secondary hrp conjugated antibodies  (Millipore)


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  • 99
    Name:
    HRP Conjugated Phosphotyrosine specific Antibody Concentrate
    Description:

    Catalog Number:
    rabtyroe
    Price:
    None
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    Structured Review

    Millipore secondary hrp conjugated antibodies

    https://www.bioz.com/result/secondary hrp conjugated antibodies/product/Millipore
    Average 99 stars, based on 25 article reviews
    Price from $9.99 to $1999.99
    secondary hrp conjugated antibodies - by Bioz Stars, 2020-07
    99/100 stars

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    Related Articles

    Immunoprecipitation:

    Article Title: MARQUIS: A Multiplex Method for Absolute Quantification of Peptides and Post-Translational Modifications
    Article Snippet: .. Immunoprecipitation 70ul protein G agarose beads (calbiochem IP08) were rinsed in 400ul IP Buffer (100mM tris, 0.3% NP-40, pH 7.4) prior to an 8 hour incubation with a cocktail of three phosphotyrosine-specific antibodies (12µg 4G10 (Millipore),12ug PT66 (Sigma), and 12ugPY100 (CST)) in 200ml IP Buffer. ..

    Incubation:

    Article Title: MARQUIS: A Multiplex Method for Absolute Quantification of Peptides and Post-Translational Modifications
    Article Snippet: .. Immunoprecipitation 70ul protein G agarose beads (calbiochem IP08) were rinsed in 400ul IP Buffer (100mM tris, 0.3% NP-40, pH 7.4) prior to an 8 hour incubation with a cocktail of three phosphotyrosine-specific antibodies (12µg 4G10 (Millipore),12ug PT66 (Sigma), and 12ugPY100 (CST)) in 200ml IP Buffer. ..

    Article Title: Modulatory Effects of a Novel Cyclized Peptide in Reducing the Expression of Markers Linked to Alzheimer's Disease
    Article Snippet: .. The next day, the membranes were rinsed 5 times for 5 min with TBST and incubated at RT for one h with gentle agitation with secondary antibodies both HRP conjugated, goat anti-mouse (1:2,000) (Sigma-Aldrich, A9309, Germany) and goat anti-rabbit (1:5,000) (Abcam, ab6721, UK). ..

    Article Title: Mesenchyme homeobox 1 mediates transforming growth factor-β (TGF-β)–induced smooth muscle cell differentiation from mouse mesenchymal progenitors
    Article Snippet: .. Then, HRP-conjugated or immunofluorescent secondary antibodies were incubated with the membranes for 1 h. The protein expression levels were detected with enhanced chemiluminescence (EMD Millipore) or scanned by Odyssey fluorescence scanner (LI-COR Biosciences) ( ). .. α-SMA or SM22α luciferase promoter constructs were transfected into 10T1/2 cells with or without other plasmids using Lipofectamine LTX (Life Technologies) as described previously ( ).

    Expressing:

    Article Title: Mesenchyme homeobox 1 mediates transforming growth factor-β (TGF-β)–induced smooth muscle cell differentiation from mouse mesenchymal progenitors
    Article Snippet: .. Then, HRP-conjugated or immunofluorescent secondary antibodies were incubated with the membranes for 1 h. The protein expression levels were detected with enhanced chemiluminescence (EMD Millipore) or scanned by Odyssey fluorescence scanner (LI-COR Biosciences) ( ). .. α-SMA or SM22α luciferase promoter constructs were transfected into 10T1/2 cells with or without other plasmids using Lipofectamine LTX (Life Technologies) as described previously ( ).

    Fluorescence:

    Article Title: Mesenchyme homeobox 1 mediates transforming growth factor-β (TGF-β)–induced smooth muscle cell differentiation from mouse mesenchymal progenitors
    Article Snippet: .. Then, HRP-conjugated or immunofluorescent secondary antibodies were incubated with the membranes for 1 h. The protein expression levels were detected with enhanced chemiluminescence (EMD Millipore) or scanned by Odyssey fluorescence scanner (LI-COR Biosciences) ( ). .. α-SMA or SM22α luciferase promoter constructs were transfected into 10T1/2 cells with or without other plasmids using Lipofectamine LTX (Life Technologies) as described previously ( ).

