peroxidase streptavidin conjugate  (Thermo Fisher)


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    Structured Review

    Thermo Fisher peroxidase streptavidin conjugate
    Peroxidase Streptavidin Conjugate, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/peroxidase streptavidin conjugate/product/Thermo Fisher
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    peroxidase streptavidin conjugate - by Bioz Stars, 2020-03
    86/100 stars

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    Related Articles

    Sandwich ELISA:

    Article Title: Epithelial morphogenesis of MDCK cells in three-dimensional collagen culture is modulated by interleukin-8
    Article Snippet: Paragraph title: Sandwich enzyme-linked immunosorbent assay. ... Three washes were done before the addition of horseradish peroxidase-streptavidin conjugate (Invitrogen) at a 1:4,000 dilution for 30 min with agitation.

    Serial Dilution:

    Article Title: Epithelial morphogenesis of MDCK cells in three-dimensional collagen culture is modulated by interleukin-8
    Article Snippet: Plates were washed three times with 0.05% Tween 20 (Roche) in PBS and then blocked with 200 μl/well of 5% BSA for 2 h. After another series of washes, a serial dilution of recombinant canine IL-8 (RcIL-8; R & D Systems) was added as the standard. .. Three washes were done before the addition of horseradish peroxidase-streptavidin conjugate (Invitrogen) at a 1:4,000 dilution for 30 min with agitation.

    Incubation:

    Article Title: Chromatin retention of DNA damage sensors DDB2 and XPC through loss of p97 segregase causes genotoxicity
    Article Snippet: Briefly, DNA purified from the whole genome (DNeasy Blood and tissue kit, Qiagen) was denatured by heating (99°C, 10 min) followed by a 15-min incubation on ice. .. After washing the plates, 100 μl of a peroxidase-streptavidin conjugate (1:10’000; Invitrogen) was distributed into each well.

    Article Title: Chromatin remodeler CHD1 promotes XPC‐to‐ TFIIH handover of nucleosomal UV lesions in nucleotide excision repair
    Article Snippet: Briefly, whole‐genome DNA was extracted using the DNeasy kit (Qiagen) and denatured by heating to 99°C for 10 min, followed by a 15‐min incubation on ice. .. After washing the plates, 100 μl of a peroxidase–streptavidin conjugate (1:10,000; Invitrogen) was distributed into each well.

    Spectrophotometry:

    Article Title: Chromatin retention of DNA damage sensors DDB2 and XPC through loss of p97 segregase causes genotoxicity
    Article Snippet: After washing the plates, 100 μl of a peroxidase-streptavidin conjugate (1:10’000; Invitrogen) was distributed into each well. .. The reaction was started by adding 0.5 mg·ml−1 o-phenylenediamine, 0.007% H2 O2 and citrate-phosphate buffer (50 mM Na2 HPO4 , 24 mM citric acid, pH 5.0), stopped with 50 μl of 2 M H2 SO4 , and monitored by measuring the absorbance at 492 nm in a PLUS384 microplate spectrophotometer (Molecular Devices).

    Article Title: Chromatin remodeler CHD1 promotes XPC‐to‐ TFIIH handover of nucleosomal UV lesions in nucleotide excision repair
    Article Snippet: After washing the plates, 100 μl of a peroxidase–streptavidin conjugate (1:10,000; Invitrogen) was distributed into each well. .. The reaction was started by adding 0.5 mg/ml o‐phenylenediamine, 0.007% (vol/vol) H2 O2 , and citrate‐phosphate buffer (50 mM Na2 HPO4 , 24 mM citric acid, pH 5.0), stopped with 50 μl of 2 M H2 SO4 , and monitored by measuring the absorbance at 492 nm in a PLUS384 microplate spectrophotometer (Molecular Devices).

    Purification:

    Article Title: Chromatin retention of DNA damage sensors DDB2 and XPC through loss of p97 segregase causes genotoxicity
    Article Snippet: Briefly, DNA purified from the whole genome (DNeasy Blood and tissue kit, Qiagen) was denatured by heating (99°C, 10 min) followed by a 15-min incubation on ice. .. After washing the plates, 100 μl of a peroxidase-streptavidin conjugate (1:10’000; Invitrogen) was distributed into each well.

    Enzyme-linked Immunosorbent Assay:

    Article Title: Epithelial morphogenesis of MDCK cells in three-dimensional collagen culture is modulated by interleukin-8
    Article Snippet: These samples were spun at 12,000 rpm to remove cell debris and then stored at −80°C until use in enzyme-linked immunosorbent assays (ELISA). .. Three washes were done before the addition of horseradish peroxidase-streptavidin conjugate (Invitrogen) at a 1:4,000 dilution for 30 min with agitation.

    Concentration Assay:

    Article Title: Chromatin remodeler CHD1 promotes XPC‐to‐ TFIIH handover of nucleosomal UV lesions in nucleotide excision repair
    Article Snippet: A volume of 50 μl per well of denatured DNA (at a concentration of 4 μg/ml for 6‐4PP detection, 200 ng/ml for CPD detection) was distributed into a 96‐well microtiter plate (Greiner) coated with protamine sulfate (Sigma) and dried overnight at 37°C. .. After washing the plates, 100 μl of a peroxidase–streptavidin conjugate (1:10,000; Invitrogen) was distributed into each well.

    Generated:

    Article Title: Epithelial morphogenesis of MDCK cells in three-dimensional collagen culture is modulated by interleukin-8
    Article Snippet: Three washes were done before the addition of horseradish peroxidase-streptavidin conjugate (Invitrogen) at a 1:4,000 dilution for 30 min with agitation. .. With the use of the average absorbance values for the known concentrations of RcIL-8, a standard curve was plotted, and a best-fitting linear trend line was generated.

    Recombinant:

    Article Title: Epithelial morphogenesis of MDCK cells in three-dimensional collagen culture is modulated by interleukin-8
    Article Snippet: Plates were washed three times with 0.05% Tween 20 (Roche) in PBS and then blocked with 200 μl/well of 5% BSA for 2 h. After another series of washes, a serial dilution of recombinant canine IL-8 (RcIL-8; R & D Systems) was added as the standard. .. Three washes were done before the addition of horseradish peroxidase-streptavidin conjugate (Invitrogen) at a 1:4,000 dilution for 30 min with agitation.

