anti streptavidin hrp  (Thermo Fisher)


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    Name:
    Streptavidin HRP ELISA GD
    Description:
    Purified from Streptomyces avidinii and provided in a phosphate buffered saline solution The streptavidin avidin biotin staining and detection strategy is used when increased assay sensitivity is desired for a wide variety of systems Format Formulation HRP Conjugate ELISA grade
    Catalog Number:
    snn2004
    Price:
    None
    Applications:
    Antibodies and Secondary Detection|Cell Analysis
    Category:
    Antibodies Secondary Detection Reagents
    Buy from Supplier


    Structured Review

    Thermo Fisher anti streptavidin hrp
    Purified from Streptomyces avidinii and provided in a phosphate buffered saline solution The streptavidin avidin biotin staining and detection strategy is used when increased assay sensitivity is desired for a wide variety of systems Format Formulation HRP Conjugate ELISA grade
    https://www.bioz.com/result/anti streptavidin hrp/product/Thermo Fisher
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti streptavidin hrp - by Bioz Stars, 2020-03
    99/100 stars

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    Related Articles

    Clone Assay:

    Article Title: Epigallocatechin-3-gallate enhances ER stress-induced cancer cell apoptosis by directly targeting PARP16 activity
    Article Snippet: Plasmids, antibodies and other materials PARP16 was cloned into pGEX-4T-1 vector and confirmed by sequencing. .. Anti-phos-PERK (Thr 981) and anti-β -Tubulin antibodies were purchased from Santa-Cruz Biotechnologies (Dallas, TX, USA); anti-eIF2α and anti-phos-eIF2α (Ser 51) antibodies were purchased from Abcam (Cambridge, MA, USA), anti-PARP16 antibody was generated by ourselves; Streptavidin-HRP was obtained from Thermo Fisher (Waltham, MA, USA); BFA, ECG and EGCG were purchased from Sigma-Aldrich (St Louis, MO, USA); TUN was obtained from Cell Signaling Technologies (Beverly, MA, USA); Biotin-labeled NAD+ from Invitrogen (Carlsbad, CA, USA); glutathione Sepharose 4B resin was obtained from GE Healthcare (Pittsburgh, PA, USA).

    Centrifugation:

    Article Title: Ciliary proteins Fap43 and Fap44 interact with each other and are essential for proper cilia and flagella beating
    Article Snippet: After centrifugation (10 min at 21,100× g at 4 °C), the axonemes were resuspended in a lysis buffer (50 mM Tris–HCl, pH 7.4, 500 mM NaCl, 0.4% SDS, 1 mM DTT), incubated at RT for 1 h, and then centrifuged at 8000× g at 4 °C. .. After washing (6 × 5 min with washing buffer: 15 mM Tris–HCl, pH 7.4 150 mM NaCl, 0.1% SDS, 0.3 mM DTT) at 4 °C, resin-bound proteins were analyzed on SDS-PAGE gel, followed by western blot with anti-streptavidin–HRP (Thermo Scientific, Rockford, IL, USA), diluted 1:40,000, or by mass spectrometry (Laboratory of Mass Spectrometry, Institute of Biochemistry and Biophysics, PAS, Warsaw, Poland).

    Immunocytochemistry:

    Article Title: Activity-regulated trafficking of the palmitoyl-acyl transferase DHHC5
    Article Snippet: Antibodies and complementary DNA constructs Primary antibodies used were as follows: δ-catenin (1:500 western blot (WB), 5 μg immunoprecipitation (IP); BD Transduction Laboratories 611536), N-cadherin (1:500; BD Transduction Laboratories 610921), PSD-95 for immunocytochemistry (ICC; 1:500; Abcam ab2723), PSD-95 for IP and WB (5 μg, 1:500; Calbiochem CP35), Gephyrin (1:500; Synaptic Systems 147 011), GFP for IP (10 μl; Synaptic Systems 132 002), GFP for WB (1:1,000; Roche 11814460001), DHHC5 (1:500 ICC, 1:1,000 WB, 1 μg IP; Sigma Prestige HPA014670), TfR (1:500; Millipore GR09L), VPS-35 (1:500; Abnova H000055737-M02), GluA1 (1:1,000; Millipore 05-855R), haemagglutinin (HA) for ICC (1:500; Cell Signaling Technology C29F4), HA for IP and WB (5 μg, 1:500; Covance MMS-101P), VGlut1 (1:500; Millipore AB5905), Fyn for WB and ICC (1:250, 1:500; BD Transduction Laboratories 610163), Fyn for IP (5 μg; Life Technologies MA5-13134), non-phosphorylated Y420 Fyn (1:1,000; Cell Signaling Technologies 2102S), STEP61 (1:1,000; Life Technologies MA1-16746), phospho-tyrosine (phY; 1:1,000 WB, 5 μg IP; Millipore 4G10/05-321), AP2μ (1:500; Thermo Scientific PA5-20745) and β-actin (1:1,000; Sigma A1978). .. Secondary antibodies used were as follows: IgG-horseradish peroxidase (HRP; 1:5,000; BioRad mouse 170-6516 and rabbit 170-6515), IgY-HRP (1:5,000; LifeSpan BioSciences LS-C86499), Streptavidin-HRP (1:5,000; Thermo Scientific 21126) and Alexa-Fluor 568 goat anti-mouse and 633 goat anti-rabbit (1:1,000; Life Technologies A-11004 and A-21070, respectively).

