fluorescein-conjugated streptavidin Search Results


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  • 99
    Vector Laboratories fluorescein conjugated streptavidin
    Retinal uptake of biotin-labeled P-IQACRG. Ocular tissues were harvested 8 hours after intravitreal injection of 150 pmol biotin-P-IQACRG, P-IQACRG (without biotin label, which served as a negative control), or biotin alone. ( A ) Avidin-affinity precipitates of the vitreous and retina were separated on SDS-PAGE, and blots were incubated with peroxidase-conjugated avidin. After biotin-P-IQACRG injection, blots displayed a band corresponding to the biotinylated molecule ( n = 5 experiments; bar on right indicates 3.5 kDa). ( B ) Localization of P-IQACRG in the retina. In animals receiving biotin-P-IQACRG, <t>fluorescein-streptavidin</t> histochemistry revealed the presence of biotinylated peptide in the inner retina, with the strongest signal in the ganglion cell layer ( left ). In eyes injected with P-IQACRG alone, we observed little if any signal ( center ). Eyes injected with biotin alone displayed restricted distribution of avidin fluorescence around the inner limiting membrane ( right ). GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; ONL, outer nuclear layer. ( C ) When avidin histology ( green ) was combined with MAP2 immunostaining for neurons ( red ), deconvolution microscopy revealed MAP2-positive neurons in the GCL that contained the biotinylated peptide in the cytosol ( yellow , marked by arrowheads ). Cell nuclei were counterstained with Hoechst 33342 ( blue ). Scale bars, 50 μm; n ≥ 4 experiments for each.
    Fluorescein Conjugated Streptavidin, supplied by Vector Laboratories, used in various techniques. Bioz Stars score: 99/100, based on 173 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Thermo Fisher streptavidin fluorescein conjugate
    Compilation of the photobleaching data (errors bars are SD ) corresponding to all the studied molecules (fluorescein, FITC, <t>streptavidin-fluorescein,</t> FD4, FD70, FD250S, and eGFP), inserted into vesicles between 10 and 100 μ m in diameter, under a laser power from 2.6 μ W to 103 μ W. eGFP data ( squares ) falls in the fast diffusion regime (5
    Streptavidin Fluorescein Conjugate, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    PerkinElmer streptavidin fluorescein isothiocyanate conjugated
    Compilation of the photobleaching data (errors bars are SD ) corresponding to all the studied molecules (fluorescein, FITC, <t>streptavidin-fluorescein,</t> FD4, FD70, FD250S, and eGFP), inserted into vesicles between 10 and 100 μ m in diameter, under a laser power from 2.6 μ W to 103 μ W. eGFP data ( squares ) falls in the fast diffusion regime (5
    Streptavidin Fluorescein Isothiocyanate Conjugated, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Thermo Fisher streptavidin conjugated to fluorescein
    Dynamics of protein S-alkylation by aHNE in RKO cells. (A) Heatmap of ratios of changes of all detected cysteine S-alkylation events shows that most adducts turn over rapidly in a time-dependent manner in cells. Lower the measured ratio (L/H) indicates more rapid turnover. (B) XIC are shown for changes in S-alkylated peptides from FAM120A protein in RKO cells, with the profiles for light- and heavy- labeled peptides in red and blue, respectively. The mean measured ratios were calculated from three biological replicate experiments and are displayed below the individual chromatograms, respectively. (C) Turnover of alkylation is not affected by proteasome inhibition. RKO cells were pretreated with (red) or without MG132 (black), followed by aHNE treatment with or without 1 and 4 h recovery periods. Proteins alkylated by aHNE were labeled with azido-biotin and detected by Western blotting with fluorescein-conjugated <t>streptavidin.</t> Data were presented as mean values ± SD, n = 3 biological replicates per group. A representative Western blot is shown in Figure S10 in the Supporting Information . (D) Distributions of the measured ratios of dynamic aHNE-cysteine adduction in vitro (red) and in situ (white).
    Streptavidin Conjugated To Fluorescein, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    GE Healthcare fluorescein conjugated streptavidin