    Software:

    Article Title: Epithelial morphogenesis of MDCK cells in three-dimensional collagen culture is modulated by interleukin-8
    Article Snippet: Three washes were done before the addition of horseradish peroxidase-streptavidin conjugate (Invitrogen) at a 1:4,000 dilution for 30 min with agitation. .. The chromogenic reaction was stopped with sulfuric acid, and the optical absorbance of each sample well was measured for 1 s at 450 nm with Wallac 1420 software on a Perkin Elmer Plate Reader.

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    Thermo Fisher horseradish peroxidase conjugated streptavidin
    Depletion of glypican-1 stimulates the endocytosis of PrP C . SH-SY5Y cells expressing wild type PrP C were treated with either control or glypican-1 siRNA and then incubated for 60 h. Cells were surface biotinylated and incubated in OptiMEM for 1 h at 37°C. Where indicated, cells were treated with trypsin to remove remaining cell surface PrP C . Cells were then lysed and total PrP C immunoprecipitated from the sample using antibody 3F4. ( A ) Samples were subjected to western blot analysis and the biotin-labelled PrP C fraction was detected with peroxidase-conjugated <t>streptavidin.</t> ( B ) Densitometric analysis (mean ± s.e.m.) of multiple blots from three separate experiments in (A) is shown. ( C ) Expression of glypican-1 (in lysate samples treated with heparinase I and heparinase III) and PrP C in the cell lysates from (A). β-actin was used as a loading control. ( D ) SH-SY5Y cells expressing PrP C were treated with either control siRNA or glypican-1 siRNA and then allowed to reach confluence for 48 h. Cells were subsequently surface biotinylated and incubated in OptiMEM for 1 h at 37°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. ( E ) Densitometric analysis of the proportion of total PrP C present in the detergent soluble fractions of the plasma membrane after siRNA treatment from three independent experiments. ( F ) SH-SY5Y cells expressing PrP C were seeded onto glass coverslips and grown to 50% confluency. Cells were fixed, and then incubated with anti-PrP antibody 3F4 and a glypican-1 polyclonal antibody. Finally, cells were incubated with Alexa488-conjugated rabbit anti-mouse and Alexa594-conjugated goat anti-rabbit antibodies and viewed using a DeltaVision Optical Restoration Microscopy System. Images are representative of three individual experiments. Scale bars equal 10 µm. * P
    Horseradish Peroxidase Conjugated Streptavidin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 90 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/horseradish peroxidase conjugated streptavidin/product/Thermo Fisher
    Average 99 stars, based on 90 article reviews
    Price from $9.99 to $1999.99
    horseradish peroxidase conjugated streptavidin - by Bioz Stars, 2020-03
    99/100 stars
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    Depletion of glypican-1 stimulates the endocytosis of PrP C . SH-SY5Y cells expressing wild type PrP C were treated with either control or glypican-1 siRNA and then incubated for 60 h. Cells were surface biotinylated and incubated in OptiMEM for 1 h at 37°C. Where indicated, cells were treated with trypsin to remove remaining cell surface PrP C . Cells were then lysed and total PrP C immunoprecipitated from the sample using antibody 3F4. ( A ) Samples were subjected to western blot analysis and the biotin-labelled PrP C fraction was detected with peroxidase-conjugated streptavidin. ( B ) Densitometric analysis (mean ± s.e.m.) of multiple blots from three separate experiments in (A) is shown. ( C ) Expression of glypican-1 (in lysate samples treated with heparinase I and heparinase III) and PrP C in the cell lysates from (A). β-actin was used as a loading control. ( D ) SH-SY5Y cells expressing PrP C were treated with either control siRNA or glypican-1 siRNA and then allowed to reach confluence for 48 h. Cells were subsequently surface biotinylated and incubated in OptiMEM for 1 h at 37°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. ( E ) Densitometric analysis of the proportion of total PrP C present in the detergent soluble fractions of the plasma membrane after siRNA treatment from three independent experiments. ( F ) SH-SY5Y cells expressing PrP C were seeded onto glass coverslips and grown to 50% confluency. Cells were fixed, and then incubated with anti-PrP antibody 3F4 and a glypican-1 polyclonal antibody. Finally, cells were incubated with Alexa488-conjugated rabbit anti-mouse and Alexa594-conjugated goat anti-rabbit antibodies and viewed using a DeltaVision Optical Restoration Microscopy System. Images are representative of three individual experiments. Scale bars equal 10 µm. * P

    Journal: PLoS Pathogens

    Article Title: Glypican-1 Mediates Both Prion Protein Lipid Raft Association and Disease Isoform Formation

    doi: 10.1371/journal.ppat.1000666

    Figure Lengend Snippet: Depletion of glypican-1 stimulates the endocytosis of PrP C . SH-SY5Y cells expressing wild type PrP C were treated with either control or glypican-1 siRNA and then incubated for 60 h. Cells were surface biotinylated and incubated in OptiMEM for 1 h at 37°C. Where indicated, cells were treated with trypsin to remove remaining cell surface PrP C . Cells were then lysed and total PrP C immunoprecipitated from the sample using antibody 3F4. ( A ) Samples were subjected to western blot analysis and the biotin-labelled PrP C fraction was detected with peroxidase-conjugated streptavidin. ( B ) Densitometric analysis (mean ± s.e.m.) of multiple blots from three separate experiments in (A) is shown. ( C ) Expression of glypican-1 (in lysate samples treated with heparinase I and heparinase III) and PrP C in the cell lysates from (A). β-actin was used as a loading control. ( D ) SH-SY5Y cells expressing PrP C were treated with either control siRNA or glypican-1 siRNA and then allowed to reach confluence for 48 h. Cells were subsequently surface biotinylated and incubated in OptiMEM for 1 h at 37°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. ( E ) Densitometric analysis of the proportion of total PrP C present in the detergent soluble fractions of the plasma membrane after siRNA treatment from three independent experiments. ( F ) SH-SY5Y cells expressing PrP C were seeded onto glass coverslips and grown to 50% confluency. Cells were fixed, and then incubated with anti-PrP antibody 3F4 and a glypican-1 polyclonal antibody. Finally, cells were incubated with Alexa488-conjugated rabbit anti-mouse and Alexa594-conjugated goat anti-rabbit antibodies and viewed using a DeltaVision Optical Restoration Microscopy System. Images are representative of three individual experiments. Scale bars equal 10 µm. * P

    Article Snippet: Where indicated, biotin-labelled PrP was detected by subsequent immunoprecipitation of epitope-tagged PrP from the individual fractions using antibody 3F4 (Eurogentec Ltd., Southampton, U.K.) and subsequent immunoblotting using horseradish peroxidase-conjugated streptavidin (Thermo Fisher Scientific, Cramlington, U.K.).

