreversibly bound to furin (0.4 μg). The inhibitor-proteases complex can be visualized using streptavidin-HRP, which detects the biotin reporter group on the compound.

    Techniques Used: Inhibition, Recombinant, Western Blot

    17) Product Images from "Novel Adhesin from Pasteurella multocida That Binds to the Integrin-Binding Fibronectin FnIII9-10 Repeats ▿"

    Article Title: Novel Adhesin from Pasteurella multocida That Binds to the Integrin-Binding Fibronectin FnIII9-10 Repeats ▿

    Journal: Infection and Immunity

    doi: 10.1128/IAI.01349-07

    Direct binding ELISAs to determine binding of rGST-PM1665 to fibronectin or recombinant fibronectin fragments. Wells were coated with whole fibronectin (control) or FnIII fragments 1 and 2, 4 to 7, or 9 and 10. Biotinylated rGST-PM1665 (A and C) or rGST-PM1665 (B) was added to these coated wells, and the amounts of bound rGST-PM1665 were detected with streptavidin-HRP (A and C) or with anti-GST antibody, followed by an HRP-conjugated anti-goat antibody (B). The error bars indicate standard errors of the mean.
    Figure Legend Snippet: Direct binding ELISAs to determine binding of rGST-PM1665 to fibronectin or recombinant fibronectin fragments. Wells were coated with whole fibronectin (control) or FnIII fragments 1 and 2, 4 to 7, or 9 and 10. Biotinylated rGST-PM1665 (A and C) or rGST-PM1665 (B) was added to these coated wells, and the amounts of bound rGST-PM1665 were detected with streptavidin-HRP (A and C) or with anti-GST antibody, followed by an HRP-conjugated anti-goat antibody (B). The error bars indicate standard errors of the mean.

    Techniques Used: Binding Assay, Recombinant

    Ligand blotting showing binding of biotinylated rGST-PM1665 to fibronectin or fragments of fibronectin. (A) SDS-PAGE gel stained with colloidal blue. Lanes: 1, whole fibronectin; 2, Fn III 1-2 ; 3, Fn III 4-7 ; 4, GST-Fn III 8-10; , 5, Fn III 9-10 ; 6, GST-Fn III 11 . (B) Ligand blot of a gel identical to that shown in panel A. The nitrocellulose membrane was incubated with biotinylated rGST-PM1665, followed by incubation with streptavidin-HRP. Recombinant GST-PM1665 bound preferentially to the GST-FnIII 8-10 and FnIII 9-10 fragments.
    Figure Legend Snippet: Ligand blotting showing binding of biotinylated rGST-PM1665 to fibronectin or fragments of fibronectin. (A) SDS-PAGE gel stained with colloidal blue. Lanes: 1, whole fibronectin; 2, Fn III 1-2 ; 3, Fn III 4-7 ; 4, GST-Fn III 8-10; , 5, Fn III 9-10 ; 6, GST-Fn III 11 . (B) Ligand blot of a gel identical to that shown in panel A. The nitrocellulose membrane was incubated with biotinylated rGST-PM1665, followed by incubation with streptavidin-HRP. Recombinant GST-PM1665 bound preferentially to the GST-FnIII 8-10 and FnIII 9-10 fragments.

    Techniques Used: Binding Assay, SDS Page, Staining, Incubation, Recombinant

    18) Product Images from "M Protein of the Group A Streptococcus Binds to the Seventh Short Consensus Repeat of Human Complement Factor H"

    Article Title: M Protein of the Group A Streptococcus Binds to the Seventh Short Consensus Repeat of Human Complement Factor H

    Journal: Infection and Immunity

    doi:

    Chemical cross-linking of fH, H15, and H5 to M6 protein. Biotinylated M6 protein and fH, H15, or H5 were incubated in 50 mM bicarbonate buffer (pH 7.4) with or without DSP cross-linker, as described in Materials and Methods. Proteins were separated by SDS–6% PAGE under nonreducing conditions, transferred to nitrocellulose, and then incubated in streptavidin-HRP. M6 protein and cross-linked M6 protein were detected by chemiluminescence. Cross-linking of M6 protein to fH and H15 is indicated by the arrows.
    Figure Legend Snippet: Chemical cross-linking of fH, H15, and H5 to M6 protein. Biotinylated M6 protein and fH, H15, or H5 were incubated in 50 mM bicarbonate buffer (pH 7.4) with or without DSP cross-linker, as described in Materials and Methods. Proteins were separated by SDS–6% PAGE under nonreducing conditions, transferred to nitrocellulose, and then incubated in streptavidin-HRP. M6 protein and cross-linked M6 protein were detected by chemiluminescence. Cross-linking of M6 protein to fH and H15 is indicated by the arrows.