    Mass Spectrometry:

    Article Title: Ciliary proteins Fap43 and Fap44 interact with each other and are essential for proper cilia and flagella beating
    Article Snippet: .. After washing (6 × 5 min with washing buffer: 15 mM Tris–HCl, pH 7.4 150 mM NaCl, 0.1% SDS, 0.3 mM DTT) at 4 °C, resin-bound proteins were analyzed on SDS-PAGE gel, followed by western blot with anti-streptavidin–HRP (Thermo Scientific, Rockford, IL, USA), diluted 1:40,000, or by mass spectrometry (Laboratory of Mass Spectrometry, Institute of Biochemistry and Biophysics, PAS, Warsaw, Poland). .. For the entire cilia proteome, wild-type and mutant cells were deciliated as above, the cilia were collected and washed with 10 mM Tris, pH 7.4, and an equal amount of ciliary protein was run on SDS-PAGE gel and analyzed by mass spectrometry (Laboratory of Mass Spectrometry, Institute of Biochemistry and Biophysics, PAS, Warsaw, Poland).

    Blocking Assay:

    Article Title: Proangiogenic effects of soluble α-Klotho on systemic sclerosis dermal microvascular endothelial cells
    Article Snippet: After blocking non-specific site binding, slides were incubated overnight at 4 °C with rabbit monoclonal anti-human α-Klotho antibody (1:20 dilution; catalog number ab181373; Abcam) diluted in 1% bovine serum albumin (BSA) in PBS. .. Subsequently, the slides were washed three times in PBS and incubated with streptavidin peroxidase (UltraVision Large Volume Detection System Anti-Polyvalent, HRP; LabVision) for 10 minutes at room temperature.

    Article Title: Telocytes are reduced during fibrotic remodelling of the colonic wall in ulcerative colitis
    Article Snippet: After blocking non-specific site binding, slides were incubated overnight at 4°C with mouse monoclonal anti-human CD34 antibody (1:50 dilution; clone QBEnd-10, catalogue no. M7165; Dako, Glostrup, Denmark) diluted in 1% bovine serum albumin (BSA) in PBS. .. Subsequently, the slides were washed three times in PBS and incubated with streptavidin peroxidase (UltraVision Large Volume Detection System Anti-Polyvalent, HRP; LabVision) for 10 min. at room temperature.

    Article Title: Proangiogenic effects of soluble α-Klotho on systemic sclerosis dermal microvascular endothelial cells
    Article Snippet: Slides were then washed, treated with 3% H2 O2 in PBS for 15 minutes at room temperature and subsequently blocked with Ultra V block (UltraVision Large Volume Detection System Anti-Polyvalent, HRP; LabVision) for 10 minutes. .. Cells were incubated overnight at 4 °C with rabbit monoclonal anti-human α-Klotho antibody (catalog number ab181373; Abcam) at 1:20 dilution in 1% BSA in PBS, followed by incubation with biotinylated secondary antibodies and streptavidin peroxidase (UltraVision Large Volume Detection System Anti-Polyvalent, HRP; LabVision) at room temperature.

    Incubation:

    Article Title: Proangiogenic effects of soluble α-Klotho on systemic sclerosis dermal microvascular endothelial cells
    Article Snippet: .. Subsequently, the slides were washed three times in PBS and incubated with streptavidin peroxidase (UltraVision Large Volume Detection System Anti-Polyvalent, HRP; LabVision) for 10 minutes at room temperature. .. Immunoreactivity was developed using 3-amino-9-ethylcarbazole (AEC kit, catalog number TA-125-SA; LabVision) as chromogen.

    Article Title: Ferritin Blocks Inhibitory Effects of Two-Chain High Molecular Weight Kininogen (HKa) on Adhesion and Survival Signaling in Endothelial Cells
    Article Snippet: Following overnight incubation, the cells were either left untreated or treated with HKa, HGK-rich peptide, control peptide or 100 µM PD98059 (Promega) in the presence or absence of ferritin in medium containing 20 ng/ml of bFGF or 62 nM FXII (Haematologic Technologies Inc) and 10 µM ZnCl2 for 24 hours. .. The membranes were blocked in 5% BSA in Tris-buffered-saline containing 0.05% Tween-20, and probed with the following antibodies: anti-phospho-MAPK 44/42, anti-total-MAPK 44/42, anti-phospho-Paxillin Y118, anti-Paxillin, anti- phospho-FAK Y397, anti-FAK, anti-phospho-Akt (Akt S473) (Cell Signaling); anti-GAPDH (Fitzgerald Industries International, Inc.); Streptavidin-HRP (Pierce); and anti-GST (Sigma).