    Recycling of US28. HeLa CD4-US28 cells were incubated with Q4120 antibody at 37°C for 1 h to allow antibody uptake, cooled on ice, and washed in acid medium to remove the Q4120 bound to the cell surface. (A) The cells were fixed, left intact or permeabilized with saponin (right and left, respectively), and then stained with a biotin-conjugated secondary antibody to detect Q4120 followed by <t>fluorescein-streptavidin.</t> (B) The cells were reincubated with a biotin-conjugated secondary antibody for 1 h at 37°C, cooled on ice, acid washed, fixed, and stained with fluorescein-streptavidin as in A. Scale bars, 10 μm. The righthand fields for each panel show Nomarski and fluorescence images of the same field.
    Fluorescein Conjugated Streptavidin, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 90/100, based on 118 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Santa Cruz Biotechnology fluorescein conjugated streptavidin
    l ‐tetrahydropalmatine (l ‐THP) inhibits RANKL‐induced activation of NF‐κB and MAPK pathway. A, Induced RAW264.7 cells treated with l ‐THP (19.00 μg/mL, 1h) were fixed with 40% formaldehyde, washed by Triton X‐100, followed by incubation with anti‐P65 antibody, goat antimouse IgG antibody and fluorescein‐conjugated <t>streptavidin.</t> B, Induced RAW264.7 cells treated with l ‐THP (19.00 μg/mL) for 0, 15, 30 and 60 min. The levels of phosphorylated P65, P50 and IκB were evaluated by Western blotting analysis. C, Induced RAW264.7 cells treated with l ‐THP (19.00 μg/mL) for 30 min, were then collected and resuspended with a 32P‐labelled DNA duplex and run in sodium dodecyl SDS‐PAGE system for EMSA analysis of the DNA binding activity of NF‐κB. D, Induced RAW 264.7 cells were treated with l ‐THP (19.00 μg/mL) for 0, 15, 30 and 60 min, followed by Western blot analysis of the levels of phosphorylated ERK, JNK and P38. (*P
    Fluorescein Conjugated Streptavidin, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 91/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher buffer
    l ‐tetrahydropalmatine (l ‐THP) inhibits RANKL‐induced activation of NF‐κB and MAPK pathway. A, Induced RAW264.7 cells treated with l ‐THP (19.00 μg/mL, 1h) were fixed with 40% formaldehyde, washed by Triton X‐100, followed by incubation with anti‐P65 antibody, goat antimouse IgG antibody and fluorescein‐conjugated <t>streptavidin.</t> B, Induced RAW264.7 cells treated with l ‐THP (19.00 μg/mL) for 0, 15, 30 and 60 min. The levels of phosphorylated P65, P50 and IκB were evaluated by Western blotting analysis. C, Induced RAW264.7 cells treated with l ‐THP (19.00 μg/mL) for 30 min, were then collected and resuspended with a 32P‐labelled DNA duplex and run in sodium dodecyl SDS‐PAGE system for EMSA analysis of the DNA binding activity of NF‐κB. D, Induced RAW 264.7 cells were treated with l ‐THP (19.00 μg/mL) for 0, 15, 30 and 60 min, followed by Western blot analysis of the levels of phosphorylated ERK, JNK and P38. (*P
    Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 29794 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Agilent technologies fluorescein conjugated streptavidin
    l ‐tetrahydropalmatine (l ‐THP) inhibits RANKL‐induced activation of NF‐κB and MAPK pathway. A, Induced RAW264.7 cells treated with l ‐THP (19.00 μg/mL, 1h) were fixed with 40% formaldehyde, washed by Triton X‐100, followed by incubation with anti‐P65 antibody, goat antimouse IgG antibody and fluorescein‐conjugated <t>streptavidin.</t> B, Induced RAW264.7 cells treated with l ‐THP (19.00 μg/mL) for 0, 15, 30 and 60 min. The levels of phosphorylated P65, P50 and IκB were evaluated by Western blotting analysis. C, Induced RAW264.7 cells treated with l ‐THP (19.00 μg/mL) for 30 min, were then collected and resuspended with a 32P‐labelled DNA duplex and run in sodium dodecyl SDS‐PAGE system for EMSA analysis of the DNA binding activity of NF‐κB. D, Induced RAW 264.7 cells were treated with l ‐THP (19.00 μg/mL) for 0, 15, 30 and 60 min, followed by Western blot analysis of the levels of phosphorylated ERK, JNK and P38. (*P