    Techniques Used: Incubation, Polyacrylamide Gel Electrophoresis

    19) Product Images from "The mechanistic basis of protection by non-neutralizing anti-alphavirus antibodies"

    Article Title: The mechanistic basis of protection by non-neutralizing anti-alphavirus antibodies

    Journal: Cell reports

    doi: 10.1016/j.celrep.2021.108962

    Binding of non-neutralizing anti-MAYV mAbs to virions and recombinant E2 protein (A and B) Anti-MAYV mAbs were tested for neutralization of MAYV on Vero (A) and C2C12 myoblast (B) cells. Serial dilutions of the indicated mAbs were incubated with 10 2 FFU of MAYV-BeH407 and then added to the indicated cells. Viral foci are plotted relative to a no mAb control. The neutralizing mAb MAY-117 was used as a positive control, and an irrelevant mIgG2c mAb was used as a negative isotype control (mean and SD of two experiments performed in triplicate). (C and D) Binding to MAYV virions (C) or recombinant MAYV E2 protein (D) by ELISA. Virions were captured with a humanized mAb to MAYV, and recombinant MAYV E2 protein was bound directly to microtiter plates. Bound murine mAbs were detected with an horseradish peroxidase (HRP)-conjugated secondary antibody. Data are expressed as OD values relative to the 10-μg/ml sample (mean and SD of two experiments performed in triplicate). (E) MAYV mAbs were competed for binding to MAYV (strain BeH407) by ELISA. Virus was captured on plates using a humanized anti-MAYV mAb. Captured virion was incubated with 10 μg/ml of the indicated mAb (first antibody). Antibody-virus complexes were incubated with 10 ng/ml of the indicated mAb labeled with biotin (second antibody). Binding was detected using streptavidin HRP and is indicated by color from high (red) to low (blue). Data are presented relative to a control with no first antibody and are representative of two experiments.
    Figure Legend Snippet: Binding of non-neutralizing anti-MAYV mAbs to virions and recombinant E2 protein (A and B) Anti-MAYV mAbs were tested for neutralization of MAYV on Vero (A) and C2C12 myoblast (B) cells. Serial dilutions of the indicated mAbs were incubated with 10 2 FFU of MAYV-BeH407 and then added to the indicated cells. Viral foci are plotted relative to a no mAb control. The neutralizing mAb MAY-117 was used as a positive control, and an irrelevant mIgG2c mAb was used as a negative isotype control (mean and SD of two experiments performed in triplicate). (C and D) Binding to MAYV virions (C) or recombinant MAYV E2 protein (D) by ELISA. Virions were captured with a humanized mAb to MAYV, and recombinant MAYV E2 protein was bound directly to microtiter plates. Bound murine mAbs were detected with an horseradish peroxidase (HRP)-conjugated secondary antibody. Data are expressed as OD values relative to the 10-μg/ml sample (mean and SD of two experiments performed in triplicate). (E) MAYV mAbs were competed for binding to MAYV (strain BeH407) by ELISA. Virus was captured on plates using a humanized anti-MAYV mAb. Captured virion was incubated with 10 μg/ml of the indicated mAb (first antibody). Antibody-virus complexes were incubated with 10 ng/ml of the indicated mAb labeled with biotin (second antibody). Binding was detected using streptavidin HRP and is indicated by color from high (red) to low (blue). Data are presented relative to a control with no first antibody and are representative of two experiments.

    Techniques Used: Binding Assay, Recombinant, Neutralization, Incubation, Positive Control, Enzyme-linked Immunosorbent Assay, Labeling

    20) Product Images from "Identification of Group B Streptococcus Capsule Type by Use of a Dual Phenotypic/Genotypic Assay"

    Article Title: Identification of Group B Streptococcus Capsule Type by Use of a Dual Phenotypic/Genotypic Assay

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.00300-17

    Recognition of GBS CPSs by the lectin EIA. Crude GBS CPS preparations (Ia, Ib, and II to IX) were incubated with L. flavus lectin specific for Neu5Ac. Three concentrations of biotin- L. flavus lectin (LFA) were used (10, 1, and 0.1 μg/ml), while 1 μg of HRP-labeled streptavidin/ml was used. Data are expressed as mean OD 450 values with the standard deviations (SD) for at least three independent experiments.
    Figure Legend Snippet: Recognition of GBS CPSs by the lectin EIA. Crude GBS CPS preparations (Ia, Ib, and II to IX) were incubated with L. flavus lectin specific for Neu5Ac. Three concentrations of biotin- L. flavus lectin (LFA) were used (10, 1, and 0.1 μg/ml), while 1 μg of HRP-labeled streptavidin/ml was used. Data are expressed as mean OD 450 values with the standard deviations (SD) for at least three independent experiments.