    Article Title: Telocytes are reduced during fibrotic remodelling of the colonic wall in ulcerative colitis
    Article Snippet: .. Subsequently, the slides were washed three times in PBS and incubated with streptavidin peroxidase (UltraVision Large Volume Detection System Anti-Polyvalent, HRP; LabVision) for 10 min. at room temperature. .. Immunoreactivity was developed using 3-amino-9-ethylcarbazole (AEC kit, catalogue no. TA-125-SA; LabVision) as chromogen (brownish-red colour).

    Article Title: Ciliary proteins Fap43 and Fap44 interact with each other and are essential for proper cilia and flagella beating
    Article Snippet: The collected supernatant was diluted with an equal volume of 50 mM Tris–HCl buffer, pH 7.4, and incubated overnight with 100 μl of streptavidin-coupled Dynabeads (Dynabeads™ M-280 Streptavidin, Thermo Fisher Scientific, Waltham, MA, USA) at 4 °C. .. After washing (6 × 5 min with washing buffer: 15 mM Tris–HCl, pH 7.4 150 mM NaCl, 0.1% SDS, 0.3 mM DTT) at 4 °C, resin-bound proteins were analyzed on SDS-PAGE gel, followed by western blot with anti-streptavidin–HRP (Thermo Scientific, Rockford, IL, USA), diluted 1:40,000, or by mass spectrometry (Laboratory of Mass Spectrometry, Institute of Biochemistry and Biophysics, PAS, Warsaw, Poland).

    Article Title: Proangiogenic effects of soluble α-Klotho on systemic sclerosis dermal microvascular endothelial cells
    Article Snippet: .. Cells were incubated overnight at 4 °C with rabbit monoclonal anti-human α-Klotho antibody (catalog number ab181373; Abcam) at 1:20 dilution in 1% BSA in PBS, followed by incubation with biotinylated secondary antibodies and streptavidin peroxidase (UltraVision Large Volume Detection System Anti-Polyvalent, HRP; LabVision) at room temperature. .. Immunoreactivity was developed with 3-amino-9-ethylcarbazole (AEC kit; LabVision).

    Article Title: TRPC3 Activation by Erythropoietin Is Modulated by TRPC6
    Article Snippet: .. Blots were incubated with anti-TRPC3 (1:400, Alomone Labs, Jerusalem, Israel), anti-TRPC3 (C) targeted to the human C-terminal sequence RRRRLQDIEMGMGNSKSRLN (1:1000, Bethyl Laboratories, Inc., Montgomery, TX) , anti-TRPC3 (N) targeted to the murine N-terminal sequence LNGDLESAEPLERHGHKASL (1:1000, Bethyl Laboratories, Inc.) , anti-TRPC6 (1:250, Alomone Labs; 1:500, Abcam, Inc., Cambridge, MA), anti-V5-HRP (1:10,000, Invitrogen), anti-FLAG (1:1000, Sigma), anti-PLCγ (1:1000, SC-81, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-Epo-R (1:1000, SC697, Santa Cruz Biotechnology, Inc.), anti-actin (1:10,000, Sigma), anti-tubulin (1:10,000, Sigma) antibodies, or streptavidin-HRP (Pierce). .. Blots were washed and incubated with the appropriate horseradish peroxidase (HRP)-conjugated antibodies (1:2000).

    Activity Assay:

    Article Title: Proangiogenic effects of soluble α-Klotho on systemic sclerosis dermal microvascular endothelial cells
    Article Snippet: Immunohistochemical analysis After deparaffinization and rehydration, skin sections (5 μm thick) were boiled for 10 minutes in sodium citrate buffer (10 mM, pH 6.0) for antigen retrieval and treated with 3% H2 O2 in methanol for 15 minutes at room temperature to block endogenous peroxidase activity. .. Subsequently, the slides were washed three times in PBS and incubated with streptavidin peroxidase (UltraVision Large Volume Detection System Anti-Polyvalent, HRP; LabVision) for 10 minutes at room temperature.

    Article Title: Telocytes are reduced during fibrotic remodelling of the colonic wall in ulcerative colitis
    Article Snippet: After deparaffinization, tissue sections (5 μm thick) were boiled for 10 min. in sodium citrate buffer (10 mM, pH 6.0) for antigen retrieval and treated with 3% H2 O2 in methanol for 15 min. at room temperature to block endogenous peroxidase activity. .. Subsequently, the slides were washed three times in PBS and incubated with streptavidin peroxidase (UltraVision Large Volume Detection System Anti-Polyvalent, HRP; LabVision) for 10 min. at room temperature.