    Fluorescein Conjugated Streptavidin, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 90/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Immunotec fluorescein conjugated streptavidin
    l ‐tetrahydropalmatine (l ‐THP) inhibits RANKL‐induced activation of NF‐κB and MAPK pathway. A, Induced RAW264.7 cells treated with l ‐THP (19.00 μg/mL, 1h) were fixed with 40% formaldehyde, washed by Triton X‐100, followed by incubation with anti‐P65 antibody, goat antimouse IgG antibody and fluorescein‐conjugated <t>streptavidin.</t> B, Induced RAW264.7 cells treated with l ‐THP (19.00 μg/mL) for 0, 15, 30 and 60 min. The levels of phosphorylated P65, P50 and IκB were evaluated by Western blotting analysis. C, Induced RAW264.7 cells treated with l ‐THP (19.00 μg/mL) for 30 min, were then collected and resuspended with a 32P‐labelled DNA duplex and run in sodium dodecyl SDS‐PAGE system for EMSA analysis of the DNA binding activity of NF‐κB. D, Induced RAW 264.7 cells were treated with l ‐THP (19.00 μg/mL) for 0, 15, 30 and 60 min, followed by Western blot analysis of the levels of phosphorylated ERK, JNK and P38. (*P
    Fluorescein Conjugated Streptavidin, supplied by Immunotec, used in various techniques. Bioz Stars score: 90/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Oncogene Science Inc fluorescein conjugated streptavidin
    l ‐tetrahydropalmatine (l ‐THP) inhibits RANKL‐induced activation of NF‐κB and MAPK pathway. A, Induced RAW264.7 cells treated with l ‐THP (19.00 μg/mL, 1h) were fixed with 40% formaldehyde, washed by Triton X‐100, followed by incubation with anti‐P65 antibody, goat antimouse IgG antibody and fluorescein‐conjugated <t>streptavidin.</t> B, Induced RAW264.7 cells treated with l ‐THP (19.00 μg/mL) for 0, 15, 30 and 60 min. The levels of phosphorylated P65, P50 and IκB were evaluated by Western blotting analysis. C, Induced RAW264.7 cells treated with l ‐THP (19.00 μg/mL) for 30 min, were then collected and resuspended with a 32P‐labelled DNA duplex and run in sodium dodecyl SDS‐PAGE system for EMSA analysis of the DNA binding activity of NF‐κB. D, Induced RAW 264.7 cells were treated with l ‐THP (19.00 μg/mL) for 0, 15, 30 and 60 min, followed by Western blot analysis of the levels of phosphorylated ERK, JNK and P38. (*P
    Fluorescein Conjugated Streptavidin, supplied by Oncogene Science Inc, used in various techniques. Bioz Stars score: 90/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    GE Healthcare streptavidin fluorescein conjugate
    Immunoanalysis of the ovarian cancer cell line, OVCAR-3. (A) Three subpopulations of OVCAR cells were identified by the cell sorter. They were CD133 + (65.2%), CD133 + /CD117 + (42.4%) and CD133 − (8.9%). (B) PAR-1 immunocytochemistry. OVCAR cells were grown on glass bottom chamber slides, fixed and successively incubated with PAR-1 antibody, appropriate biotinylated secondary antibody and <t>FITC-streptavidin.</t> Isotype antidodies were used in parallel and the nuclei were DAPI-labeled. Initial magnification, ×400. Scale bar represents 10 μ m.
    Streptavidin Fluorescein Conjugate, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 88/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Vector Laboratories fluorescein streptavidin conjugate
    Immunoanalysis of the ovarian cancer cell line, OVCAR-3. (A) Three subpopulations of OVCAR cells were identified by the cell sorter. They were CD133 + (65.2%), CD133 + /CD117 + (42.4%) and CD133 − (8.9%). (B) PAR-1 immunocytochemistry. OVCAR cells were grown on glass bottom chamber slides, fixed and successively incubated with PAR-1 antibody, appropriate biotinylated secondary antibody and <t>FITC-streptavidin.</t> Isotype antidodies were used in parallel and the nuclei were DAPI-labeled. Initial magnification, ×400. Scale bar represents 10 μ m.
    Fluorescein Streptavidin Conjugate, supplied by Vector Laboratories, used in various techniques. Bioz Stars score: 94/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher fluorescein conjugated alexa streptavidin
