peroxidase conjugated secondary antibodies  (Millipore)


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    Structured Review

    Millipore peroxidase conjugated secondary antibodies
    Peroxidase Conjugated Secondary Antibodies, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 120 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/peroxidase conjugated secondary antibodies/product/Millipore
    Average 99 stars, based on 120 article reviews
    Price from $9.99 to $1999.99
    peroxidase conjugated secondary antibodies - by Bioz Stars, 2020-07
    99/100 stars

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    Western Blot:

    Article Title: Identification and characterization of the first endogenous phospholipase A2 inhibitor from a non-venomous tropical snake,Boa constrictor (Serpentes: Boidae)
    Article Snippet: .. Western blotting was revealed with rabbit anti-CNF IgG (0.5 mg/mL), followed by commercial anti-rabbit IgG-peroxidase antibody (A0545, Sigma) at a 1:5000 dilution. ..

    Incubation:

    Article Title: DNA Subtraction of In Vivo Selected Phage Repertoires for Efficient Peptide Pathology Biomarker Identification in Neuroinflammation Multiple Sclerosis Model
    Article Snippet: .. After five washes with PBS, they were incubated with goat antirabbit horseradish peroxidase-labeled antibody (1:1,000; Sigma-Aldrich A0545) in TBS-BSA (30 minutes at RT). .. After intensive washing, the binding of phages to protein extracts on the dot-blot membranes was revealed by 4-chloro-1-naphthol (Sigma-Aldrich) in TBS in the presence of 0.3% H2 O2 .

    other:

    Article Title: SCFFBW7-mediated degradation of Brg1 suppresses gastric cancer metastasis
    Article Snippet: Polyclonal anti-FLAG antibody (F2425, 1:1000), monoclonal anti-FLAG antibody (F-3165, 1:1000), anti-Vinculin antibody (V9131, 1:5000), peroxidase-conjugated anti-mouse secondary antibody (A4416, 1:3000), peroxidase-conjugated anti-rabbit secondary antibody (A4914, 1:3000) and CK1 inhibitor IC261 were purchased from Sigma.

    Article Title: Systemic antibody response to nano-size calcium phospate biocompatible adjuvant adsorbed HEV-71 killed vaccine
    Article Snippet: Samples collection The blood samples were withdrawn from rabbit ear venous at intervals (0, 1, 3, 5, 7, and 9 weeks); the samples were allowed to clot normally at room temperature for 2 hours, then were centrifuged and serum was collected and preserved at -80℃ for determining of HEV-71 postvaccination IgG and IgM antibodies in an in-house developed using goat anti-rabbit-horseradish peroxidase (HRP) conjugate IgG (A0545, Sigma) and goat anti-rabbit IgM-alkaline phosphate (402004, AbD Serotec, Dusseldorf, Germany).

    Immunofluorescence:

    Article Title: Recruitment of VPS33A to HOPS by VPS16 Is Required for Lysosome Fusion with Endosomes and Autophagosomes
    Article Snippet: .. Antibodies The antibodies used were: Polyclonal rabbit anti-VPS18 and polyclonal rabbit anti-VPS33A [both as described ( )], polyclonal rabbit anti-Actin (A2066; Sigma-Aldrich), monoclonal mouse anti-HA (HA.11, MMS-101R; Covance), polyclonal rabbit anti-GFP , monoclonal mouse anti-LC3 (4E12, M152-3, MBL, used for immunofluorescence staining), polyclonal HRP conjugated goat-anti-rabbit IgG (A9169; Sigma-Aldrich) and polyclonal HRP conjugated rabbit-anti-mouse IgG (A9044; Sigma-Aldrich). .. We thank Folma Buss and David Tumbarello for HeLaM cells stably expressing mRFP-GFP-LC3, Reiner Schulte and Michal Maj for help with FACS analysis, Sally Gray for technical assistance and David Owen for discussing experiments and critical reading of the manuscript.

    Staining:

    Article Title: Recruitment of VPS33A to HOPS by VPS16 Is Required for Lysosome Fusion with Endosomes and Autophagosomes
    Article Snippet: .. Antibodies The antibodies used were: Polyclonal rabbit anti-VPS18 and polyclonal rabbit anti-VPS33A [both as described ( )], polyclonal rabbit anti-Actin (A2066; Sigma-Aldrich), monoclonal mouse anti-HA (HA.11, MMS-101R; Covance), polyclonal rabbit anti-GFP , monoclonal mouse anti-LC3 (4E12, M152-3, MBL, used for immunofluorescence staining), polyclonal HRP conjugated goat-anti-rabbit IgG (A9169; Sigma-Aldrich) and polyclonal HRP conjugated rabbit-anti-mouse IgG (A9044; Sigma-Aldrich). .. We thank Folma Buss and David Tumbarello for HeLaM cells stably expressing mRFP-GFP-LC3, Reiner Schulte and Michal Maj for help with FACS analysis, Sally Gray for technical assistance and David Owen for discussing experiments and critical reading of the manuscript.