    Article Title: Isolation of secreted proteins from Drosophila ovaries and embryos through in vivo BirA-mediated biotinylation
    Article Snippet: Paragraph title: BirA activity assay ... The biotinylated proteins were detected using streptavidin-HRP (Thermo Scientific) according to Hung et al., 2016 [ ] with the modification of additional rinsing steps and imaged as described above.

    Expressing:

    Article Title: Ciliary proteins Fap43 and Fap44 interact with each other and are essential for proper cilia and flagella beating
    Article Snippet: To identify potential Fap43p or Fap44p-interacting proteins using proximity-dependent labeling, cells expressing either Fap43p–HA–BirA* or Fap44p–HA–BirA* were grown in SPP medium to a density of 2 × 105 cells/ml, starved overnight in 10 mM Tris–HCl buffer, pH 7.5, and incubated in the same buffer supplied with 50 μM biotin for 2–4 h at 30 °C. .. After washing (6 × 5 min with washing buffer: 15 mM Tris–HCl, pH 7.4 150 mM NaCl, 0.1% SDS, 0.3 mM DTT) at 4 °C, resin-bound proteins were analyzed on SDS-PAGE gel, followed by western blot with anti-streptavidin–HRP (Thermo Scientific, Rockford, IL, USA), diluted 1:40,000, or by mass spectrometry (Laboratory of Mass Spectrometry, Institute of Biochemistry and Biophysics, PAS, Warsaw, Poland).

    Article Title: Isolation of secreted proteins from Drosophila ovaries and embryos through in vivo BirA-mediated biotinylation
    Article Snippet: Negative controls consisted of extracts lacking secBirA expression and/or added MBP-AviTag. .. The biotinylated proteins were detected using streptavidin-HRP (Thermo Scientific) according to Hung et al., 2016 [ ] with the modification of additional rinsing steps and imaged as described above.

    BIA-KA:

    Article Title: Ferritin Blocks Inhibitory Effects of Two-Chain High Molecular Weight Kininogen (HKa) on Adhesion and Survival Signaling in Endothelial Cells
    Article Snippet: Cells were lysed in Triton X-100 (TX-100) lysis buffer (50 mM Tris, pH 7.5, 150 mM sodium chloride, 0.5% TX-100) supplemented with protease and phosphatase inhibitor cocktails (Roche) and protein concentration of the clarified samples determined using BCA protein assay kit (Pierce). .. The membranes were blocked in 5% BSA in Tris-buffered-saline containing 0.05% Tween-20, and probed with the following antibodies: anti-phospho-MAPK 44/42, anti-total-MAPK 44/42, anti-phospho-Paxillin Y118, anti-Paxillin, anti- phospho-FAK Y397, anti-FAK, anti-phospho-Akt (Akt S473) (Cell Signaling); anti-GAPDH (Fitzgerald Industries International, Inc.); Streptavidin-HRP (Pierce); and anti-GST (Sigma).

    Modification:

    Article Title: Isolation of secreted proteins from Drosophila ovaries and embryos through in vivo BirA-mediated biotinylation
    Article Snippet: .. The biotinylated proteins were detected using streptavidin-HRP (Thermo Scientific) according to Hung et al., 2016 [ ] with the modification of additional rinsing steps and imaged as described above. .. Visualization of biotinylated proteins in ovarian and embryonic extracts Ovarian and embryonic extracts were generated, and SDS-PAGE gels run and blotted as described above for the BirA Western blot.

    Western Blot:

    Article Title: ?-Catenin Phosphorylated at Serine 45 Is Spatially Uncoupled from ?-Catenin Phosphorylated in the GSK3 Domain: Implications for Signaling
    Article Snippet: .. Lysates were immunoprecipitated with antibodies as indicated, and Western blots were performed using streptavidin-HRP (Invitrogen). .. Phospho-peptide mapping Cytosolic β-catenin was affinity precipitated with 100 µg GST-ICAT from SW480 controls cell lysate (approximately 50 mg total protein) and subjected to SDS-PAGE.