    Immunoanalysis of the ovarian cancer cell line, OVCAR-3. (A) Three subpopulations of OVCAR cells were identified by the cell sorter. They were CD133 + (65.2%), CD133 + /CD117 + (42.4%) and CD133 − (8.9%). (B) PAR-1 immunocytochemistry. OVCAR cells were grown on glass bottom chamber slides, fixed and successively incubated with PAR-1 antibody, appropriate biotinylated secondary antibody and <t>FITC-streptavidin.</t> Isotype antidodies were used in parallel and the nuclei were DAPI-labeled. Initial magnification, ×400. Scale bar represents 10 μ m.
    Fluorescein Conjugated Alexa Streptavidin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare fitc conjugated streptavidin streptavidin fluorescein
    Immunoanalysis of the ovarian cancer cell line, OVCAR-3. (A) Three subpopulations of OVCAR cells were identified by the cell sorter. They were CD133 + (65.2%), CD133 + /CD117 + (42.4%) and CD133 − (8.9%). (B) PAR-1 immunocytochemistry. OVCAR cells were grown on glass bottom chamber slides, fixed and successively incubated with PAR-1 antibody, appropriate biotinylated secondary antibody and <t>FITC-streptavidin.</t> Isotype antidodies were used in parallel and the nuclei were DAPI-labeled. Initial magnification, ×400. Scale bar represents 10 μ m.
    Fitc Conjugated Streptavidin Streptavidin Fluorescein, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Agilent technologies fluorescein isothiocyanate conjugated streptavidin
    Immunoanalysis of the ovarian cancer cell line, OVCAR-3. (A) Three subpopulations of OVCAR cells were identified by the cell sorter. They were CD133 + (65.2%), CD133 + /CD117 + (42.4%) and CD133 − (8.9%). (B) PAR-1 immunocytochemistry. OVCAR cells were grown on glass bottom chamber slides, fixed and successively incubated with PAR-1 antibody, appropriate biotinylated secondary antibody and <t>FITC-streptavidin.</t> Isotype antidodies were used in parallel and the nuclei were DAPI-labeled. Initial magnification, ×400. Scale bar represents 10 μ m.
    Fluorescein Isothiocyanate Conjugated Streptavidin, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 93/100, based on 48 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    GE Healthcare fluorescein isothiocyanate conjugated streptavidin
    Immunoanalysis of the ovarian cancer cell line, OVCAR-3. (A) Three subpopulations of OVCAR cells were identified by the cell sorter. They were CD133 + (65.2%), CD133 + /CD117 + (42.4%) and CD133 − (8.9%). (B) PAR-1 immunocytochemistry. OVCAR cells were grown on glass bottom chamber slides, fixed and successively incubated with PAR-1 antibody, appropriate biotinylated secondary antibody and <t>FITC-streptavidin.</t> Isotype antidodies were used in parallel and the nuclei were DAPI-labeled. Initial magnification, ×400. Scale bar represents 10 μ m.
    Fluorescein Isothiocyanate Conjugated Streptavidin, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 89/100, based on 42 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Retinal uptake of biotin-labeled P-IQACRG. Ocular tissues were harvested 8 hours after intravitreal injection of 150 pmol biotin-P-IQACRG, P-IQACRG (without biotin label, which served as a negative control), or biotin alone. ( A ) Avidin-affinity precipitates of the vitreous and retina were separated on SDS-PAGE, and blots were incubated with peroxidase-conjugated avidin. After biotin-P-IQACRG injection, blots displayed a band corresponding to the biotinylated molecule ( n = 5 experiments; bar on right indicates 3.5 kDa). ( B ) Localization of P-IQACRG in the retina. In animals receiving biotin-P-IQACRG, fluorescein-streptavidin histochemistry revealed the presence of biotinylated peptide in the inner retina, with the strongest signal in the ganglion cell layer ( left ). In eyes injected with P-IQACRG alone, we observed little if any signal ( center ). Eyes injected with biotin alone displayed restricted distribution of avidin fluorescence around the inner limiting membrane ( right ). GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; ONL, outer nuclear layer. ( C ) When avidin histology ( green ) was combined with MAP2 immunostaining for neurons ( red ), deconvolution microscopy revealed MAP2-positive neurons in the GCL that contained the biotinylated peptide in the cytosol ( yellow , marked by arrowheads ). Cell nuclei were counterstained with Hoechst 33342 ( blue ). Scale bars, 50 μm; n ≥ 4 experiments for each.