    Western Blot:

    Article Title: Stimulation of Murine Intestinal Secretion by Daily Genistein Injections: Gender-dependent Differences
    Article Snippet: .. Western blot antibodies: mAb to CFTR [CF3] (Abcam, Cambridge, MA), anti-mouse IgG, HRP conjugated (Upstate/Millipore, Temecula, CA), Anti-Actin (Millipore, Temecula, CA) and anti-mouse IgG, HRP conjugated (Upstate/ Millipore, Temecula, CA). ..

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  • 99
    Millipore horseradish peroxidase hrp conjugated anti human igg secondary antibody
    Binding of T-DM1 to cell surface CKAP5 is independent of HER2 ( A ) The levels of endogenous HER2 and CKAP5 in the WCL of the indicated cell lines analyzed by Western blot. ( B and C ) Binding of trastuzumab and T-DM1 to the breast cancer cells overexpressing HER2 ( B ) or cells expressing low levels of HER2 ( C ). The indicated cell lines were incubated with 100 μg/ml trastuzumab or T-DM1 in the culture media either with or without serum at 37°C for 1 hr. Amount of trastuzumab and T-DM1 in WCL were determined by Western blot analysis using anti-human <t>IgG</t> conjugated with <t>HRP.</t> ( D ) T-DM1 is not co-localized with LAMP-1 in THLE2 cells. THLE2 cells were incubated with 100 μg/ml trastuzumab or T-DM1 for 1 hour at 37°C, and subsequently were fixed for fluorescent immunostaining of trastuzumab or T-DM1 (green) and LAMP-1 (red). Nucleus was stained with DAPI (blue). Images were taken by a Zeiss LSM880 confocal microscope. Scale bar, 20 μm. ( E ) T-DM1 is co-localities with LAMP-1 in JIMT1 cells. JIMT1 cells were incubated with 100 μg/ml T-DM1 for 1 hour at 37°C, and subsequently were fixed for fluorescent immunostaining of T-DM1 (green) and LAMP-1 (red). Nucleus was stained with DAPI (blue). Images were taken by a Zeiss LSM880 confocal microscope. Scale bar, 20 μm. ( F ) Bright field images showing cytoplasmic vacuolization induced by T-DM1 in MDA-MB-468 cells that do not express HER2. Scale bar, 40 μm. ( G ) Cell growth profiles of MDA-MB-468 cells. Cells were treated with indicated dose. Specifically, 2.5 × 10 4 cells/well were seeded in 12-well plates and incubated in the cell culture media containing either 5 μg/ml or 10 μg/ml of T-DM1. On the indicated days, the cells were trypsinized and counted.
    Horseradish Peroxidase Hrp Conjugated Anti Human Igg Secondary Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/horseradish peroxidase hrp conjugated anti human igg secondary antibody/product/Millipore
    Average 99 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    horseradish peroxidase hrp conjugated anti human igg secondary antibody - by Bioz Stars, 2020-07
    99/100 stars
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    98
    Millipore hrp conjugated streptavidin
    Discovery and validation of SLRP–Hsp47 interactions. A , far-Western blotting to detect potential intracellular SLRP interactants. Lysates of chondrocyte-like ATDC5 cells were run on SDS-PAGE, and the proteins were transferred onto a nitrocellulose membrane. Membrane strips were blocked with BSA and incubated with biotinylated proteins: BSA (negative control), decorin (DCN), fibromodulin (FM), or lumican (LUM). Binding was detected with <t>streptavidin-HRP</t> and peroxidase substrate. The arrow marks a presumed common interactant of DCN, FM, and LUM. The corresponding region was cut out from the SDS-polyacrylamide gel and analyzed by MS. B , co-immunoprecipitation assays to detect potential SLRP–Hsp47 interactions. Lysates from ATDC5 cells were used alone (−) or mixed with 1 μg of recombinant DCN, His-tagged FM, or His-tagged LUM. The lysates were incubated with decorin rabbit anti-serum ( IP : DCN ) or mouse anti-His antibodies ( IP : his-tag ) and with Protein A– or Protein G–Sepharose, respectively. Co-precipitated proteins were run on SDS-PAGE and immunoblotted for Hsp47 ( WB : HSP47 ). The capture of SLRPs was validated with the corresponding antisera ( WB : DCN , FM , LUM ). C , pulldown assays to validate SLRP–Hsp47 interactions. Magnetic beads were coated with BSA or Hsp47 (bait) and incubated with biotinylated DCN, FM, or LUM (all prey). The beads were washed, and the eluates were run on SDS-PAGE. The binding was detected by blotting with streptavidin-HRP.
    Hrp Conjugated Streptavidin, supplied by Millipore, used in various techniques. Bioz Stars score: 98/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hrp conjugated streptavidin/product/Millipore
    Average 98 stars, based on 36 article reviews
    Price from $9.99 to $1999.99
    hrp conjugated streptavidin - by Bioz Stars, 2020-07
    98/100 stars
      Buy from Supplier