    Techniques Used: Enzyme-linked Immunosorbent Assay, IA, Incubation, Labeling

    21) Product Images from "Role of Nse1 Subunit of SMC5/6 Complex as a Ubiquitin Ligase"

    Article Title: Role of Nse1 Subunit of SMC5/6 Complex as a Ubiquitin Ligase

    Journal: Cells

    doi: 10.3390/cells11010165

    S. pombe Nse1 promotes in vitro ubiquitination together with Ubc13/Mms2. ( A ) Nse1 stimulates ubiquitin-chain formation when combined with Ubc13/Mms2. S. pombe E1 (Uba1), indicated E2s, biotinylated ubiquitin, ATP, and MgCl 2 were incubated in the presence or absence of Nse1/3/4 trimer for 1 h at 37 °C. The mixture was separated on 12% SDS–PAGE followed by Western blotting and visualization of biotinylated ubiquitin using Streptavidin-HRP. Numbers on the left indicate molecular weights of protein standards (in kDa). ( B ) Nse1/3/4 trimer promotes ubiquitination more efficiently than Nse1/3 dimer or Nse1 alone, and deletion of its vRING domain ablates this activity. In vitro ubiquitination assay was performed using E1, Ubc13/Mms2, biotinylated ubiquitin, indicated E3, and analyzed as in ( A ).
    Figure Legend Snippet: S. pombe Nse1 promotes in vitro ubiquitination together with Ubc13/Mms2. ( A ) Nse1 stimulates ubiquitin-chain formation when combined with Ubc13/Mms2. S. pombe E1 (Uba1), indicated E2s, biotinylated ubiquitin, ATP, and MgCl 2 were incubated in the presence or absence of Nse1/3/4 trimer for 1 h at 37 °C. The mixture was separated on 12% SDS–PAGE followed by Western blotting and visualization of biotinylated ubiquitin using Streptavidin-HRP. Numbers on the left indicate molecular weights of protein standards (in kDa). ( B ) Nse1/3/4 trimer promotes ubiquitination more efficiently than Nse1/3 dimer or Nse1 alone, and deletion of its vRING domain ablates this activity. In vitro ubiquitination assay was performed using E1, Ubc13/Mms2, biotinylated ubiquitin, indicated E3, and analyzed as in ( A ).

    Techniques Used: In Vitro, Incubation, SDS Page, Western Blot, Activity Assay, Ubiquitin Assay

    22) Product Images from "Novel Inhibitors and Activity-Based Probes Targeting Trypsin-Like Serine Proteases"

    Article Title: Novel Inhibitors and Activity-Based Probes Targeting Trypsin-Like Serine Proteases

    Journal: Frontiers in Chemistry

    doi: 10.3389/fchem.2022.782608

    NAP858 functions as an activity probe (AP) for TLPs. Proteases, matriptase, HAT and thrombin, were treated (±) NAP858 (±Cbz-Arg P (OPh) 2 ), for 30 min at 37°C. Samples were resolved by SDS-PAGE under reducing conditions with streptavidin-HRP used to visualise the inhibitor-protease complex.
    Figure Legend Snippet: NAP858 functions as an activity probe (AP) for TLPs. Proteases, matriptase, HAT and thrombin, were treated (±) NAP858 (±Cbz-Arg P (OPh) 2 ), for 30 min at 37°C. Samples were resolved by SDS-PAGE under reducing conditions with streptavidin-HRP used to visualise the inhibitor-protease complex.

    Techniques Used: Activity Assay, HAT Assay, SDS Page

    NAP858 and NAP897 function as activity-based probes (ABPs) for trypsin. Samples of trypsin were treated with either (A) NAP858 (+) or (B) NAP897 (+), used at a concentration of 50 μM, for 30 min, at 37°C. For each blot a vehicle control sample was included (−/−) as well as a further sample of trypsin pre-treated with Cbz-Arg P (OPh) 2 (+) prior to incubation with the APs. (C) Solutions containing ascending amounts of trypsin (0.01–0.5 μg/ml) were also incubated with NAP858 (+), under exactly the same conditions as indicated for panels (A) and (B) , above. All treated samples were reduced prior to SDS-PAGE. Western blotting analysis was then performed which allowed visualisation of the biotinylated inhibitor-protease complex using streptavidin-HRP.
    Figure Legend Snippet: NAP858 and NAP897 function as activity-based probes (ABPs) for trypsin. Samples of trypsin were treated with either (A) NAP858 (+) or (B) NAP897 (+), used at a concentration of 50 μM, for 30 min, at 37°C. For each blot a vehicle control sample was included (−/−) as well as a further sample of trypsin pre-treated with Cbz-Arg P (OPh) 2 (+) prior to incubation with the APs. (C) Solutions containing ascending amounts of trypsin (0.01–0.5 μg/ml) were also incubated with NAP858 (+), under exactly the same conditions as indicated for panels (A) and (B) , above. All treated samples were reduced prior to SDS-PAGE. Western blotting analysis was then performed which allowed visualisation of the biotinylated inhibitor-protease complex using streptavidin-HRP.