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: Protection of Retinal Ganglion Cells by Caspase Substrate-Binding Peptide IQACRG from N-Methyl-d-Aspartate Receptor-Mediated Excitotoxicity

    doi: 10.1167/iovs.09-4102

    Figure Lengend Snippet: Retinal uptake of biotin-labeled P-IQACRG. Ocular tissues were harvested 8 hours after intravitreal injection of 150 pmol biotin-P-IQACRG, P-IQACRG (without biotin label, which served as a negative control), or biotin alone. ( A ) Avidin-affinity precipitates of the vitreous and retina were separated on SDS-PAGE, and blots were incubated with peroxidase-conjugated avidin. After biotin-P-IQACRG injection, blots displayed a band corresponding to the biotinylated molecule ( n = 5 experiments; bar on right indicates 3.5 kDa). ( B ) Localization of P-IQACRG in the retina. In animals receiving biotin-P-IQACRG, fluorescein-streptavidin histochemistry revealed the presence of biotinylated peptide in the inner retina, with the strongest signal in the ganglion cell layer ( left ). In eyes injected with P-IQACRG alone, we observed little if any signal ( center ). Eyes injected with biotin alone displayed restricted distribution of avidin fluorescence around the inner limiting membrane ( right ). GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; ONL, outer nuclear layer. ( C ) When avidin histology ( green ) was combined with MAP2 immunostaining for neurons ( red ), deconvolution microscopy revealed MAP2-positive neurons in the GCL that contained the biotinylated peptide in the cytosol ( yellow , marked by arrowheads ). Cell nuclei were counterstained with Hoechst 33342 ( blue ). Scale bars, 50 μm; n ≥ 4 experiments for each.

    Article Snippet: To visualize biotin-P-IQACRG, sections were reacted with fluorescein-conjugated streptavidin (Vector Laboratories, Burlingame, CA; 1:300 dilution).

    Techniques: Labeling, Injection, Negative Control, Avidin-Biotin Assay, SDS Page, Incubation, Fluorescence, Immunostaining, Microscopy

    Activation of murine T-lymphocytes leads to up-regulation in the binding of siglec-E to cell surface proteins in a sialic acid-dependent manner. A , T-lymphocytes were activated with anti-CD3 and anti-CD28 for 24 h and then cultured for up to 4 days in the presence of IL-2. Cells harvested at each time point were incubated with fluorophore-conjugated CD4 ( left panel ) and CD8 antibodies ( right panel ) together with a pre-complex of FITC-conjugated goat anti-human Fc and siglec-Fc. Mean Fluorescence Intensity ( MFI ) of sialic acid-dependent binding by FACS is shown following subtraction of MFI values for sialidase-treated cells. B , for the detection of MAL and SNA binding, harvested cells corresponding to each time point were incubated with biotinylated lectin followed by a secondary incubation with FITC-conjugated streptavidin. C , splenocytes harvested at 24 h were untreated ( open histograms ) or pretreated with V. cholerae sialidase ( left panel ) or proteinase K ( right panel ) (shaded histograms) and labeled with siglec-E-Fc/anti-Fc-FITC complexes. Data are representative of at least three independent experiments.

    Journal: The Journal of Biological Chemistry

    Article Title: Early Murine T-lymphocyte Activation Is Accompanied by a Switch from N-Glycolyl- to N-Acetyl-neuraminic Acid and Generation of Ligands for Siglec-E *

    doi: 10.1074/jbc.M111.243410

    Figure Lengend Snippet: Activation of murine T-lymphocytes leads to up-regulation in the binding of siglec-E to cell surface proteins in a sialic acid-dependent manner. A , T-lymphocytes were activated with anti-CD3 and anti-CD28 for 24 h and then cultured for up to 4 days in the presence of IL-2. Cells harvested at each time point were incubated with fluorophore-conjugated CD4 ( left panel ) and CD8 antibodies ( right panel ) together with a pre-complex of FITC-conjugated goat anti-human Fc and siglec-Fc. Mean Fluorescence Intensity ( MFI ) of sialic acid-dependent binding by FACS is shown following subtraction of MFI values for sialidase-treated cells. B , for the detection of MAL and SNA binding, harvested cells corresponding to each time point were incubated with biotinylated lectin followed by a secondary incubation with FITC-conjugated streptavidin. C , splenocytes harvested at 24 h were untreated ( open histograms ) or pretreated with V. cholerae sialidase ( left panel ) or proteinase K ( right panel ) (shaded histograms) and labeled with siglec-E-Fc/anti-Fc-FITC complexes. Data are representative of at least three independent experiments.