    94
    Millipore hrp conjugated secondary antibodies
    Discovery and validation of SLRP–Hsp47 interactions. A , far-Western blotting to detect potential intracellular SLRP interactants. Lysates of chondrocyte-like ATDC5 cells were run on SDS-PAGE, and the proteins were transferred onto a nitrocellulose membrane. Membrane strips were blocked with BSA and incubated with biotinylated proteins: BSA (negative control), decorin (DCN), fibromodulin (FM), or lumican (LUM). Binding was detected with <t>streptavidin-HRP</t> and peroxidase substrate. The arrow marks a presumed common interactant of DCN, FM, and LUM. The corresponding region was cut out from the SDS-polyacrylamide gel and analyzed by MS. B , co-immunoprecipitation assays to detect potential SLRP–Hsp47 interactions. Lysates from ATDC5 cells were used alone (−) or mixed with 1 μg of recombinant DCN, His-tagged FM, or His-tagged LUM. The lysates were incubated with decorin rabbit anti-serum ( IP : DCN ) or mouse anti-His antibodies ( IP : his-tag ) and with Protein A– or Protein G–Sepharose, respectively. Co-precipitated proteins were run on SDS-PAGE and immunoblotted for Hsp47 ( WB : HSP47 ). The capture of SLRPs was validated with the corresponding antisera ( WB : DCN , FM , LUM ). C , pulldown assays to validate SLRP–Hsp47 interactions. Magnetic beads were coated with BSA or Hsp47 (bait) and incubated with biotinylated DCN, FM, or LUM (all prey). The beads were washed, and the eluates were run on SDS-PAGE. The binding was detected by blotting with streptavidin-HRP.
    Hrp Conjugated Secondary Antibodies, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 556 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hrp conjugated secondary antibodies/product/Millipore
    Average 94 stars, based on 556 article reviews
    Price from $9.99 to $1999.99
    hrp conjugated secondary antibodies - by Bioz Stars, 2020-07
    94/100 stars
      Buy from Supplier

    Image Search Results


    Binding of T-DM1 to cell surface CKAP5 is independent of HER2 ( A ) The levels of endogenous HER2 and CKAP5 in the WCL of the indicated cell lines analyzed by Western blot. ( B and C ) Binding of trastuzumab and T-DM1 to the breast cancer cells overexpressing HER2 ( B ) or cells expressing low levels of HER2 ( C ). The indicated cell lines were incubated with 100 μg/ml trastuzumab or T-DM1 in the culture media either with or without serum at 37°C for 1 hr. Amount of trastuzumab and T-DM1 in WCL were determined by Western blot analysis using anti-human IgG conjugated with HRP. ( D ) T-DM1 is not co-localized with LAMP-1 in THLE2 cells. THLE2 cells were incubated with 100 μg/ml trastuzumab or T-DM1 for 1 hour at 37°C, and subsequently were fixed for fluorescent immunostaining of trastuzumab or T-DM1 (green) and LAMP-1 (red). Nucleus was stained with DAPI (blue). Images were taken by a Zeiss LSM880 confocal microscope. Scale bar, 20 μm. ( E ) T-DM1 is co-localities with LAMP-1 in JIMT1 cells. JIMT1 cells were incubated with 100 μg/ml T-DM1 for 1 hour at 37°C, and subsequently were fixed for fluorescent immunostaining of T-DM1 (green) and LAMP-1 (red). Nucleus was stained with DAPI (blue). Images were taken by a Zeiss LSM880 confocal microscope. Scale bar, 20 μm. ( F ) Bright field images showing cytoplasmic vacuolization induced by T-DM1 in MDA-MB-468 cells that do not express HER2. Scale bar, 40 μm. ( G ) Cell growth profiles of MDA-MB-468 cells. Cells were treated with indicated dose. Specifically, 2.5 × 10 4 cells/well were seeded in 12-well plates and incubated in the cell culture media containing either 5 μg/ml or 10 μg/ml of T-DM1. On the indicated days, the cells were trypsinized and counted.