    Article Title: Ciliary proteins Fap43 and Fap44 interact with each other and are essential for proper cilia and flagella beating
    Article Snippet: .. After washing (6 × 5 min with washing buffer: 15 mM Tris–HCl, pH 7.4 150 mM NaCl, 0.1% SDS, 0.3 mM DTT) at 4 °C, resin-bound proteins were analyzed on SDS-PAGE gel, followed by western blot with anti-streptavidin–HRP (Thermo Scientific, Rockford, IL, USA), diluted 1:40,000, or by mass spectrometry (Laboratory of Mass Spectrometry, Institute of Biochemistry and Biophysics, PAS, Warsaw, Poland). .. For the entire cilia proteome, wild-type and mutant cells were deciliated as above, the cilia were collected and washed with 10 mM Tris, pH 7.4, and an equal amount of ciliary protein was run on SDS-PAGE gel and analyzed by mass spectrometry (Laboratory of Mass Spectrometry, Institute of Biochemistry and Biophysics, PAS, Warsaw, Poland).

    Article Title: Activity-regulated trafficking of the palmitoyl-acyl transferase DHHC5
    Article Snippet: Antibodies and complementary DNA constructs Primary antibodies used were as follows: δ-catenin (1:500 western blot (WB), 5 μg immunoprecipitation (IP); BD Transduction Laboratories 611536), N-cadherin (1:500; BD Transduction Laboratories 610921), PSD-95 for immunocytochemistry (ICC; 1:500; Abcam ab2723), PSD-95 for IP and WB (5 μg, 1:500; Calbiochem CP35), Gephyrin (1:500; Synaptic Systems 147 011), GFP for IP (10 μl; Synaptic Systems 132 002), GFP for WB (1:1,000; Roche 11814460001), DHHC5 (1:500 ICC, 1:1,000 WB, 1 μg IP; Sigma Prestige HPA014670), TfR (1:500; Millipore GR09L), VPS-35 (1:500; Abnova H000055737-M02), GluA1 (1:1,000; Millipore 05-855R), haemagglutinin (HA) for ICC (1:500; Cell Signaling Technology C29F4), HA for IP and WB (5 μg, 1:500; Covance MMS-101P), VGlut1 (1:500; Millipore AB5905), Fyn for WB and ICC (1:250, 1:500; BD Transduction Laboratories 610163), Fyn for IP (5 μg; Life Technologies MA5-13134), non-phosphorylated Y420 Fyn (1:1,000; Cell Signaling Technologies 2102S), STEP61 (1:1,000; Life Technologies MA1-16746), phospho-tyrosine (phY; 1:1,000 WB, 5 μg IP; Millipore 4G10/05-321), AP2μ (1:500; Thermo Scientific PA5-20745) and β-actin (1:1,000; Sigma A1978). .. Secondary antibodies used were as follows: IgG-horseradish peroxidase (HRP; 1:5,000; BioRad mouse 170-6516 and rabbit 170-6515), IgY-HRP (1:5,000; LifeSpan BioSciences LS-C86499), Streptavidin-HRP (1:5,000; Thermo Scientific 21126) and Alexa-Fluor 568 goat anti-mouse and 633 goat anti-rabbit (1:1,000; Life Technologies A-11004 and A-21070, respectively).

    Immunohistochemistry:

    Article Title: Proangiogenic effects of soluble α-Klotho on systemic sclerosis dermal microvascular endothelial cells
    Article Snippet: Paragraph title: Immunohistochemical analysis ... Subsequently, the slides were washed three times in PBS and incubated with streptavidin peroxidase (UltraVision Large Volume Detection System Anti-Polyvalent, HRP; LabVision) for 10 minutes at room temperature.

    Article Title: Telocytes are reduced during fibrotic remodelling of the colonic wall in ulcerative colitis
    Article Snippet: Paragraph title: Immunoperoxidase-based immunohistochemistry ... Subsequently, the slides were washed three times in PBS and incubated with streptavidin peroxidase (UltraVision Large Volume Detection System Anti-Polyvalent, HRP; LabVision) for 10 min. at room temperature.

    Concentration Assay:

    Article Title: Proangiogenic effects of soluble α-Klotho on systemic sclerosis dermal microvascular endothelial cells
    Article Snippet: Subsequently, the slides were washed three times in PBS and incubated with streptavidin peroxidase (UltraVision Large Volume Detection System Anti-Polyvalent, HRP; LabVision) for 10 minutes at room temperature. .. Sections not exposed to primary antibodies or incubated with isotype-matched and concentration-matched non-immune rabbit IgG (Sigma-Aldrich, St. Louis, MO, USA) were included as negative controls for antibody specificity.

    Article Title: Telocytes are reduced during fibrotic remodelling of the colonic wall in ulcerative colitis
    Article Snippet: Subsequently, the slides were washed three times in PBS and incubated with streptavidin peroxidase (UltraVision Large Volume Detection System Anti-Polyvalent, HRP; LabVision) for 10 min. at room temperature. .. Sections not exposed to primary antibodies or incubated with isotype-matched and concentration-matched non-immune mouse IgG (Sigma-Aldrich, St. Louis, MO, USA) were included as negative controls for antibody specificity.