    Techniques: Expressing, Incubation, Immunoprecipitation, Western Blot, Gradient Centrifugation, Microscopy

    Depletion of glypican-1 does not affect cell division or surface levels of PrP C . ( A ) ScN2a cells were seeded into 96 well plates and treated with transfection reagent only or incubated with either control siRNA or one of the four siRNAs targeted to glypican-1. Those experiments exceeding 48 h were dosed with a second treatment of the indicated siRNAs. Cells were then rinsed with PBS and fixed with 70% (v/v) ethanol. Plates were allowed to dry, stained with Hoescht 33342 and the fluorescence measured. ( B ) ScN2a cells were treated with control or glypican-1 siRNA. After 96 h, cell monolayers were labelled with a membrane impermeable biotin reagent. Biotin-labelled cell surface PrP was detected by immunoprecipitation using 6D11 and subsequent immunoblotting using HRP-conjugated streptavidin. Total PrP and PK-resistant PrP (PrP Sc ) were detected by immunoblotting using antibody 6D11. ( C ) Densitometric analysis of the proportion of the relative amount of biotinylated cell surface PrP in the absence or presence of glypican-1 siRNA from three independent experiments.

    Journal: PLoS Pathogens

    Article Title: Glypican-1 Mediates Both Prion Protein Lipid Raft Association and Disease Isoform Formation

    doi: 10.1371/journal.ppat.1000666

    Figure Lengend Snippet: Depletion of glypican-1 does not affect cell division or surface levels of PrP C . ( A ) ScN2a cells were seeded into 96 well plates and treated with transfection reagent only or incubated with either control siRNA or one of the four siRNAs targeted to glypican-1. Those experiments exceeding 48 h were dosed with a second treatment of the indicated siRNAs. Cells were then rinsed with PBS and fixed with 70% (v/v) ethanol. Plates were allowed to dry, stained with Hoescht 33342 and the fluorescence measured. ( B ) ScN2a cells were treated with control or glypican-1 siRNA. After 96 h, cell monolayers were labelled with a membrane impermeable biotin reagent. Biotin-labelled cell surface PrP was detected by immunoprecipitation using 6D11 and subsequent immunoblotting using HRP-conjugated streptavidin. Total PrP and PK-resistant PrP (PrP Sc ) were detected by immunoblotting using antibody 6D11. ( C ) Densitometric analysis of the proportion of the relative amount of biotinylated cell surface PrP in the absence or presence of glypican-1 siRNA from three independent experiments.

    Article Snippet: Where indicated, biotin-labelled PrP was detected by subsequent immunoprecipitation of epitope-tagged PrP from the individual fractions using antibody 3F4 (Eurogentec Ltd., Southampton, U.K.) and subsequent immunoblotting using horseradish peroxidase-conjugated streptavidin (Thermo Fisher Scientific, Cramlington, U.K.).

    Techniques: Transfection, Incubation, Staining, Fluorescence, Immunoprecipitation

    Heparin stimulates the endocytosis of PrP C in a dose-dependent manner and displaces it from detergent-resistant lipid rafts. ( A ) SH-SY5Y cells expressing PrP C were surface biotinylated and then incubated for 1 h at 37°C in the absence or presence of various concentrations of heparin diluted in OptiMEM. Prior to lysis cells were, where indicated, incubated with trypsin to digest cell surface PrP C . Cells were then lysed and PrP C immunoprecipitated from the sample using antibody 3F4. Samples were subjected to SDS PAGE and western blot analysis and the biotin-labelled PrP C detected with peroxidase-conjugated streptavidin. ( B ) Densitometric analysis of multiple blots from four separate experiments as described in (A) is shown. ( C ) SH-SY5Y cells expressing PrP C were surface biotinylated and then incubated in the absence or presence of 50 µM heparin prepared in OptiMEM for 1 h at 37°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. PrP C was immunoprecipitated from equal volumes of each gradient fraction using 3F4 and subjected to SDS-PAGE and western blotting. The gradient fractions from both the untreated and heparin treated cells were analysed on the same SDS gel and immunoblotted under identical conditions. The biotin-labelled PrP C was detected with peroxidase-conjugated streptavidin. Flotillin-1 and transferrin receptor (TfR) were detected by immunoblotting as markers for DRM and detergent-soluble fractions, respectively. ( D ) Densitometric analysis of the proportion of total PrP C in the detergent soluble fractions of the plasma membrane. ( E ) Untransfected SH-SY5Y cells and SH-SY5Y cells expressing either PrP C or PrP-TM were grown to confluence and then incubated for 1 h in the presence or absence of 50 µM heparin prepared in OptiMEM. Media samples were collected and concentrated and cells harvested and lysed. Cell lysate samples were immunoblotted for PrP C using antibody 3F4, with β-actin used as a loading control. ( F ) Quantification of PrP C and PrP-TM levels after treatment of cells with heparin as in (E). Experiments were performed in triplicate and repeated on three occasions. * P