    Journal: Oncotarget

    Article Title: Payload of T-DM1 binds to cell surface cytoskeleton-associated protein 5 to mediate cytotoxicity of hepatocytes

    doi: 10.18632/oncotarget.26461

    Figure Lengend Snippet: Binding of T-DM1 to cell surface CKAP5 is independent of HER2 ( A ) The levels of endogenous HER2 and CKAP5 in the WCL of the indicated cell lines analyzed by Western blot. ( B and C ) Binding of trastuzumab and T-DM1 to the breast cancer cells overexpressing HER2 ( B ) or cells expressing low levels of HER2 ( C ). The indicated cell lines were incubated with 100 μg/ml trastuzumab or T-DM1 in the culture media either with or without serum at 37°C for 1 hr. Amount of trastuzumab and T-DM1 in WCL were determined by Western blot analysis using anti-human IgG conjugated with HRP. ( D ) T-DM1 is not co-localized with LAMP-1 in THLE2 cells. THLE2 cells were incubated with 100 μg/ml trastuzumab or T-DM1 for 1 hour at 37°C, and subsequently were fixed for fluorescent immunostaining of trastuzumab or T-DM1 (green) and LAMP-1 (red). Nucleus was stained with DAPI (blue). Images were taken by a Zeiss LSM880 confocal microscope. Scale bar, 20 μm. ( E ) T-DM1 is co-localities with LAMP-1 in JIMT1 cells. JIMT1 cells were incubated with 100 μg/ml T-DM1 for 1 hour at 37°C, and subsequently were fixed for fluorescent immunostaining of T-DM1 (green) and LAMP-1 (red). Nucleus was stained with DAPI (blue). Images were taken by a Zeiss LSM880 confocal microscope. Scale bar, 20 μm. ( F ) Bright field images showing cytoplasmic vacuolization induced by T-DM1 in MDA-MB-468 cells that do not express HER2. Scale bar, 40 μm. ( G ) Cell growth profiles of MDA-MB-468 cells. Cells were treated with indicated dose. Specifically, 2.5 × 10 4 cells/well were seeded in 12-well plates and incubated in the cell culture media containing either 5 μg/ml or 10 μg/ml of T-DM1. On the indicated days, the cells were trypsinized and counted.

    Article Snippet: The WCL was subjected to Western blot analysis to determine the amounts of trastuzumab and T-DM1 using horseradish peroxidase (HRP) conjugated anti-human IgG secondary antibody (EMD Millipore).

    Techniques: Binding Assay, Western Blot, Expressing, Incubation, Immunostaining, Staining, Microscopy, Multiple Displacement Amplification, Cell Culture

    Binding of T-DM1 to cell surface CKAP5 is independent of HER2 ( A ) The levels of endogenous HER2 and CKAP5 in the WCL of the indicated cell lines analyzed by Western blot. ( B and C ) Binding of trastuzumab and T-DM1 to the breast cancer cells overexpressing HER2 ( B ) or cells expressing low levels of HER2 ( C ). The indicated cell lines were incubated with 100 μg/ml trastuzumab or T-DM1 in the culture media either with or without serum at 37°C for 1 hr. Amount of trastuzumab and T-DM1 in WCL were determined by Western blot analysis using anti-human IgG conjugated with HRP. ( D ) T-DM1 is not co-localized with LAMP-1 in THLE2 cells. THLE2 cells were incubated with 100 μg/ml trastuzumab or T-DM1 for 1 hour at 37°C, and subsequently were fixed for fluorescent immunostaining of trastuzumab or T-DM1 (green) and LAMP-1 (red). Nucleus was stained with DAPI (blue). Images were taken by a Zeiss LSM880 confocal microscope. Scale bar, 20 μm. ( E ) T-DM1 is co-localities with LAMP-1 in JIMT1 cells. JIMT1 cells were incubated with 100 μg/ml T-DM1 for 1 hour at 37°C, and subsequently were fixed for fluorescent immunostaining of T-DM1 (green) and LAMP-1 (red). Nucleus was stained with DAPI (blue). Images were taken by a Zeiss LSM880 confocal microscope. Scale bar, 20 μm. ( F ) Bright field images showing cytoplasmic vacuolization induced by T-DM1 in MDA-MB-468 cells that do not express HER2. Scale bar, 40 μm. ( G ) Cell growth profiles of MDA-MB-468 cells. Cells were treated with indicated dose. Specifically, 2.5 × 10 4 cells/well were seeded in 12-well plates and incubated in the cell culture media containing either 5 μg/ml or 10 μg/ml of T-DM1. On the indicated days, the cells were trypsinized and counted.