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    Millipore anti hcn1
    Effects of the ethanol withdrawal procedure on the hyperpolarization-activated cyclic nucleotide-gated cation channel <t>(HCN1)</t> and brain-derived neurotrophic factor (BDNF) protein or gene level changes in the nucleus accumbens (NAc). (A) The expression of BDNF BDNF mRNA in the NAc. (B) The expression of BDNF-positive cells in the NAc. (C) Expression of BDNF positive cells in the NAc. (D) The number of BDNF-positive cells in the Hip. (E) The expression of HCN1 mRNA in the NAc. (F) The expression of HCN1-positive cells in the NAc. (G) Expression of HCN1 positive cells in the NAc. (H) The number of HCN1-positive cells in the NAc. The BDNF- and HCN1-positive cells in the NAc are represented by photomicrographs (400×). Data are mean ± standard error. * P
    Anti Hcn1, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti hcn1/product/Millipore
    Average 95 stars, based on 3 article reviews
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    anti hcn1 - by Bioz Stars, 2020-07
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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 218 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 218 article reviews
    Price from $9.99 to $1999.99
    horseradish peroxidase hrp conjugated anti human igg secondary antibody - by Bioz Stars, 2020-07
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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
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    Image Search Results


    Effects of the ethanol withdrawal procedure on the hyperpolarization-activated cyclic nucleotide-gated cation channel (HCN1) and brain-derived neurotrophic factor (BDNF) protein or gene level changes in the nucleus accumbens (NAc). (A) The expression of BDNF BDNF mRNA in the NAc. (B) The expression of BDNF-positive cells in the NAc. (C) Expression of BDNF positive cells in the NAc. (D) The number of BDNF-positive cells in the Hip. (E) The expression of HCN1 mRNA in the NAc. (F) The expression of HCN1-positive cells in the NAc. (G) Expression of HCN1 positive cells in the NAc. (H) The number of HCN1-positive cells in the NAc. The BDNF- and HCN1-positive cells in the NAc are represented by photomicrographs (400×). Data are mean ± standard error. * P

    Journal: Frontiers in Psychiatry

    Article Title: Synaptic Ultrastructure Might Be Involved in HCN1-Related BDNF mRNA in Withdrawal-Anxiety After Ethanol Dependence

    doi: 10.3389/fpsyt.2018.00215

    Figure Lengend Snippet: Effects of the ethanol withdrawal procedure on the hyperpolarization-activated cyclic nucleotide-gated cation channel (HCN1) and brain-derived neurotrophic factor (BDNF) protein or gene level changes in the nucleus accumbens (NAc). (A) The expression of BDNF BDNF mRNA in the NAc. (B) The expression of BDNF-positive cells in the NAc. (C) Expression of BDNF positive cells in the NAc. (D) The number of BDNF-positive cells in the Hip. (E) The expression of HCN1 mRNA in the NAc. (F) The expression of HCN1-positive cells in the NAc. (G) Expression of HCN1 positive cells in the NAc. (H) The number of HCN1-positive cells in the NAc. The BDNF- and HCN1-positive cells in the NAc are represented by photomicrographs (400×). Data are mean ± standard error. * P

    Article Snippet: The next day, the membrane was washed three times, 10 min each time, with PBS-T and incubated at room temperature with secondary antibodies, including anti-HCN1 [Peroxidase-Conjugated Affinipure Goat Anti-mouse IgG (H + L), 1:200] and anti-BDNF [Peroxidase-Conjugated Affinipure Goat Anti-Rabbit IgG (H + L), 1:200], and reacted with the Immobilon western chemiluminescent HRP substrate (WBKLS0500; Millipore, Bedford, MA, USA) for the color reaction.