    Journal: PLoS Pathogens

    Article Title: Glypican-1 Mediates Both Prion Protein Lipid Raft Association and Disease Isoform Formation

    doi: 10.1371/journal.ppat.1000666

    Figure Lengend Snippet: Heparin stimulates the endocytosis of PrP C in a dose-dependent manner and displaces it from detergent-resistant lipid rafts. ( A ) SH-SY5Y cells expressing PrP C were surface biotinylated and then incubated for 1 h at 37°C in the absence or presence of various concentrations of heparin diluted in OptiMEM. Prior to lysis cells were, where indicated, incubated with trypsin to digest cell surface PrP C . Cells were then lysed and PrP C immunoprecipitated from the sample using antibody 3F4. Samples were subjected to SDS PAGE and western blot analysis and the biotin-labelled PrP C detected with peroxidase-conjugated streptavidin. ( B ) Densitometric analysis of multiple blots from four separate experiments as described in (A) is shown. ( C ) SH-SY5Y cells expressing PrP C were surface biotinylated and then incubated in the absence or presence of 50 µM heparin prepared in OptiMEM for 1 h at 37°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. PrP C was immunoprecipitated from equal volumes of each gradient fraction using 3F4 and subjected to SDS-PAGE and western blotting. The gradient fractions from both the untreated and heparin treated cells were analysed on the same SDS gel and immunoblotted under identical conditions. The biotin-labelled PrP C was detected with peroxidase-conjugated streptavidin. Flotillin-1 and transferrin receptor (TfR) were detected by immunoblotting as markers for DRM and detergent-soluble fractions, respectively. ( D ) Densitometric analysis of the proportion of total PrP C in the detergent soluble fractions of the plasma membrane. ( E ) Untransfected SH-SY5Y cells and SH-SY5Y cells expressing either PrP C or PrP-TM were grown to confluence and then incubated for 1 h in the presence or absence of 50 µM heparin prepared in OptiMEM. Media samples were collected and concentrated and cells harvested and lysed. Cell lysate samples were immunoblotted for PrP C using antibody 3F4, with β-actin used as a loading control. ( F ) Quantification of PrP C and PrP-TM levels after treatment of cells with heparin as in (E). Experiments were performed in triplicate and repeated on three occasions. * P

    Article Snippet: Where indicated, biotin-labelled PrP was detected by subsequent immunoprecipitation of epitope-tagged PrP from the individual fractions using antibody 3F4 (Eurogentec Ltd., Southampton, U.K.) and subsequent immunoblotting using horseradish peroxidase-conjugated streptavidin (Thermo Fisher Scientific, Cramlington, U.K.).

    Techniques: Expressing, Incubation, Lysis, Immunoprecipitation, SDS Page, Western Blot, Gradient Centrifugation, SDS-Gel

    Depletion of glypican-1 inhibits the association of PrP-TM with DRMs. SH-SY5Y cells expressing PrP-TM were treated with either control siRNA or siRNA targeted to glypican-1 and then allowed to reach confluence for 48 h. Cells were subsequently surface biotinylated and incubated in OptiMEM for 1 h at 37°C in the presence of Tyrphostin A23 to block endocytosis. The media was removed and the cells washed in phosphate-buffered saline prior to homogenisation in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. ( A ) Quantification of glypican-1 and PrP-TM expression in cell lysates. To detect glypican-1, cell lysate samples were treated with heparinase I and heparinase III prior to electrophoresis as described in the materials and methods section. ( B ) PrP-TM was immunoprecipitated from equal volumes of each gradient fraction using 3F4 and then subjected to western blotting with peroxidase-conjugated streptavidin. Flotillin-1 and transferrin receptor (TfR) were detected by immunoblotting as markers for DRM and detergent-soluble fractions, respectively. ( C ) Densitometric analysis of the proportion of total PrP-TM present in the detergent soluble fractions of the plasma membrane after siRNA treatment from multiple blots from three independent experiments. * P

    Journal: PLoS Pathogens

    Article Title: Glypican-1 Mediates Both Prion Protein Lipid Raft Association and Disease Isoform Formation

    doi: 10.1371/journal.ppat.1000666

    Figure Lengend Snippet: Depletion of glypican-1 inhibits the association of PrP-TM with DRMs. SH-SY5Y cells expressing PrP-TM were treated with either control siRNA or siRNA targeted to glypican-1 and then allowed to reach confluence for 48 h. Cells were subsequently surface biotinylated and incubated in OptiMEM for 1 h at 37°C in the presence of Tyrphostin A23 to block endocytosis. The media was removed and the cells washed in phosphate-buffered saline prior to homogenisation in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. ( A ) Quantification of glypican-1 and PrP-TM expression in cell lysates. To detect glypican-1, cell lysate samples were treated with heparinase I and heparinase III prior to electrophoresis as described in the materials and methods section. ( B ) PrP-TM was immunoprecipitated from equal volumes of each gradient fraction using 3F4 and then subjected to western blotting with peroxidase-conjugated streptavidin. Flotillin-1 and transferrin receptor (TfR) were detected by immunoblotting as markers for DRM and detergent-soluble fractions, respectively. ( C ) Densitometric analysis of the proportion of total PrP-TM present in the detergent soluble fractions of the plasma membrane after siRNA treatment from multiple blots from three independent experiments. * P

    Article Snippet: Where indicated, biotin-labelled PrP was detected by subsequent immunoprecipitation of epitope-tagged PrP from the individual fractions using antibody 3F4 (Eurogentec Ltd., Southampton, U.K.) and subsequent immunoblotting using horseradish peroxidase-conjugated streptavidin (Thermo Fisher Scientific, Cramlington, U.K.).

    Techniques: Expressing, Incubation, Blocking Assay, Homogenization, Gradient Centrifugation, Electrophoresis, Immunoprecipitation, Western Blot

    The association of PrP-TM with DRMs is disrupted by treatment of cells with either heparin or bacterial PI-PLC. SH-SY5Y cells expressing PrP-TM were surface biotinylated and then ( A ) incubated in the absence or presence of 50 µM heparin prepared in OptiMEM for 1 h at 37°C or ( B ) incubated in the absence or presence of 1 U/ml bacterial PI-PLC for 1 h at 4°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. PrP-TM was immunoprecipitated from equal volumes of each gradient fraction using 3F4 and subjected to western blotting. The biotin-labelled PrP-TM fraction was detected with peroxidase-conjugated streptavidin. Flotillin-1 and transferrin receptor (TfR) were detected by immunoblotting as markers for DRM and detergent-soluble fractions respectively. ( C ) Densitometric analysis of the proportion of total PrP-TM present in the detergent soluble fractions of the plasma membrane after heparin and PI-PLC treatment. Experiments were performed in triplicate and repeated on three occasions. * P