    Article Title: Proangiogenic effects of soluble α-Klotho on systemic sclerosis dermal microvascular endothelial cells
    Article Snippet: Cells were incubated overnight at 4 °C with rabbit monoclonal anti-human α-Klotho antibody (catalog number ab181373; Abcam) at 1:20 dilution in 1% BSA in PBS, followed by incubation with biotinylated secondary antibodies and streptavidin peroxidase (UltraVision Large Volume Detection System Anti-Polyvalent, HRP; LabVision) at room temperature. .. Irrelevant isotype-matched and concentration-matched rabbit IgG (Sigma-Aldrich) were used as negative controls.

    Protease Inhibitor:

    Article Title: Isolation of secreted proteins from Drosophila ovaries and embryos through in vivo BirA-mediated biotinylation
    Article Snippet: BirA activity assay Dissected ovaries and dechorionated embryos (0–4 hour old) were homogenized in reaction buffer [(50mM Tris, pH 8.1, 500mM potassium glutamate, 0.1% Tween-20, 1X protease inhibitor cocktail cOmplete, EDTA-free (Roche)], then centrifuged at 13,500 rpm for 15 minutes at 4°C. .. The biotinylated proteins were detected using streptavidin-HRP (Thermo Scientific) according to Hung et al., 2016 [ ] with the modification of additional rinsing steps and imaged as described above.

    Cell Culture:

    Article Title: Interferon-? Activates Transglutaminase 2 via a Phosphatidylinositol-3-Kinase-Dependent Pathway: Implications for Celiac Sprue Therapy S⃞
    Article Snippet: .. Cell culture medium, antibiotics, trypsin-EDTA, sterile PBS, goat anti-rabbit (H-L) and goat anti-mouse (H-L) secondary antibodies, streptavidin Alexa Fluor 647, streptavidin-HRP, and goat anti-mouse HRP were from Invitrogen (Carlsbad, CA). .. Primary antibodies mouse anti-TG2 (CUB 7402 + TG100) and rabbit anti-E-cadherin were from Thermo Fisher Scientific (Waltham, MA) and Cell Signaling Technology (Danvers, MA), respectively.

    Generated:

    Article Title: Epigallocatechin-3-gallate enhances ER stress-induced cancer cell apoptosis by directly targeting PARP16 activity
    Article Snippet: .. Anti-phos-PERK (Thr 981) and anti-β -Tubulin antibodies were purchased from Santa-Cruz Biotechnologies (Dallas, TX, USA); anti-eIF2α and anti-phos-eIF2α (Ser 51) antibodies were purchased from Abcam (Cambridge, MA, USA), anti-PARP16 antibody was generated by ourselves; Streptavidin-HRP was obtained from Thermo Fisher (Waltham, MA, USA); BFA, ECG and EGCG were purchased from Sigma-Aldrich (St Louis, MO, USA); TUN was obtained from Cell Signaling Technologies (Beverly, MA, USA); Biotin-labeled NAD+ from Invitrogen (Carlsbad, CA, USA); glutathione Sepharose 4B resin was obtained from GE Healthcare (Pittsburgh, PA, USA). ..

    Article Title: Activity-regulated trafficking of the palmitoyl-acyl transferase DHHC5
    Article Snippet: Secondary antibodies used were as follows: IgG-horseradish peroxidase (HRP; 1:5,000; BioRad mouse 170-6516 and rabbit 170-6515), IgY-HRP (1:5,000; LifeSpan BioSciences LS-C86499), Streptavidin-HRP (1:5,000; Thermo Scientific 21126) and Alexa-Fluor 568 goat anti-mouse and 633 goat anti-rabbit (1:1,000; Life Technologies A-11004 and A-21070, respectively). .. GFP–δ-catenin and RFP–δ-catenin were generated as previously described .

    other:

    Article Title: Isolation of secreted proteins from Drosophila ovaries and embryos through in vivo BirA-mediated biotinylation
    Article Snippet: Biotinylated proteins were detected using streptavidin-HRP and imaged as described above.

    Protein Concentration:

    Article Title: Ferritin Blocks Inhibitory Effects of Two-Chain High Molecular Weight Kininogen (HKa) on Adhesion and Survival Signaling in Endothelial Cells
    Article Snippet: Cells were lysed in Triton X-100 (TX-100) lysis buffer (50 mM Tris, pH 7.5, 150 mM sodium chloride, 0.5% TX-100) supplemented with protease and phosphatase inhibitor cocktails (Roche) and protein concentration of the clarified samples determined using BCA protein assay kit (Pierce). .. The membranes were blocked in 5% BSA in Tris-buffered-saline containing 0.05% Tween-20, and probed with the following antibodies: anti-phospho-MAPK 44/42, anti-total-MAPK 44/42, anti-phospho-Paxillin Y118, anti-Paxillin, anti- phospho-FAK Y397, anti-FAK, anti-phospho-Akt (Akt S473) (Cell Signaling); anti-GAPDH (Fitzgerald Industries International, Inc.); Streptavidin-HRP (Pierce); and anti-GST (Sigma).