    Techniques Used: Activity Assay, Concentration Assay, Incubation, SDS Page, Western Blot

    ABP of cockroach extracts for trypsin-like proteases using NAP858 (A) Cockroach extract (480 μg) was treated (±) with NAP858 (± heat inactivation at 95°C, for 10 min), for 30 min, at 37°C or (B) cockroach extract (3 mg) was pretreated with neutroavidin agarose beads to remove endogenous biotinylated proteins and the supernatant probed (±) with NAP858 (±Cbz-Arg P (OPh) 2 ), for 30 min, at 37°C. Purification of the labelled proteins was achieved by incubation with a fresh batch of washed neutravidin agarose beads. Bound proteins were extracted from beads using SDS-containing reducing treatment buffer. In both cases (A,B) samples were subsequently labelled and visualised using streptavidin-HRP.
    Figure Legend Snippet: ABP of cockroach extracts for trypsin-like proteases using NAP858 (A) Cockroach extract (480 μg) was treated (±) with NAP858 (± heat inactivation at 95°C, for 10 min), for 30 min, at 37°C or (B) cockroach extract (3 mg) was pretreated with neutroavidin agarose beads to remove endogenous biotinylated proteins and the supernatant probed (±) with NAP858 (±Cbz-Arg P (OPh) 2 ), for 30 min, at 37°C. Purification of the labelled proteins was achieved by incubation with a fresh batch of washed neutravidin agarose beads. Bound proteins were extracted from beads using SDS-containing reducing treatment buffer. In both cases (A,B) samples were subsequently labelled and visualised using streptavidin-HRP.

    Techniques Used: Purification, Incubation

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    Vector Laboratories streptavidin horseradish peroxidase
    Prolectin acts as a cell adhesion molecule ( A ) Expression of fucosylated markers on normal CHO cells or CHO cells engineered to express fucosylated antigens tested by flow cytometry. Cells were introduced in the channels of a BioFlux microfluidic system coated with <t>prolectin/streptavidin</t> tetramers under a pressure of 0.1 Dyn/cm 2 . Unbound cells were washed away and pictures of the attached cells were taken at 10x magnification with a Leica DMI 6000B microscope. Bound cells were counted on three different segments along the channels and the mean cell densities and SEM are represented on the histogram. ( B ) DU-145 cells were introduced in the channels under a pressure of 0.1 Dyn/cm 2 and in presence or 25 mM D-galactose or L-fucose. Unbound cells were washed away and pictures of the attached cells were taken. Bound cells were counted in the whole channels. The results of five independent experiments are shown. Due to the high variation between experiments, the results are shown separately. Similar experiments were performed with the other tumor cell lines MCF-7, OVCAR-3 and HT-29. ( C ) MCF10A-LXSN and MCF10A-Kras(v12) cells were treated with dimethyl sulfoxide (DMSO, green), 5 μM kifunensin (orange), or 400 μM 2F-fucose (blue) during four days and stained with prolectin by flow cytometry as described above. Control cells and cells treated with the inhibitors were injected into prolectin-coated BioFlux channels. Unbound cells were washed away and pictures of the attached cells were taken. Bound cells were counted in the whole channels. ( D ) Prolectin expression was tested on Rat6-Neo and Rat6-CTLY11 fibroblasts by flow cytometry. Percentages of “positive” cells are indicated. Rat6-Neo and Rat6-CTLY11 or Rat6-CTLY11 in the presence of 25 mM galactose or fucose were injected in BioFlux channels covered with a layer of DU-145 cells under a pressure of 0.05 Dyn/cm 2 . Films of 1 minute (600 pictures) were acquired on a Leica DMI 6000B microscope using the Metamorph software. Chronophotographic images, corresponding to 10 pictures segments of the films, were constructed using the FiJi software and distances covered by individual cells were measured. Student tests were performed to assess statistical significance.
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    Prolectin acts as a cell adhesion molecule ( A ) Expression of fucosylated markers on normal CHO cells or CHO cells engineered to express fucosylated antigens tested by flow cytometry. Cells were introduced in the channels of a BioFlux microfluidic system coated with prolectin/streptavidin tetramers under a pressure of 0.1 Dyn/cm 2 . Unbound cells were washed away and pictures of the attached cells were taken at 10x magnification with a Leica DMI 6000B microscope. Bound cells were counted on three different segments along the channels and the mean cell densities and SEM are represented on the histogram. ( B ) DU-145 cells were introduced in the channels under a pressure of 0.1 Dyn/cm 2 and in presence or 25 mM D-galactose or L-fucose. Unbound cells were washed away and pictures of the attached cells were taken. Bound cells were counted in the whole channels. The results of five independent experiments are shown. Due to the high variation between experiments, the results are shown separately. Similar experiments were performed with the other tumor cell lines MCF-7, OVCAR-3 and HT-29. ( C ) MCF10A-LXSN and MCF10A-Kras(v12) cells were treated with dimethyl sulfoxide (DMSO, green), 5 μM kifunensin (orange), or 400 μM 2F-fucose (blue) during four days and stained with prolectin by flow cytometry as described above. Control cells and cells treated with the inhibitors were injected into prolectin-coated BioFlux channels. Unbound cells were washed away and pictures of the attached cells were taken. Bound cells were counted in the whole channels. ( D ) Prolectin expression was tested on Rat6-Neo and Rat6-CTLY11 fibroblasts by flow cytometry. Percentages of “positive” cells are indicated. Rat6-Neo and Rat6-CTLY11 or Rat6-CTLY11 in the presence of 25 mM galactose or fucose were injected in BioFlux channels covered with a layer of DU-145 cells under a pressure of 0.05 Dyn/cm 2 . Films of 1 minute (600 pictures) were acquired on a Leica DMI 6000B microscope using the Metamorph software. Chronophotographic images, corresponding to 10 pictures segments of the films, were constructed using the FiJi software and distances covered by individual cells were measured. Student tests were performed to assess statistical significance.