    Techniques: Derivative Assay, Expressing

    Effects of the ethanol withdrawal procedure on the hyperpolarization-activated cyclic nucleotide-gated cation channel (HCN1) and brain-derived neurotrophic factor (BDNF) protein or gene level changes in the hippocampus (Hip). (A) The expression of BDNF BDNF mRNA in the Hip. (B) The expression of BDNF-positive cells in the Hip. (C) Expression of BDNF positive cells in the Hip. (D) The number of BDNF-positive cells in the Hip. (E) The expression of HCN1 mRNA in the Hip. (F) The expression of HCN1-positive cells in the Hip. (G) Expression of HCN1 positive cells in Hip. (H) The number of HCN1-positive cells in the Hip. The BDNF- and HCN1-positive cells in the Hip are represented by photomicrographs (400×). Data are mean ± standard error. * P

    Journal: Frontiers in Psychiatry

    Article Title: Synaptic Ultrastructure Might Be Involved in HCN1-Related BDNF mRNA in Withdrawal-Anxiety After Ethanol Dependence

    doi: 10.3389/fpsyt.2018.00215

    Figure Lengend Snippet: Effects of the ethanol withdrawal procedure on the hyperpolarization-activated cyclic nucleotide-gated cation channel (HCN1) and brain-derived neurotrophic factor (BDNF) protein or gene level changes in the hippocampus (Hip). (A) The expression of BDNF BDNF mRNA in the Hip. (B) The expression of BDNF-positive cells in the Hip. (C) Expression of BDNF positive cells in the Hip. (D) The number of BDNF-positive cells in the Hip. (E) The expression of HCN1 mRNA in the Hip. (F) The expression of HCN1-positive cells in the Hip. (G) Expression of HCN1 positive cells in Hip. (H) The number of HCN1-positive cells in the Hip. The BDNF- and HCN1-positive cells in the Hip are represented by photomicrographs (400×). Data are mean ± standard error. * P

    Article Snippet: The next day, the membrane was washed three times, 10 min each time, with PBS-T and incubated at room temperature with secondary antibodies, including anti-HCN1 [Peroxidase-Conjugated Affinipure Goat Anti-mouse IgG (H + L), 1:200] and anti-BDNF [Peroxidase-Conjugated Affinipure Goat Anti-Rabbit IgG (H + L), 1:200], and reacted with the Immobilon western chemiluminescent HRP substrate (WBKLS0500; Millipore, Bedford, MA, USA) for the color reaction.

    Techniques: Derivative Assay, Expressing

    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

    Specificity of synaptic protein antibodies used for sandwich ELISAs. Supernatants from RIPA buffer extracted rat brain were subjected to SDS-PAGE, blotted to PVDF membrane, and probed with monoclonal ( lanes 1–4) and polyclonal ( lanes 5–8) antibodies. Different amounts of total protein were added to each gel. For synaptophysin lanes 1, 2, 5, 6 contain 10 µg and lanes 3, 4, 7, 8 contain 2 µg total protein. For SNAP-25 and PSD-95 lanes 1, 2, 5, 6 contain 15 µg and lanes 3, 4, 7, 8 contain 5 µg total protein. For GFAP lanes 1, 2, 5, 6 contain 1 µg and lanes 3, 4, 7, 8 contain 0.2 µg total protein. Blots were developed using horseradish peroxidase conjugated secondary antibodies with a chemiluminescent substrate. Blots were exposed to film for various lengths of time and developed using standard techniques. Representative data are shown from an experiment that was repeated twice with similar results

    Journal: Experimental brain research. Experimentelle Hirnforschung. Experimentation cerebrale

    Article Title: Panel of synaptic protein ELISAs for evaluating neurological phenotype

    doi: 10.1007/s00221-010-2182-x

    Figure Lengend Snippet: Specificity of synaptic protein antibodies used for sandwich ELISAs. Supernatants from RIPA buffer extracted rat brain were subjected to SDS-PAGE, blotted to PVDF membrane, and probed with monoclonal ( lanes 1–4) and polyclonal ( lanes 5–8) antibodies. Different amounts of total protein were added to each gel. For synaptophysin lanes 1, 2, 5, 6 contain 10 µg and lanes 3, 4, 7, 8 contain 2 µg total protein. For SNAP-25 and PSD-95 lanes 1, 2, 5, 6 contain 15 µg and lanes 3, 4, 7, 8 contain 5 µg total protein. For GFAP lanes 1, 2, 5, 6 contain 1 µg and lanes 3, 4, 7, 8 contain 0.2 µg total protein. Blots were developed using horseradish peroxidase conjugated secondary antibodies with a chemiluminescent substrate. Blots were exposed to film for various lengths of time and developed using standard techniques. Representative data are shown from an experiment that was repeated twice with similar results

    Article Snippet: Blots were developed using horseradish peroxidase conjugated secondary antibodies (Millipore) at the following dilutions: goat anti-mouse IgG 1:3,000, goat anti-rabbit IgG 1:5,000 and rabbit anti-sheep IgG 1:1,000 (Invitrogen) and Super-signal chemiluminescent substrate (Pierce Chemical, Thermo Scientific, Rockford, IL).

    Techniques: SDS Page

    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