    Sequencing:

    Article Title: Epigallocatechin-3-gallate enhances ER stress-induced cancer cell apoptosis by directly targeting PARP16 activity
    Article Snippet: Plasmids, antibodies and other materials PARP16 was cloned into pGEX-4T-1 vector and confirmed by sequencing. .. Anti-phos-PERK (Thr 981) and anti-β -Tubulin antibodies were purchased from Santa-Cruz Biotechnologies (Dallas, TX, USA); anti-eIF2α and anti-phos-eIF2α (Ser 51) antibodies were purchased from Abcam (Cambridge, MA, USA), anti-PARP16 antibody was generated by ourselves; Streptavidin-HRP was obtained from Thermo Fisher (Waltham, MA, USA); BFA, ECG and EGCG were purchased from Sigma-Aldrich (St Louis, MO, USA); TUN was obtained from Cell Signaling Technologies (Beverly, MA, USA); Biotin-labeled NAD+ from Invitrogen (Carlsbad, CA, USA); glutathione Sepharose 4B resin was obtained from GE Healthcare (Pittsburgh, PA, USA).

    Article Title: TRPC3 Activation by Erythropoietin Is Modulated by TRPC6
    Article Snippet: .. Blots were incubated with anti-TRPC3 (1:400, Alomone Labs, Jerusalem, Israel), anti-TRPC3 (C) targeted to the human C-terminal sequence RRRRLQDIEMGMGNSKSRLN (1:1000, Bethyl Laboratories, Inc., Montgomery, TX) , anti-TRPC3 (N) targeted to the murine N-terminal sequence LNGDLESAEPLERHGHKASL (1:1000, Bethyl Laboratories, Inc.) , anti-TRPC6 (1:250, Alomone Labs; 1:500, Abcam, Inc., Cambridge, MA), anti-V5-HRP (1:10,000, Invitrogen), anti-FLAG (1:1000, Sigma), anti-PLCγ (1:1000, SC-81, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-Epo-R (1:1000, SC697, Santa Cruz Biotechnology, Inc.), anti-actin (1:10,000, Sigma), anti-tubulin (1:10,000, Sigma) antibodies, or streptavidin-HRP (Pierce). .. Blots were washed and incubated with the appropriate horseradish peroxidase (HRP)-conjugated antibodies (1:2000).

    Affinity Purification:

    Article Title: Liver ubiquitome uncovers nutrient-stress-mediated trafficking and secretion of complement C3
    Article Snippet: Tandem affinity purification of ubiquitylated proteins in denaturing conditions (8 M urea) were performed as described in Meierhofer et al. .. The following antibodies were used: anti-ubiquitin (P4D1; Santa Cruz Biotechnology, Dallas, TX, USA), anti-Iκ B (CST), anti-GAPDH (G9545; Sigma Aldrich), streptavidin-HRP (Thermo Fisher Scientific, Waltham, MA, USA), anti-C3a (BD), and anti-p-IRE1α (Thermo Fisher Scientific).

    Binding Assay:

    Article Title: Proangiogenic effects of soluble α-Klotho on systemic sclerosis dermal microvascular endothelial cells
    Article Snippet: After blocking non-specific site binding, slides were incubated overnight at 4 °C with rabbit monoclonal anti-human α-Klotho antibody (1:20 dilution; catalog number ab181373; Abcam) diluted in 1% bovine serum albumin (BSA) in PBS. .. Subsequently, the slides were washed three times in PBS and incubated with streptavidin peroxidase (UltraVision Large Volume Detection System Anti-Polyvalent, HRP; LabVision) for 10 minutes at room temperature.

    Article Title: Telocytes are reduced during fibrotic remodelling of the colonic wall in ulcerative colitis
    Article Snippet: After blocking non-specific site binding, slides were incubated overnight at 4°C with mouse monoclonal anti-human CD34 antibody (1:50 dilution; clone QBEnd-10, catalogue no. M7165; Dako, Glostrup, Denmark) diluted in 1% bovine serum albumin (BSA) in PBS. .. Subsequently, the slides were washed three times in PBS and incubated with streptavidin peroxidase (UltraVision Large Volume Detection System Anti-Polyvalent, HRP; LabVision) for 10 min. at room temperature.

    Article Title: Isolation of secreted proteins from Drosophila ovaries and embryos through in vivo BirA-mediated biotinylation
    Article Snippet: Activity assays were carried out at 37°C for 2 hours in 36 μl of reaction buffer containing 300 μg extracted ovarian/embryonic protein, 0.325 μg/μl Maltose Binding Protein (MBP)-AviTag substrate (Avidity, L.L.C.), 8.3 mM ATP and 42 μM biotin. .. The biotinylated proteins were detected using streptavidin-HRP (Thermo Scientific) according to Hung et al., 2016 [ ] with the modification of additional rinsing steps and imaged as described above.