    Journal: Oncotarget

    Article Title: Carcinoma-associated fucosylated antigens are markers of the epithelial state and can contribute to cell adhesion through CLEC17A (Prolectin)

    doi: 10.18632/oncotarget.7476

    Figure Lengend Snippet: Prolectin acts as a cell adhesion molecule ( A ) Expression of fucosylated markers on normal CHO cells or CHO cells engineered to express fucosylated antigens tested by flow cytometry. Cells were introduced in the channels of a BioFlux microfluidic system coated with prolectin/streptavidin tetramers under a pressure of 0.1 Dyn/cm 2 . Unbound cells were washed away and pictures of the attached cells were taken at 10x magnification with a Leica DMI 6000B microscope. Bound cells were counted on three different segments along the channels and the mean cell densities and SEM are represented on the histogram. ( B ) DU-145 cells were introduced in the channels under a pressure of 0.1 Dyn/cm 2 and in presence or 25 mM D-galactose or L-fucose. Unbound cells were washed away and pictures of the attached cells were taken. Bound cells were counted in the whole channels. The results of five independent experiments are shown. Due to the high variation between experiments, the results are shown separately. Similar experiments were performed with the other tumor cell lines MCF-7, OVCAR-3 and HT-29. ( C ) MCF10A-LXSN and MCF10A-Kras(v12) cells were treated with dimethyl sulfoxide (DMSO, green), 5 μM kifunensin (orange), or 400 μM 2F-fucose (blue) during four days and stained with prolectin by flow cytometry as described above. Control cells and cells treated with the inhibitors were injected into prolectin-coated BioFlux channels. Unbound cells were washed away and pictures of the attached cells were taken. Bound cells were counted in the whole channels. ( D ) Prolectin expression was tested on Rat6-Neo and Rat6-CTLY11 fibroblasts by flow cytometry. Percentages of “positive” cells are indicated. Rat6-Neo and Rat6-CTLY11 or Rat6-CTLY11 in the presence of 25 mM galactose or fucose were injected in BioFlux channels covered with a layer of DU-145 cells under a pressure of 0.05 Dyn/cm 2 . Films of 1 minute (600 pictures) were acquired on a Leica DMI 6000B microscope using the Metamorph software. Chronophotographic images, corresponding to 10 pictures segments of the films, were constructed using the FiJi software and distances covered by individual cells were measured. Student tests were performed to assess statistical significance.

    Article Snippet: Biotinylated-prolectin monomers were incubated with streptavidin-horseradish peroxidase (strepatvidin-HRP; Vector laboratories, Burlingame, CA) or streptavidin-phycoerythrin (streptavidin-PE; BD Pharmingen) at a molar ratio of 1:5 in TBS-BSA, 10 mM CaCl2 for 1–2 h at room temperature.