    Journal: Oncotarget

    Article Title: Payload of T-DM1 binds to cell surface cytoskeleton-associated protein 5 to mediate cytotoxicity of hepatocytes

    doi: 10.18632/oncotarget.26461

    Figure Lengend Snippet: Binding of T-DM1 to cell surface CKAP5 is independent of HER2 ( A ) The levels of endogenous HER2 and CKAP5 in the WCL of the indicated cell lines analyzed by Western blot. ( B and C ) Binding of trastuzumab and T-DM1 to the breast cancer cells overexpressing HER2 ( B ) or cells expressing low levels of HER2 ( C ). The indicated cell lines were incubated with 100 μg/ml trastuzumab or T-DM1 in the culture media either with or without serum at 37°C for 1 hr. Amount of trastuzumab and T-DM1 in WCL were determined by Western blot analysis using anti-human IgG conjugated with HRP. ( D ) T-DM1 is not co-localized with LAMP-1 in THLE2 cells. THLE2 cells were incubated with 100 μg/ml trastuzumab or T-DM1 for 1 hour at 37°C, and subsequently were fixed for fluorescent immunostaining of trastuzumab or T-DM1 (green) and LAMP-1 (red). Nucleus was stained with DAPI (blue). Images were taken by a Zeiss LSM880 confocal microscope. Scale bar, 20 μm. ( E ) T-DM1 is co-localities with LAMP-1 in JIMT1 cells. JIMT1 cells were incubated with 100 μg/ml T-DM1 for 1 hour at 37°C, and subsequently were fixed for fluorescent immunostaining of T-DM1 (green) and LAMP-1 (red). Nucleus was stained with DAPI (blue). Images were taken by a Zeiss LSM880 confocal microscope. Scale bar, 20 μm. ( F ) Bright field images showing cytoplasmic vacuolization induced by T-DM1 in MDA-MB-468 cells that do not express HER2. Scale bar, 40 μm. ( G ) Cell growth profiles of MDA-MB-468 cells. Cells were treated with indicated dose. Specifically, 2.5 × 10 4 cells/well were seeded in 12-well plates and incubated in the cell culture media containing either 5 μg/ml or 10 μg/ml of T-DM1. On the indicated days, the cells were trypsinized and counted.

    Article Snippet: The WCL was subjected to Western blot analysis to determine the amounts of trastuzumab and T-DM1 using horseradish peroxidase (HRP) conjugated anti-human IgG secondary antibody (EMD Millipore).

    Techniques: Binding Assay, Western Blot, Expressing, Incubation, Immunostaining, Staining, Microscopy, Multiple Displacement Amplification, Cell Culture