    Journal: PLoS Pathogens

    Article Title: Glypican-1 Mediates Both Prion Protein Lipid Raft Association and Disease Isoform Formation

    doi: 10.1371/journal.ppat.1000666

    Figure Lengend Snippet: The association of PrP-TM with DRMs is disrupted by treatment of cells with either heparin or bacterial PI-PLC. SH-SY5Y cells expressing PrP-TM were surface biotinylated and then ( A ) incubated in the absence or presence of 50 µM heparin prepared in OptiMEM for 1 h at 37°C or ( B ) incubated in the absence or presence of 1 U/ml bacterial PI-PLC for 1 h at 4°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. PrP-TM was immunoprecipitated from equal volumes of each gradient fraction using 3F4 and subjected to western blotting. The biotin-labelled PrP-TM fraction was detected with peroxidase-conjugated streptavidin. Flotillin-1 and transferrin receptor (TfR) were detected by immunoblotting as markers for DRM and detergent-soluble fractions respectively. ( C ) Densitometric analysis of the proportion of total PrP-TM present in the detergent soluble fractions of the plasma membrane after heparin and PI-PLC treatment. Experiments were performed in triplicate and repeated on three occasions. * P

    Article Snippet: Where indicated, biotin-labelled PrP was detected by subsequent immunoprecipitation of epitope-tagged PrP from the individual fractions using antibody 3F4 (Eurogentec Ltd., Southampton, U.K.) and subsequent immunoblotting using horseradish peroxidase-conjugated streptavidin (Thermo Fisher Scientific, Cramlington, U.K.).

    Techniques: Planar Chromatography, Expressing, Incubation, Gradient Centrifugation, Immunoprecipitation, Western Blot

    Oxidative modification of identified proteins in planta upon H 2 O 2 treatment. Transgenic plants expressing the protein of interest fused with the FLAG tag were vacuum infiltrated with either water (mock) or 5 mM H 2 O 2 . For analysis of AtCIAPIN1, eEF1α, and AtPTP1, free thiols in the total protein were labeled with BIAM during protein extraction. For analysis of AtNAP1;1 and AtPDIL1-1, free thiols in the samples were first alkylated by IAM. Samples were then treated with DTT and newly generated free thiols were labeled by BIAM. After that, FLAG-tagged protein from each sample was affinity purified, separated by SDS-PAGE, and detected by HRP-Conjugated Streptavidin (to determine the amount of BIAM attached to the FLAG-tagged protein) or by the anti-FLAG M2 antibody (to determine the amount of the total recombinant protein).

    Journal: Journal of Proteome Research

    Article Title: Proteomic Analysis of Early-Responsive Redox-Sensitive Proteins in Arabidopsis

    doi: 10.1021/pr200918f

    Figure Lengend Snippet: Oxidative modification of identified proteins in planta upon H 2 O 2 treatment. Transgenic plants expressing the protein of interest fused with the FLAG tag were vacuum infiltrated with either water (mock) or 5 mM H 2 O 2 . For analysis of AtCIAPIN1, eEF1α, and AtPTP1, free thiols in the total protein were labeled with BIAM during protein extraction. For analysis of AtNAP1;1 and AtPDIL1-1, free thiols in the samples were first alkylated by IAM. Samples were then treated with DTT and newly generated free thiols were labeled by BIAM. After that, FLAG-tagged protein from each sample was affinity purified, separated by SDS-PAGE, and detected by HRP-Conjugated Streptavidin (to determine the amount of BIAM attached to the FLAG-tagged protein) or by the anti-FLAG M2 antibody (to determine the amount of the total recombinant protein).

    Article Snippet: Immunoprecipitated protein was separated by SDS-PAGE and immunoblotted with either anti-FLAG M2-Peroxidase (HRP) antibody (Sigma) or Horseradish Peroxidase-Conjugated Streptavidin (Thermo Scientific).

    Techniques: Modification, Transgenic Assay, Expressing, FLAG-tag, Labeling, Protein Extraction, Generated, Affinity Purification, SDS Page, Recombinant

    Comparison of histone extraction protocols, and specificity testing of streptavidin and anti-biotin Panel A: Nuclear histones were extracted from Jurkat cells (lanes 1, 5, and 9) or HeLa cells (lanes 3, 7, and 11) by using HCl; for comparison histones were extracted from Jurkat cells (lanes 2, 6, and 10) or HeLa cells (lanes 4, 8, and 12) by using H 2 SO 4 +TCA+acetone/HCl+acetone. Histones were probed with coomassie blue (lanes 1–4) and antibodies to the C-termini in histone H3 (lanes 5–8) and H4 (lanes 9–12). Panel B: HCl extracts of Jurkat cell histones (lanes 1, 4, 7, 10, and 13), recombinant human histone H4 (lanes 2, 5, 8, 11, and 14), and chemically biotinylated histone H4 (lanes 3, 6, 9, 12, and 15) were probed with streptavidin without biotin competitor (lanes 1–3) and with 5 mM free biotin (lanes 4–6), and with anti-biotin without biotin competitor (lanes 7–9) and with 5 mM free biotin (lanes 10–12), and with coomassie blue (lanes 13–15).

    Journal: Molecular genetics and metabolism

    Article Title: Biotinylation is a natural, albeit rare, modification of human histones

    doi: 10.1016/j.ymgme.2011.08.030

    Figure Lengend Snippet: Comparison of histone extraction protocols, and specificity testing of streptavidin and anti-biotin Panel A: Nuclear histones were extracted from Jurkat cells (lanes 1, 5, and 9) or HeLa cells (lanes 3, 7, and 11) by using HCl; for comparison histones were extracted from Jurkat cells (lanes 2, 6, and 10) or HeLa cells (lanes 4, 8, and 12) by using H 2 SO 4 +TCA+acetone/HCl+acetone. Histones were probed with coomassie blue (lanes 1–4) and antibodies to the C-termini in histone H3 (lanes 5–8) and H4 (lanes 9–12). Panel B: HCl extracts of Jurkat cell histones (lanes 1, 4, 7, 10, and 13), recombinant human histone H4 (lanes 2, 5, 8, 11, and 14), and chemically biotinylated histone H4 (lanes 3, 6, 9, 12, and 15) were probed with streptavidin without biotin competitor (lanes 1–3) and with 5 mM free biotin (lanes 4–6), and with anti-biotin without biotin competitor (lanes 7–9) and with 5 mM free biotin (lanes 10–12), and with coomassie blue (lanes 13–15).