    Techniques: Expressing, Flow Cytometry, Cytometry, Microscopy, Staining, Injection, Software, Construct

    The fucose binding lectin BC2L-C-Nt stains epithelial cells rather than mesenchymal cells Breast cell lines were stained with biotinylated lectins BC2L-C-Nt followed by PE-conjugated streptavidin and analyzed by flow cytometry. Representative flow cytometry histograms as well as a histogram of the mean and SEM of Mean fluorescence intensities (MFI) for three independent experiments are presented.

    Journal: Oncotarget

    Article Title: Carcinoma-associated fucosylated antigens are markers of the epithelial state and can contribute to cell adhesion through CLEC17A (Prolectin)

    doi: 10.18632/oncotarget.7476

    Figure Lengend Snippet: The fucose binding lectin BC2L-C-Nt stains epithelial cells rather than mesenchymal cells Breast cell lines were stained with biotinylated lectins BC2L-C-Nt followed by PE-conjugated streptavidin and analyzed by flow cytometry. Representative flow cytometry histograms as well as a histogram of the mean and SEM of Mean fluorescence intensities (MFI) for three independent experiments are presented.

    Article Snippet: Biotinylated-prolectin monomers were incubated with streptavidin-horseradish peroxidase (strepatvidin-HRP; Vector laboratories, Burlingame, CA) or streptavidin-phycoerythrin (streptavidin-PE; BD Pharmingen) at a molar ratio of 1:5 in TBS-BSA, 10 mM CaCl2 for 1–2 h at room temperature.

    Techniques: Binding Assay, Staining, Flow Cytometry, Cytometry, Fluorescence

    Prolectin is a human C-type lectin that recognizes similar fucosylated ligands as BC2L-C-Nt and efficiently binds to tumor cell lines and tumor tissues ( A ) Diagram of Prolectin structure (CRD: carbohydrate recognition domain; TM: transmembrane domain; IC: intra-cytoplasmic domain). ( B ) Glycan binding assay of prolectin and BC2L-C-Nt. Plates coated with a series of fucosylated HBGAs conjugated to HSA or PAA were probed with either biotinylated-BC2L-C-Nt followed by Avidin-HRP or tetramers of biotin-prolectin CRD bound to streptavidin-HRP. The results shown are the mean and SEM of three independent experiments. ( C ) Flow cytometry staining of tumor cell lines from various origins. Tetramers of biotinylated-prolectin CRD in complex with streptavidin-PE, pre-incubated in the absence of sugar or with L-fucose, were bound to the indicated cell lines, which were then analyzed by flow cytometry. The bar histogram shows the mean fluorescence intensities. Results are representative of at least two experiments. ( D ) Prolectin preferentially stains epithelial cell lines. The epithelial/mesenchymal cell lines pairs MCF10A-LXSN/MCF10A-Kras(v12) and MCF10A-Puro R /MCF10A-SNAIL were stained with prolectin as described above. Means and SEM of three independent experiments are shown ( E ) Prolectin binding to tumor tissues. Tetramers of biotinylated-prolectin CRD conjugated with streptavidin-HRP were incubated on fixed tissue sections from different tumor (T) or adjacent normal (NT) tissues. To control for the involvement of specific glycan binding in the prolectin staining, colon tumor sections were stained as above after prolectin tetramers were incubated with Ca 2+ , EDTA or Ca 2+ + L-fucose. Colon tissues had been fixed in ethanol whereas breast, lung and uterine tissues were from formalin-fixed TMAs. Magnifications of 20× are shown (the black bars represent 100 μm).

    Journal: Oncotarget

    Article Title: Carcinoma-associated fucosylated antigens are markers of the epithelial state and can contribute to cell adhesion through CLEC17A (Prolectin)

    doi: 10.18632/oncotarget.7476

    Figure Lengend Snippet: Prolectin is a human C-type lectin that recognizes similar fucosylated ligands as BC2L-C-Nt and efficiently binds to tumor cell lines and tumor tissues ( A ) Diagram of Prolectin structure (CRD: carbohydrate recognition domain; TM: transmembrane domain; IC: intra-cytoplasmic domain). ( B ) Glycan binding assay of prolectin and BC2L-C-Nt. Plates coated with a series of fucosylated HBGAs conjugated to HSA or PAA were probed with either biotinylated-BC2L-C-Nt followed by Avidin-HRP or tetramers of biotin-prolectin CRD bound to streptavidin-HRP. The results shown are the mean and SEM of three independent experiments. ( C ) Flow cytometry staining of tumor cell lines from various origins. Tetramers of biotinylated-prolectin CRD in complex with streptavidin-PE, pre-incubated in the absence of sugar or with L-fucose, were bound to the indicated cell lines, which were then analyzed by flow cytometry. The bar histogram shows the mean fluorescence intensities. Results are representative of at least two experiments. ( D ) Prolectin preferentially stains epithelial cell lines. The epithelial/mesenchymal cell lines pairs MCF10A-LXSN/MCF10A-Kras(v12) and MCF10A-Puro R /MCF10A-SNAIL were stained with prolectin as described above. Means and SEM of three independent experiments are shown ( E ) Prolectin binding to tumor tissues. Tetramers of biotinylated-prolectin CRD conjugated with streptavidin-HRP were incubated on fixed tissue sections from different tumor (T) or adjacent normal (NT) tissues. To control for the involvement of specific glycan binding in the prolectin staining, colon tumor sections were stained as above after prolectin tetramers were incubated with Ca 2+ , EDTA or Ca 2+ + L-fucose. Colon tissues had been fixed in ethanol whereas breast, lung and uterine tissues were from formalin-fixed TMAs. Magnifications of 20× are shown (the black bars represent 100 μm).