    Article Snippet: For plant lectin labeling, cells were incubated with 1 μg/ml biotinylated Maackia amurensis leukoagglutinin (MAL) or Sambucus nigra agglutinin (SNA) (Vector Labs) followed by a secondary incubation with a 1/200 dilution of FITC-conjugated streptavidin (Vector Labs) for a further 30 min. To assess cell surface NeuGc expression, cells were incubated with a 1/500 dilution of chicken anti-Neu5Gc antibody (Sialix) followed by a secondary incubation with Cy5-conjugated donkey anti-chicken antibody (Jackson Immunoresearch) according to the manufacturer's instructions.

    Techniques: Activation Assay, Binding Assay, Cell Culture, Incubation, Fluorescence, FACS, Labeling

    Compilation of the photobleaching data (errors bars are SD ) corresponding to all the studied molecules (fluorescein, FITC, streptavidin-fluorescein, FD4, FD70, FD250S, and eGFP), inserted into vesicles between 10 and 100 μ m in diameter, under a laser power from 2.6 μ W to 103 μ W. eGFP data ( squares ) falls in the fast diffusion regime (5

    Journal: Biophysical Journal

    Article Title: Continuous Photobleaching in Vesicles and Living Cells: A Measure of Diffusion and Compartmentation

    doi: 10.1529/biophysj.105.069815

    Figure Lengend Snippet: Compilation of the photobleaching data (errors bars are SD ) corresponding to all the studied molecules (fluorescein, FITC, streptavidin-fluorescein, FD4, FD70, FD250S, and eGFP), inserted into vesicles between 10 and 100 μ m in diameter, under a laser power from 2.6 μ W to 103 μ W. eGFP data ( squares ) falls in the fast diffusion regime (5

    Article Snippet: They were prepared in sucrose solutions (typically 40 mM) containing one of the following molecules with a concentration of ∼20 nM: Fluorescein-5- isothiocyanate (FITC, MW = 389.38), FITC-dextran FD4, FD70, and FD250S ( MW = 4300, 68,100, and 282,000) from Sigma (St. Louis, MO); fluorescein ( MW = 332.31) and streptavidin fluorescein conjugate ( MW = 53132) from Molecular Probes (Eugene, OR); or purified recombinant eGFP proteins from Clontech (Palo Alto, CA).

    Techniques: Diffusion-based Assay

    Dynamics of protein S-alkylation by aHNE in RKO cells. (A) Heatmap of ratios of changes of all detected cysteine S-alkylation events shows that most adducts turn over rapidly in a time-dependent manner in cells. Lower the measured ratio (L/H) indicates more rapid turnover. (B) XIC are shown for changes in S-alkylated peptides from FAM120A protein in RKO cells, with the profiles for light- and heavy- labeled peptides in red and blue, respectively. The mean measured ratios were calculated from three biological replicate experiments and are displayed below the individual chromatograms, respectively. (C) Turnover of alkylation is not affected by proteasome inhibition. RKO cells were pretreated with (red) or without MG132 (black), followed by aHNE treatment with or without 1 and 4 h recovery periods. Proteins alkylated by aHNE were labeled with azido-biotin and detected by Western blotting with fluorescein-conjugated streptavidin. Data were presented as mean values ± SD, n = 3 biological replicates per group. A representative Western blot is shown in Figure S10 in the Supporting Information . (D) Distributions of the measured ratios of dynamic aHNE-cysteine adduction in vitro (red) and in situ (white).