    Article Snippet: Controls included pre-immune serum, horseradish peroxidase-conjugated streptavidin (Thermo Scientific), and IRDye 800CW Streptavidin (LI-COR).

    Techniques: Recombinant

    Biotinylation marks can be detected in bulk extracts from human cells, using streptavidin and anti-biotin as probes Bulk extracts of histone extracts from various cell lineages were probed with streptavidin (panel A), anti-biotin (panel B), and the loading and transfer controls coomassie blue (panel C), anti-H3 (panel D), and anti-H4 (panel E). Panel F: Histones from HeLa cells were extracted with H 2 SO 4 +TCA+acetone/HCl+acetone. Ten or five microgram of histones were loaded per well. Blots were blocked with PBS containing 5% BSA. After probing with horseradish peroxidase-conjugated anti-biotin or Nutravidin, the blots were washed for 6 hrs, and exposed to autoradiography film for 1 min.

    Journal: Molecular genetics and metabolism

    Article Title: Biotinylation is a natural, albeit rare, modification of human histones

    doi: 10.1016/j.ymgme.2011.08.030

    Figure Lengend Snippet: Biotinylation marks can be detected in bulk extracts from human cells, using streptavidin and anti-biotin as probes Bulk extracts of histone extracts from various cell lineages were probed with streptavidin (panel A), anti-biotin (panel B), and the loading and transfer controls coomassie blue (panel C), anti-H3 (panel D), and anti-H4 (panel E). Panel F: Histones from HeLa cells were extracted with H 2 SO 4 +TCA+acetone/HCl+acetone. Ten or five microgram of histones were loaded per well. Blots were blocked with PBS containing 5% BSA. After probing with horseradish peroxidase-conjugated anti-biotin or Nutravidin, the blots were washed for 6 hrs, and exposed to autoradiography film for 1 min.

    Article Snippet: Controls included pre-immune serum, horseradish peroxidase-conjugated streptavidin (Thermo Scientific), and IRDye 800CW Streptavidin (LI-COR).

    Techniques: Autoradiography

    Specificity of antibodies to H3K4bio, H3K9bio and H3K18bio Panel A: Confirmation of equal loading of peptides H3K4bio, H3K9bio, and H3K18bio with streptavidin. Panel B: Transblots of peptides N 1–25 (non-biotinylated negative control), H3K4bio, H3K9bio, and H3K18bio were probed with anti-H3K4bio (left), anti-H3K9bio (middle), and anti-H3K18bio (right). Panel C: HCl extracts of histones from Jurkat cells were probed using streptavidin, anti-H3K9bio, and anti-H3K18bio; samples of biotin-free histones (“-”) were generated by using avidin agarose. Panel D: Bulk HCl extracts of histones from Jurkat cells were probed with anti-H3K9bio (top) and anti-H3K18bio (bottom) after pre-incubation of antibodies with increasing amounts of competing peptides H3K4bio, H3K9bio, and H3K18bio; controls (“C”) were prepared without peptide competitors. Note the difference in the order of peptide competitors in the two gels. For some gels, bands from the same analytical runs were electronically re-arranged to facilitate comparisons. Panel E: Peptides H3K9bio, H3K9ac, and H3K9me2 were probed with streptavidin (lanes 1, 2, 9 and 10), anti-H3K9bio (lanes 3, 4, 11 and 12), anti-H3K9ac (lanes 5 and 6), and anti-H3K9me2 (lanes 13 and 14); Ponceau S was used as loading control (lanes 7, 8, 15 and 16). Panel F: Peptides H3K18bio and H3K18ac were probed with streptavidin (lanes 1 and 2), anti-H3K18bio (lanes 3 and 4) and anti-H3K18ac (lanes 5 and 6); Ponceau S was used as loading control (lanes 7 and 8).

    Journal: Molecular genetics and metabolism

    Article Title: Biotinylation is a natural, albeit rare, modification of human histones

    doi: 10.1016/j.ymgme.2011.08.030

    Figure Lengend Snippet: Specificity of antibodies to H3K4bio, H3K9bio and H3K18bio Panel A: Confirmation of equal loading of peptides H3K4bio, H3K9bio, and H3K18bio with streptavidin. Panel B: Transblots of peptides N 1–25 (non-biotinylated negative control), H3K4bio, H3K9bio, and H3K18bio were probed with anti-H3K4bio (left), anti-H3K9bio (middle), and anti-H3K18bio (right). Panel C: HCl extracts of histones from Jurkat cells were probed using streptavidin, anti-H3K9bio, and anti-H3K18bio; samples of biotin-free histones (“-”) were generated by using avidin agarose. Panel D: Bulk HCl extracts of histones from Jurkat cells were probed with anti-H3K9bio (top) and anti-H3K18bio (bottom) after pre-incubation of antibodies with increasing amounts of competing peptides H3K4bio, H3K9bio, and H3K18bio; controls (“C”) were prepared without peptide competitors. Note the difference in the order of peptide competitors in the two gels. For some gels, bands from the same analytical runs were electronically re-arranged to facilitate comparisons. Panel E: Peptides H3K9bio, H3K9ac, and H3K9me2 were probed with streptavidin (lanes 1, 2, 9 and 10), anti-H3K9bio (lanes 3, 4, 11 and 12), anti-H3K9ac (lanes 5 and 6), and anti-H3K9me2 (lanes 13 and 14); Ponceau S was used as loading control (lanes 7, 8, 15 and 16). Panel F: Peptides H3K18bio and H3K18ac were probed with streptavidin (lanes 1 and 2), anti-H3K18bio (lanes 3 and 4) and anti-H3K18ac (lanes 5 and 6); Ponceau S was used as loading control (lanes 7 and 8).

    Article Snippet: Controls included pre-immune serum, horseradish peroxidase-conjugated streptavidin (Thermo Scientific), and IRDye 800CW Streptavidin (LI-COR).