    Article Snippet: Biotinylated-prolectin monomers were incubated with streptavidin-horseradish peroxidase (strepatvidin-HRP; Vector laboratories, Burlingame, CA) or streptavidin-phycoerythrin (streptavidin-PE; BD Pharmingen) at a molar ratio of 1:5 in TBS-BSA, 10 mM CaCl2 for 1–2 h at room temperature.

    Techniques: Binding Assay, Avidin-Biotin Assay, Flow Cytometry, Cytometry, Staining, Incubation, Fluorescence

    ER-targeted BioID2 to label secreted proteins. (A) Schematic of lentivirus expressing ER-targeted BioID2 construct. (B) Photomicrographs of A549 and A549-BioID2 cells showing co-localization of the BioID2 (HA-tagged; green) and the ER marker calnexin (red). Nuclei are stained with DAPI (blue). Scale bar is 25 microns. (C) Proof of concept demonstrating that secreted proteins are biotinylated by ER-targeted BioID2. A549 or A549 cells stable expression ER-BioID2 were transfected with a plasmid encoding a V5-SFTPA2 construct. Eighteen hours after transfection, biotin was added to some samples and the media and cell lysates were examined the next day by western blotting for SFTPA2 (V5), BioID2 (HA), or biotinylated proteins (Strep-HRP). GAPDH was a load control for cell lysates. Biotinylated proteins were detected in the media only in cells that expressed ER-BioID2 and were cultured in excess biotin. Biotinylated SFTPA2 was detected in media only when ER-BioID2 was present. (D) Experimental procedure for unbiased proteomic analysis of secreted proteins. Cells were cultured for 5 days in the presence of Dox to induce senescence followed by incubation with excess biotin for 8 h. After biotin labeling, cells were washed and fresh media was added. Twenty-four hours later, media was collected and biotinylated proteins were isolated by incubation with streptavidin-coated beads and analyzed by stain-free SDS-page (E) .

    Journal: Frontiers in Medicine

    Article Title: Transcriptional and Proteomic Characterization of Telomere-Induced Senescence in a Human Alveolar Epithelial Cell Line

    doi: 10.3389/fmed.2021.600626

    Figure Lengend Snippet: ER-targeted BioID2 to label secreted proteins. (A) Schematic of lentivirus expressing ER-targeted BioID2 construct. (B) Photomicrographs of A549 and A549-BioID2 cells showing co-localization of the BioID2 (HA-tagged; green) and the ER marker calnexin (red). Nuclei are stained with DAPI (blue). Scale bar is 25 microns. (C) Proof of concept demonstrating that secreted proteins are biotinylated by ER-targeted BioID2. A549 or A549 cells stable expression ER-BioID2 were transfected with a plasmid encoding a V5-SFTPA2 construct. Eighteen hours after transfection, biotin was added to some samples and the media and cell lysates were examined the next day by western blotting for SFTPA2 (V5), BioID2 (HA), or biotinylated proteins (Strep-HRP). GAPDH was a load control for cell lysates. Biotinylated proteins were detected in the media only in cells that expressed ER-BioID2 and were cultured in excess biotin. Biotinylated SFTPA2 was detected in media only when ER-BioID2 was present. (D) Experimental procedure for unbiased proteomic analysis of secreted proteins. Cells were cultured for 5 days in the presence of Dox to induce senescence followed by incubation with excess biotin for 8 h. After biotin labeling, cells were washed and fresh media was added. Twenty-four hours later, media was collected and biotinylated proteins were isolated by incubation with streptavidin-coated beads and analyzed by stain-free SDS-page (E) .

    Article Snippet: Detection of biotinylated proteins was accomplished by incubating membranes with streptavidin conjugated to horseradish peroxidase (Strep-HRP) and developing the membranes according to the manufacture's protocol (Vector Laboratories).