    Journal: Analytical Chemistry

    Article Title: Quantitative Chemoproteomics for Site-Specific Analysis of Protein Alkylation by 4-Hydroxy-2-Nonenal in Cells

    doi: 10.1021/ac504685y

    Figure Lengend Snippet: Dynamics of protein S-alkylation by aHNE in RKO cells. (A) Heatmap of ratios of changes of all detected cysteine S-alkylation events shows that most adducts turn over rapidly in a time-dependent manner in cells. Lower the measured ratio (L/H) indicates more rapid turnover. (B) XIC are shown for changes in S-alkylated peptides from FAM120A protein in RKO cells, with the profiles for light- and heavy- labeled peptides in red and blue, respectively. The mean measured ratios were calculated from three biological replicate experiments and are displayed below the individual chromatograms, respectively. (C) Turnover of alkylation is not affected by proteasome inhibition. RKO cells were pretreated with (red) or without MG132 (black), followed by aHNE treatment with or without 1 and 4 h recovery periods. Proteins alkylated by aHNE were labeled with azido-biotin and detected by Western blotting with fluorescein-conjugated streptavidin. Data were presented as mean values ± SD, n = 3 biological replicates per group. A representative Western blot is shown in Figure S10 in the Supporting Information . (D) Distributions of the measured ratios of dynamic aHNE-cysteine adduction in vitro (red) and in situ (white).

    Article Snippet: The collected proteins were resolved on SDS-PAGE gels and detected by either immunoblotting with fluorescein-conjugated streptavidin (Alexa Fluor 680 nm, Life Technologies, Grand Island, NY) or direct in-gel imaging of fluorescein conjugated adducts as indicated.

    Techniques: Labeling, Inhibition, Western Blot, In Vitro, In Situ

    Recycling of US28. HeLa CD4-US28 cells were incubated with Q4120 antibody at 37°C for 1 h to allow antibody uptake, cooled on ice, and washed in acid medium to remove the Q4120 bound to the cell surface. (A) The cells were fixed, left intact or permeabilized with saponin (right and left, respectively), and then stained with a biotin-conjugated secondary antibody to detect Q4120 followed by fluorescein-streptavidin. (B) The cells were reincubated with a biotin-conjugated secondary antibody for 1 h at 37°C, cooled on ice, acid washed, fixed, and stained with fluorescein-streptavidin as in A. Scale bars, 10 μm. The righthand fields for each panel show Nomarski and fluorescence images of the same field.

    Journal: Molecular Biology of the Cell

    Article Title: The Human Cytomegalovirus US28 Protein Is Located in Endocytic Vesicles and Undergoes Constitutive Endocytosis and Recycling

    doi:

    Figure Lengend Snippet: Recycling of US28. HeLa CD4-US28 cells were incubated with Q4120 antibody at 37°C for 1 h to allow antibody uptake, cooled on ice, and washed in acid medium to remove the Q4120 bound to the cell surface. (A) The cells were fixed, left intact or permeabilized with saponin (right and left, respectively), and then stained with a biotin-conjugated secondary antibody to detect Q4120 followed by fluorescein-streptavidin. (B) The cells were reincubated with a biotin-conjugated secondary antibody for 1 h at 37°C, cooled on ice, acid washed, fixed, and stained with fluorescein-streptavidin as in A. Scale bars, 10 μm. The righthand fields for each panel show Nomarski and fluorescence images of the same field.

    Article Snippet: Cells, either intact or permeabilized with 0.05% saponin, were stained with fluorescein-conjugated streptavidin (Amersham Pharmacia Biotech) for 45 min.

    Techniques: Incubation, Staining, Fluorescence

    l ‐tetrahydropalmatine (l ‐THP) inhibits RANKL‐induced activation of NF‐κB and MAPK pathway. A, Induced RAW264.7 cells treated with l ‐THP (19.00 μg/mL, 1h) were fixed with 40% formaldehyde, washed by Triton X‐100, followed by incubation with anti‐P65 antibody, goat antimouse IgG antibody and fluorescein‐conjugated streptavidin. B, Induced RAW264.7 cells treated with l ‐THP (19.00 μg/mL) for 0, 15, 30 and 60 min. The levels of phosphorylated P65, P50 and IκB were evaluated by Western blotting analysis. C, Induced RAW264.7 cells treated with l ‐THP (19.00 μg/mL) for 30 min, were then collected and resuspended with a 32P‐labelled DNA duplex and run in sodium dodecyl SDS‐PAGE system for EMSA analysis of the DNA binding activity of NF‐κB. D, Induced RAW 264.7 cells were treated with l ‐THP (19.00 μg/mL) for 0, 15, 30 and 60 min, followed by Western blot analysis of the levels of phosphorylated ERK, JNK and P38. (*P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: l‐tetrahydropalmatine suppresses osteoclastogenesis in vivo and in vitro via blocking RANK‐TRAF6 interactions and inhibiting NF‐κB and MAPK pathways, et al. l‐tetrahydropalmatine suppresses osteoclastogenesis in vivo and in vitro via blocking RANK‐TRAF6 interactions and inhibiting NF‐κB and MAPK pathways