    Techniques: Negative Control, Generated, Avidin-Biotin Assay, Incubation

    Specificity of antibodies to H4K8bio and H4K12bio Panel A: Transblots of peptides N 1–19 (non-biotinylated negative control), H4K8bio, and H4K12bio were probed with anti-H4K8bio; pre-immune serum was used as negative control. Panel B: HCl extracts of histones from Jurkat cells were probed using streptavidin and anti-H4K8bio; samples of biotin-free histones (“-”) were generated by using avidin agarose. Panel C: HCl extracts of histones from Jurkat cells were probed with anti-H4K8bio after pre-incubation of antibodies with increasing amounts of competing peptides H4K8bio and H4K12bio; controls (“C”) were prepared without peptide competitors. For some gels, bands from the same analytical runs were electronically rearranged to facilitate comparisons. Panel D: Peptides H4K8bio and H4K8ac were probed with streptavidin (lanes 1 and 2), anti-H4K8bio (lanes 3 and 4) and anti-H4K8ac (lanes 5 and 6); Ponceau S was used as loading control (lanes 7 and 8). Panel E: Peptides H4K12bio and H4K12ac were probed with streptavidin (lanes 1 and 2), anti-H4K12bio (lanes 3 and 4) and anti-H4K12ac (lanes 5 and 6); Ponceau S was used as loading control (lanes 7 and 8).

    Journal: Molecular genetics and metabolism

    Article Title: Biotinylation is a natural, albeit rare, modification of human histones

    doi: 10.1016/j.ymgme.2011.08.030

    Figure Lengend Snippet: Specificity of antibodies to H4K8bio and H4K12bio Panel A: Transblots of peptides N 1–19 (non-biotinylated negative control), H4K8bio, and H4K12bio were probed with anti-H4K8bio; pre-immune serum was used as negative control. Panel B: HCl extracts of histones from Jurkat cells were probed using streptavidin and anti-H4K8bio; samples of biotin-free histones (“-”) were generated by using avidin agarose. Panel C: HCl extracts of histones from Jurkat cells were probed with anti-H4K8bio after pre-incubation of antibodies with increasing amounts of competing peptides H4K8bio and H4K12bio; controls (“C”) were prepared without peptide competitors. For some gels, bands from the same analytical runs were electronically rearranged to facilitate comparisons. Panel D: Peptides H4K8bio and H4K8ac were probed with streptavidin (lanes 1 and 2), anti-H4K8bio (lanes 3 and 4) and anti-H4K8ac (lanes 5 and 6); Ponceau S was used as loading control (lanes 7 and 8). Panel E: Peptides H4K12bio and H4K12ac were probed with streptavidin (lanes 1 and 2), anti-H4K12bio (lanes 3 and 4) and anti-H4K12ac (lanes 5 and 6); Ponceau S was used as loading control (lanes 7 and 8).

    Article Snippet: Controls included pre-immune serum, horseradish peroxidase-conjugated streptavidin (Thermo Scientific), and IRDye 800CW Streptavidin (LI-COR).

    Techniques: Negative Control, Generated, Avidin-Biotin Assay, Incubation

    Validation of the biotin depletion and repletion protocol Panel A: Jurkat cells after a 2-wk depletion in biotin-deficient medium (0.025 nM, lane 1) compared with cells cultured in medium containing a physiological concentration of biotin (0.25 nM) (lane 2), and cells after a 1-wk repletion in medium containing a pharmacological concentration of biotin (10 nM) (lane 3). Biotinylated carboxylases were probed using streptavidin (SA). Equal expression, loading, and transfer of carboxylases was confirmed using anti-PC and anti-PCC. Panel B: As described for panel A, but HeLa cells (lanes 4–6) were substituted for Jurkat cells.

    Journal: Molecular genetics and metabolism

    Article Title: Biotinylation is a natural, albeit rare, modification of human histones

    doi: 10.1016/j.ymgme.2011.08.030

    Figure Lengend Snippet: Validation of the biotin depletion and repletion protocol Panel A: Jurkat cells after a 2-wk depletion in biotin-deficient medium (0.025 nM, lane 1) compared with cells cultured in medium containing a physiological concentration of biotin (0.25 nM) (lane 2), and cells after a 1-wk repletion in medium containing a pharmacological concentration of biotin (10 nM) (lane 3). Biotinylated carboxylases were probed using streptavidin (SA). Equal expression, loading, and transfer of carboxylases was confirmed using anti-PC and anti-PCC. Panel B: As described for panel A, but HeLa cells (lanes 4–6) were substituted for Jurkat cells.

    Article Snippet: Controls included pre-immune serum, horseradish peroxidase-conjugated streptavidin (Thermo Scientific), and IRDye 800CW Streptavidin (LI-COR).

    Techniques: Cell Culture, Concentration Assay, Expressing, Periodic Counter-current Chromatography

    Nuclear CD44 associates with STAT3 and functions to modulate transcription. (A) Confocal microscopy of H1299 cells cultured in serum-free medium for 24 h and treated with control IgG or H-3 for 1 h. (B) Nuclear (N) and cytosolic (C) fractions were prepared from AZ521/mock and AZ521/CD44 cells and immunoprecipitated followed by Western blotting. (C) Nuclear (Nuc) and cytosolic (Cyto) fractions were immunoprecipitated followed by Western blotting. (D) Nuclear extracts were prepared from the parental H1299 and HT29 cells or cell clones stably harboring lentivirus-encoded control (Cont) shRNA or shRNA targeting CD44 and immunoprecipitated followed by Western blotting. (E) Confocal microscopy of AZ521/CD44 cells with anti-STAT3 (in red) after incubation with control IgG or H-3 for 1 and 2.5 h. (F) AZ521/CD44 cells were incubated with biotin-conjugated H-3 at 4°C. After removal to 37°C for 1 h, cytosolic and nuclear fractions and immunoprecipitates from streptavidin beads were analyzed by Western blotting. (G) Nuclear extracts were prepared from AZ521/CD44 (top) and AZ521/CD44(NLS) mutant (bottom) cells after cross-linking with 1% formaldehyde. ChIP was performed using anti-CD44 and anti-STAT3. PCR amplification of designated regions within cyclin D1 promoter was performed. (H) Nuclear extracts were prepared from AZ521/mock and AZ521/CD44 s cells cultured in serum-free medium for 24 h. EMSA was performed with biotin-labeled double-stranded oligonucleotide probes corresponding to various regions of cyclin D1 promoter in the presence and absence of anti-CD44, anti-STAT3, or anti-p300 antibodies. Shifted and supershifted complexes are indicated by arrowheads. A nonspecific band is indicated by an asterisk. Ab, antibody. (I) Reporter assays were performed in AZ521 cells and AZ521 cell clones stably harboring lentivirus-encoded control shRNA or shRNA targeting STAT3 or p300 using the pFR-Luc reporter plasmid containing five copies of GAL4 DNA–binding sites. Data are presented as the means ± SD and were derived from at least three independent experiments. *, P