    CKAP5 localizes on the cell surface and is targeted by T-DM1 ( A ) Fluorescence immunostaining showing that CKAP5 is co-localized with actin at the cell edges and concentrated at lamellipodia (white arrows) in THLE2 cells. The cells were permeabilized for immunostaining. Scale bar, 20 μm. ( B , C and D ) Fluorescence immunostaining showing membrane staining of CKAP5 and co-localization of CKAP5 with actin ( B ), PI(3)P ( C ) and PI(4,5)P2 ( D ) on membrane ruffling (white arrows) in THLE2 cells. The cells were not permeabilized for immunostaining. Scale bar, 20 μm. ( E ) Subcellular fractionation of THLE2 cells shows that CKAP5 is found in cytosol, organelle and plasma membrane. ( F ) Knock-down efficiency of CKAP5 expression is evaluated on Western blot. CKAP5 protein expression was about 73% decreased in CKAP5 siRNA-treated THLE2 cells compared with that of control siRNA-treated THLE2 cells. ( G ) Flow cytometric histogram showing that CKAP5 is detected on THLE2 cell surface, and THLE2 cells, in which CKAP5 expression is silenced by siRNA, have lower level of CKAP5 expression than that of control cells. ( H ) Immunostaining of membrane bound T-DM1 and its co-localization with CKAP5 at lamellipodia (red arrowheads) in THLE2 cells. Scale bar, 20 μm. ( I ) T-DM1 binds to CKAP5 from the plasma membrane fraction of THLE2 cells. After incubating 250 μg/ml trastuzumab and T-DM1 with THLE2 cells in HBSS at RT for 1 hour, cell surface proteins were cross-linked by DTSSP and the WCLs were subjected to subcellular fractionation into cytosol and plasma membrane (PM) fraction. PM fraction was solubilized and then subjected to immunoprecipitation. ( J ) T-DM1 specifically binds to the cell surface. The cells were first incubated with 100 μg/ml trastuzumab or T-DM1 in the culture media at 37°C for 1 hr. The cells were then washed with either PBS or low pH stripping buffer containing 0.2 M acetic acid / 0.5 M NaCl (pH 3.0 or 4.0) for 15 secs. Amount of cell-bound trastuzumab and T-DM1 in WCL was detected by Western blot analysis using anti-human IgG conjugated with HRP. ( K ) Amount of cell-bound T-DM1 is examined in WCL of CHO-K1 cells transfected with either control siRNA or CKAP5 siRNA. The experimental procedures are essentially the same as that described in Figure 2J , except that the cells were transfected with indicated siRNA and were not treated with low pH buffer.

    Journal: Oncotarget

    Article Title: Payload of T-DM1 binds to cell surface cytoskeleton-associated protein 5 to mediate cytotoxicity of hepatocytes

    doi: 10.18632/oncotarget.26461

    Figure Lengend Snippet: CKAP5 localizes on the cell surface and is targeted by T-DM1 ( A ) Fluorescence immunostaining showing that CKAP5 is co-localized with actin at the cell edges and concentrated at lamellipodia (white arrows) in THLE2 cells. The cells were permeabilized for immunostaining. Scale bar, 20 μm. ( B , C and D ) Fluorescence immunostaining showing membrane staining of CKAP5 and co-localization of CKAP5 with actin ( B ), PI(3)P ( C ) and PI(4,5)P2 ( D ) on membrane ruffling (white arrows) in THLE2 cells. The cells were not permeabilized for immunostaining. Scale bar, 20 μm. ( E ) Subcellular fractionation of THLE2 cells shows that CKAP5 is found in cytosol, organelle and plasma membrane. ( F ) Knock-down efficiency of CKAP5 expression is evaluated on Western blot. CKAP5 protein expression was about 73% decreased in CKAP5 siRNA-treated THLE2 cells compared with that of control siRNA-treated THLE2 cells. ( G ) Flow cytometric histogram showing that CKAP5 is detected on THLE2 cell surface, and THLE2 cells, in which CKAP5 expression is silenced by siRNA, have lower level of CKAP5 expression than that of control cells. ( H ) Immunostaining of membrane bound T-DM1 and its co-localization with CKAP5 at lamellipodia (red arrowheads) in THLE2 cells. Scale bar, 20 μm. ( I ) T-DM1 binds to CKAP5 from the plasma membrane fraction of THLE2 cells. After incubating 250 μg/ml trastuzumab and T-DM1 with THLE2 cells in HBSS at RT for 1 hour, cell surface proteins were cross-linked by DTSSP and the WCLs were subjected to subcellular fractionation into cytosol and plasma membrane (PM) fraction. PM fraction was solubilized and then subjected to immunoprecipitation. ( J ) T-DM1 specifically binds to the cell surface. The cells were first incubated with 100 μg/ml trastuzumab or T-DM1 in the culture media at 37°C for 1 hr. The cells were then washed with either PBS or low pH stripping buffer containing 0.2 M acetic acid / 0.5 M NaCl (pH 3.0 or 4.0) for 15 secs. Amount of cell-bound trastuzumab and T-DM1 in WCL was detected by Western blot analysis using anti-human IgG conjugated with HRP. ( K ) Amount of cell-bound T-DM1 is examined in WCL of CHO-K1 cells transfected with either control siRNA or CKAP5 siRNA. The experimental procedures are essentially the same as that described in Figure 2J , except that the cells were transfected with indicated siRNA and were not treated with low pH buffer.