    doi: 10.1111/jcmm.14790

    Figure Lengend Snippet: l ‐tetrahydropalmatine (l ‐THP) inhibits RANKL‐induced activation of NF‐κB and MAPK pathway. A, Induced RAW264.7 cells treated with l ‐THP (19.00 μg/mL, 1h) were fixed with 40% formaldehyde, washed by Triton X‐100, followed by incubation with anti‐P65 antibody, goat antimouse IgG antibody and fluorescein‐conjugated streptavidin. B, Induced RAW264.7 cells treated with l ‐THP (19.00 μg/mL) for 0, 15, 30 and 60 min. The levels of phosphorylated P65, P50 and IκB were evaluated by Western blotting analysis. C, Induced RAW264.7 cells treated with l ‐THP (19.00 μg/mL) for 30 min, were then collected and resuspended with a 32P‐labelled DNA duplex and run in sodium dodecyl SDS‐PAGE system for EMSA analysis of the DNA binding activity of NF‐κB. D, Induced RAW 264.7 cells were treated with l ‐THP (19.00 μg/mL) for 0, 15, 30 and 60 min, followed by Western blot analysis of the levels of phosphorylated ERK, JNK and P38. (*P

    Article Snippet: In short, cells were fixed with 40% formaldehyde, then washed by Triton X‐100, followed by incubation with anti‐P65 or anti‐F‐actin antibody, goat antimouse IgG antibody and fluorescein‐conjugated streptavidin (Santa Cruz).

    Techniques: Activation Assay, Incubation, Western Blot, SDS Page, Binding Assay, Activity Assay

    Immunoanalysis of the ovarian cancer cell line, OVCAR-3. (A) Three subpopulations of OVCAR cells were identified by the cell sorter. They were CD133 + (65.2%), CD133 + /CD117 + (42.4%) and CD133 − (8.9%). (B) PAR-1 immunocytochemistry. OVCAR cells were grown on glass bottom chamber slides, fixed and successively incubated with PAR-1 antibody, appropriate biotinylated secondary antibody and FITC-streptavidin. Isotype antidodies were used in parallel and the nuclei were DAPI-labeled. Initial magnification, ×400. Scale bar represents 10 μ m.

    Journal: International Journal of Oncology

    Article Title: Endothelial protein C receptor expressed by ovarian cancer cells as a possible biomarker of cancer onset

    doi: 10.3892/ijo.2012.1492

    Figure Lengend Snippet: Immunoanalysis of the ovarian cancer cell line, OVCAR-3. (A) Three subpopulations of OVCAR cells were identified by the cell sorter. They were CD133 + (65.2%), CD133 + /CD117 + (42.4%) and CD133 − (8.9%). (B) PAR-1 immunocytochemistry. OVCAR cells were grown on glass bottom chamber slides, fixed and successively incubated with PAR-1 antibody, appropriate biotinylated secondary antibody and FITC-streptavidin. Isotype antidodies were used in parallel and the nuclei were DAPI-labeled. Initial magnification, ×400. Scale bar represents 10 μ m.

    Article Snippet: Reagents Reagents were obtained from the following sources: primary antibody AF2245 against EPCR (R & D Systems, Minneapolis, MN, USA); primary antibody ATAP2 against PAR-1 (Invitrogen, Carlsbad, CA, USA); biotinylated anti-rabbit, anti-mouse and anti-goat IgG, streptavidin-fluorescein conjugate (Amersham, Buckinghamshire, UK); rabbit anti-goat HRP (DakoCytomation, Glostrup, Denmark); phycoerythrin-coupled anti-P-gp antibody (Millipore, Billerica, MA, USA); human recombinant aPC (Lilly, Suresnes, France); U0126 and wortmannin (Calbiochem, San Diego, CA, USA).

    Techniques: Immunocytochemistry, Incubation, Labeling