    Journal: The Journal of Cell Biology

    Article Title: Acetylation and activation of STAT3 mediated by nuclear translocation of CD44

    doi: 10.1083/jcb.200812060

    Figure Lengend Snippet: Nuclear CD44 associates with STAT3 and functions to modulate transcription. (A) Confocal microscopy of H1299 cells cultured in serum-free medium for 24 h and treated with control IgG or H-3 for 1 h. (B) Nuclear (N) and cytosolic (C) fractions were prepared from AZ521/mock and AZ521/CD44 cells and immunoprecipitated followed by Western blotting. (C) Nuclear (Nuc) and cytosolic (Cyto) fractions were immunoprecipitated followed by Western blotting. (D) Nuclear extracts were prepared from the parental H1299 and HT29 cells or cell clones stably harboring lentivirus-encoded control (Cont) shRNA or shRNA targeting CD44 and immunoprecipitated followed by Western blotting. (E) Confocal microscopy of AZ521/CD44 cells with anti-STAT3 (in red) after incubation with control IgG or H-3 for 1 and 2.5 h. (F) AZ521/CD44 cells were incubated with biotin-conjugated H-3 at 4°C. After removal to 37°C for 1 h, cytosolic and nuclear fractions and immunoprecipitates from streptavidin beads were analyzed by Western blotting. (G) Nuclear extracts were prepared from AZ521/CD44 (top) and AZ521/CD44(NLS) mutant (bottom) cells after cross-linking with 1% formaldehyde. ChIP was performed using anti-CD44 and anti-STAT3. PCR amplification of designated regions within cyclin D1 promoter was performed. (H) Nuclear extracts were prepared from AZ521/mock and AZ521/CD44 s cells cultured in serum-free medium for 24 h. EMSA was performed with biotin-labeled double-stranded oligonucleotide probes corresponding to various regions of cyclin D1 promoter in the presence and absence of anti-CD44, anti-STAT3, or anti-p300 antibodies. Shifted and supershifted complexes are indicated by arrowheads. A nonspecific band is indicated by an asterisk. Ab, antibody. (I) Reporter assays were performed in AZ521 cells and AZ521 cell clones stably harboring lentivirus-encoded control shRNA or shRNA targeting STAT3 or p300 using the pFR-Luc reporter plasmid containing five copies of GAL4 DNA–binding sites. Data are presented as the means ± SD and were derived from at least three independent experiments. *, P

    Article Snippet: DNA–protein complexes were fractionated by PAGE and visualized by horseradish peroxidase–conjugated streptavidin using the LightShift Chemiluminescent EMSA kit (Thermo Fisher Scientific).

    Techniques: Confocal Microscopy, Cell Culture, Immunoprecipitation, Western Blot, Clone Assay, Stable Transfection, shRNA, Incubation, Mutagenesis, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Amplification, Labeling, Plasmid Preparation, Binding Assay, Derivative Assay

    Nuclear localization of full-length CD44. (A) Immunohistochemistry of nuclear CD44 in human gastric mucosal (a) and cancerous (b–d) tissues using anti-CD44 v 6 antibody, with nuclei counterstained in hematoxylin. (B) Western blot analyses of nuclear (Nuc) and cytosolic (Cyto) fractions of human gastric (GC), colon (CRC), and lung (NSCLC) cancer cell lines. NUGC, Nagoya University gastric cancer. (C and D) HT29 (C) and H1299 (D) cells were surface labeled with biotin at 4°C followed by incubation at 37°C in the presence of OPN (C) and H-3 (D). The nuclear fraction was incubated with streptavidin beads and subjected to Western blotting. (B–D) Molecular mass is shown in kilodaltons. (E) H1299 and AZ521/CD44 cells were suspended in medium for 30 min, replated on dishes for 1 h, and immunostained using H-3 (top) and anti-myc (bottom) antibodies. Representative images taken by confocal laser microscopy are shown. (F) H1299 cells were incubated with biotin-conjugated control IgG or H-3 at 4°C followed by further incubation at 37°C for 60 min. After removing surface-retained biotin, cells were fixed and stained by avidin. Bars: (A) 100 µm; (E) 10 µm; (F) 25 µm.

    Journal: The Journal of Cell Biology

    Article Title: Acetylation and activation of STAT3 mediated by nuclear translocation of CD44

    doi: 10.1083/jcb.200812060

    Figure Lengend Snippet: Nuclear localization of full-length CD44. (A) Immunohistochemistry of nuclear CD44 in human gastric mucosal (a) and cancerous (b–d) tissues using anti-CD44 v 6 antibody, with nuclei counterstained in hematoxylin. (B) Western blot analyses of nuclear (Nuc) and cytosolic (Cyto) fractions of human gastric (GC), colon (CRC), and lung (NSCLC) cancer cell lines. NUGC, Nagoya University gastric cancer. (C and D) HT29 (C) and H1299 (D) cells were surface labeled with biotin at 4°C followed by incubation at 37°C in the presence of OPN (C) and H-3 (D). The nuclear fraction was incubated with streptavidin beads and subjected to Western blotting. (B–D) Molecular mass is shown in kilodaltons. (E) H1299 and AZ521/CD44 cells were suspended in medium for 30 min, replated on dishes for 1 h, and immunostained using H-3 (top) and anti-myc (bottom) antibodies. Representative images taken by confocal laser microscopy are shown. (F) H1299 cells were incubated with biotin-conjugated control IgG or H-3 at 4°C followed by further incubation at 37°C for 60 min. After removing surface-retained biotin, cells were fixed and stained by avidin. Bars: (A) 100 µm; (E) 10 µm; (F) 25 µm.

    Article Snippet: DNA–protein complexes were fractionated by PAGE and visualized by horseradish peroxidase–conjugated streptavidin using the LightShift Chemiluminescent EMSA kit (Thermo Fisher Scientific).

    Techniques: Immunohistochemistry, Western Blot, Labeling, Incubation, Microscopy, Staining, Avidin-Biotin Assay