    Article Snippet: The WCL was subjected to Western blot analysis to determine the amounts of trastuzumab and T-DM1 using horseradish peroxidase (HRP) conjugated anti-human IgG secondary antibody (EMD Millipore).

    Techniques: Fluorescence, Immunostaining, Staining, Fractionation, Expressing, Western Blot, Flow Cytometry, Immunoprecipitation, Incubation, Stripping Membranes, Transfection

    Discovery and validation of SLRP–Hsp47 interactions. A , far-Western blotting to detect potential intracellular SLRP interactants. Lysates of chondrocyte-like ATDC5 cells were run on SDS-PAGE, and the proteins were transferred onto a nitrocellulose membrane. Membrane strips were blocked with BSA and incubated with biotinylated proteins: BSA (negative control), decorin (DCN), fibromodulin (FM), or lumican (LUM). Binding was detected with streptavidin-HRP and peroxidase substrate. The arrow marks a presumed common interactant of DCN, FM, and LUM. The corresponding region was cut out from the SDS-polyacrylamide gel and analyzed by MS. B , co-immunoprecipitation assays to detect potential SLRP–Hsp47 interactions. Lysates from ATDC5 cells were used alone (−) or mixed with 1 μg of recombinant DCN, His-tagged FM, or His-tagged LUM. The lysates were incubated with decorin rabbit anti-serum ( IP : DCN ) or mouse anti-His antibodies ( IP : his-tag ) and with Protein A– or Protein G–Sepharose, respectively. Co-precipitated proteins were run on SDS-PAGE and immunoblotted for Hsp47 ( WB : HSP47 ). The capture of SLRPs was validated with the corresponding antisera ( WB : DCN , FM , LUM ). C , pulldown assays to validate SLRP–Hsp47 interactions. Magnetic beads were coated with BSA or Hsp47 (bait) and incubated with biotinylated DCN, FM, or LUM (all prey). The beads were washed, and the eluates were run on SDS-PAGE. The binding was detected by blotting with streptavidin-HRP.

    Journal: The Journal of Biological Chemistry

    Article Title: The endoplasmic reticulum–resident collagen chaperone Hsp47 interacts with and promotes the secretion of decorin, fibromodulin, and lumican

    doi: 10.1074/jbc.RA117.000758

    Figure Lengend Snippet: Discovery and validation of SLRP–Hsp47 interactions. A , far-Western blotting to detect potential intracellular SLRP interactants. Lysates of chondrocyte-like ATDC5 cells were run on SDS-PAGE, and the proteins were transferred onto a nitrocellulose membrane. Membrane strips were blocked with BSA and incubated with biotinylated proteins: BSA (negative control), decorin (DCN), fibromodulin (FM), or lumican (LUM). Binding was detected with streptavidin-HRP and peroxidase substrate. The arrow marks a presumed common interactant of DCN, FM, and LUM. The corresponding region was cut out from the SDS-polyacrylamide gel and analyzed by MS. B , co-immunoprecipitation assays to detect potential SLRP–Hsp47 interactions. Lysates from ATDC5 cells were used alone (−) or mixed with 1 μg of recombinant DCN, His-tagged FM, or His-tagged LUM. The lysates were incubated with decorin rabbit anti-serum ( IP : DCN ) or mouse anti-His antibodies ( IP : his-tag ) and with Protein A– or Protein G–Sepharose, respectively. Co-precipitated proteins were run on SDS-PAGE and immunoblotted for Hsp47 ( WB : HSP47 ). The capture of SLRPs was validated with the corresponding antisera ( WB : DCN , FM , LUM ). C , pulldown assays to validate SLRP–Hsp47 interactions. Magnetic beads were coated with BSA or Hsp47 (bait) and incubated with biotinylated DCN, FM, or LUM (all prey). The beads were washed, and the eluates were run on SDS-PAGE. The binding was detected by blotting with streptavidin-HRP.

    Article Snippet: Then the membrane was incubated with HRP-conjugated streptavidin diluted 1:50,000 in blocking buffer and incubated for 1 h, washed 3 × 10 min in TBST, and developed using Luminata forte (Millipore) and a CCD camera.

    Techniques: Far Western Blot, SDS Page, Incubation, Negative Control, Binding Assay, Mass Spectrometry, Immunoprecipitation, Recombinant, Western Blot, Magnetic Beads