    Techniques: Expressing, Construct, Marker, Staining, Transfection, Plasmid Preparation, Western Blot, Cell Culture, Incubation, Labeling, Isolation, SDS Page

    Dd NHE1 groups with plasma membrane NHEs and localizes to the leading edge of chemotaxing cells. (A) Dictyostelium NHE1 ( Dd NHE1; AA052201 ), the nine known mammalian NHEs ( Hs NHE1–9; P19634 , Q9UBY0 , P48764 , XM_371544.2, Q14940 , Q92581 , Q96T83 , Q9Y2E8 , and Q8IVB4 , respectively), and three Drosophila NHEs ( Dm NHE1–3; Q8SZX8 , Q9VIF9, and Q81PJ4 , respectively) were aligned using ClustalW; the resulting tree diagram is shown. An asterisk (plasma membrane) or plus (intracellular membranes) denotes NHEs confirmed to have the indicated localization. (B) Localization of Dd NHE1-HA in chemotaxing cells, as determined by immunolabeling with anti-HA antibodies, was visualized by confocal microscopy. The three-dimensional projection of two representative cells is shown. White asterisks indicate direction of the point source of cAMP delivered by a micropipette. (C) Surface biotinylation followed by immunoprecipitating the indicated proteins and immunoblotting with HRP:Streptavidin-confirmed Dd NHE1-HA is at the plasma membrane. Immunoprecipitation of the plasma membrane cAMP receptor 1 (cAR1) in cells expressing Dd NHE1-HA and the GFP-tagged PH domain of CRAC (GFP-PH CRAC ) expressed in Ax2 cells were included as positive and negative controls, respectively. Immunoblot of total PH CRAC :GFP is shown separately (right).

    Journal: The Journal of Cell Biology

    Article Title: A developmentally regulated Na-H exchanger in Dictyostelium discoideum is necessary for cell polarity during chemotaxis

    doi: 10.1083/jcb.200412145

    Figure Lengend Snippet: Dd NHE1 groups with plasma membrane NHEs and localizes to the leading edge of chemotaxing cells. (A) Dictyostelium NHE1 ( Dd NHE1; AA052201 ), the nine known mammalian NHEs ( Hs NHE1–9; P19634 , Q9UBY0 , P48764 , XM_371544.2, Q14940 , Q92581 , Q96T83 , Q9Y2E8 , and Q8IVB4 , respectively), and three Drosophila NHEs ( Dm NHE1–3; Q8SZX8 , Q9VIF9, and Q81PJ4 , respectively) were aligned using ClustalW; the resulting tree diagram is shown. An asterisk (plasma membrane) or plus (intracellular membranes) denotes NHEs confirmed to have the indicated localization. (B) Localization of Dd NHE1-HA in chemotaxing cells, as determined by immunolabeling with anti-HA antibodies, was visualized by confocal microscopy. The three-dimensional projection of two representative cells is shown. White asterisks indicate direction of the point source of cAMP delivered by a micropipette. (C) Surface biotinylation followed by immunoprecipitating the indicated proteins and immunoblotting with HRP:Streptavidin-confirmed Dd NHE1-HA is at the plasma membrane. Immunoprecipitation of the plasma membrane cAMP receptor 1 (cAR1) in cells expressing Dd NHE1-HA and the GFP-tagged PH domain of CRAC (GFP-PH CRAC ) expressed in Ax2 cells were included as positive and negative controls, respectively. Immunoblot of total PH CRAC :GFP is shown separately (right).

    Article Snippet: Membranes were blocked with 5% milk and probed with HRP/Streptavidin (1:1000; Vector Laboratories).

    Techniques: Immunolabeling, Confocal Microscopy, Immunoprecipitation, Expressing

    Cross-competition between HMAbs . A cross-competition assay was performed to determine whether the three HMAbs and MMAb 4G2 recognized overlapping or non-overlapping sties on DENV-1 E protein. Purified MMAb 4G2 and HMAbs were incubated with biotinylated HMAbs, washed, and the presence of biotinylated antibodies was detected using streptavidin.

    Journal: Virology Journal

    Article Title: Neutralizing and non-neutralizing monoclonal antibodies against dengue virus E protein derived from a naturally infected patient

    doi: 10.1186/1743-422X-7-28

    Figure Lengend Snippet: Cross-competition between HMAbs . A cross-competition assay was performed to determine whether the three HMAbs and MMAb 4G2 recognized overlapping or non-overlapping sties on DENV-1 E protein. Purified MMAb 4G2 and HMAbs were incubated with biotinylated HMAbs, washed, and the presence of biotinylated antibodies was detected using streptavidin.

    Article Snippet: Bound biotinylated HMAb was detected with horseradish peroxidase streptavidin (Vector, Burlingame, CA ).

    Techniques: Competitive Binding Assay, Purification, Incubation