    Mutagenesis:

    Article Title: Ciliary proteins Fap43 and Fap44 interact with each other and are essential for proper cilia and flagella beating
    Article Snippet: After washing (6 × 5 min with washing buffer: 15 mM Tris–HCl, pH 7.4 150 mM NaCl, 0.1% SDS, 0.3 mM DTT) at 4 °C, resin-bound proteins were analyzed on SDS-PAGE gel, followed by western blot with anti-streptavidin–HRP (Thermo Scientific, Rockford, IL, USA), diluted 1:40,000, or by mass spectrometry (Laboratory of Mass Spectrometry, Institute of Biochemistry and Biophysics, PAS, Warsaw, Poland). .. For the entire cilia proteome, wild-type and mutant cells were deciliated as above, the cilia were collected and washed with 10 mM Tris, pH 7.4, and an equal amount of ciliary protein was run on SDS-PAGE gel and analyzed by mass spectrometry (Laboratory of Mass Spectrometry, Institute of Biochemistry and Biophysics, PAS, Warsaw, Poland).

    Transfection:

    Article Title: TRPC3 Activation by Erythropoietin Is Modulated by TRPC6
    Article Snippet: Blots were incubated with anti-TRPC3 (1:400, Alomone Labs, Jerusalem, Israel), anti-TRPC3 (C) targeted to the human C-terminal sequence RRRRLQDIEMGMGNSKSRLN (1:1000, Bethyl Laboratories, Inc., Montgomery, TX) , anti-TRPC3 (N) targeted to the murine N-terminal sequence LNGDLESAEPLERHGHKASL (1:1000, Bethyl Laboratories, Inc.) , anti-TRPC6 (1:250, Alomone Labs; 1:500, Abcam, Inc., Cambridge, MA), anti-V5-HRP (1:10,000, Invitrogen), anti-FLAG (1:1000, Sigma), anti-PLCγ (1:1000, SC-81, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-Epo-R (1:1000, SC697, Santa Cruz Biotechnology, Inc.), anti-actin (1:10,000, Sigma), anti-tubulin (1:10,000, Sigma) antibodies, or streptavidin-HRP (Pierce). .. To examine the interaction of TRPC3 with TRPC6, PLCγ, or Epo-R, HEK 293T cells were transfected with hTRPC3 (in pcDNA3.1/V5-His TOPO), hTRPC6 (in pCMV tag), TRPC3/TRPC6 chimeras in either vector, hEpo-R (in pcDNA3), rat PLCγ1 (in pcDNA3), or combinations of these vectors.

    Microscopy:

    Article Title: Proangiogenic effects of soluble α-Klotho on systemic sclerosis dermal microvascular endothelial cells
    Article Snippet: Subsequently, the slides were washed three times in PBS and incubated with streptavidin peroxidase (UltraVision Large Volume Detection System Anti-Polyvalent, HRP; LabVision) for 10 minutes at room temperature. .. Skin sections were finally counterstained with Mayer’s hematoxylin (Bio-Optica, Milan, Italy), washed, mounted in an aqueous mounting medium and observed under a Leica DM4000 B microscope (Leica Microsystems).

    Article Title: Telocytes are reduced during fibrotic remodelling of the colonic wall in ulcerative colitis
    Article Snippet: Subsequently, the slides were washed three times in PBS and incubated with streptavidin peroxidase (UltraVision Large Volume Detection System Anti-Polyvalent, HRP; LabVision) for 10 min. at room temperature. .. Sections were finally counterstained with haematoxylin, washed, mounted in an aqueous mounting medium and observed under a Leica DM4000 B microscope equipped with fully automated transmitted light axes (Leica Microsystems).

    Labeling:

    Article Title: ?-Catenin Phosphorylated at Serine 45 Is Spatially Uncoupled from ?-Catenin Phosphorylated in the GSK3 Domain: Implications for Signaling
    Article Snippet: In brief, cells were rinsed in PBS supplemented with 0.1 mM CaCl2 and 1 mM MgCl2 (PBS++ ), labeled in the dark on ice for 20 minutes with 1 mg/mL EZ-link Sulfo-NHS-LC-biotin (Pierce) in a solution of 10 mM triethanolamine, pH 8, 2 mM CaCl2 , and 150 mM NaCl. .. Lysates were immunoprecipitated with antibodies as indicated, and Western blots were performed using streptavidin-HRP (Invitrogen).

    Article Title: Ciliary proteins Fap43 and Fap44 interact with each other and are essential for proper cilia and flagella beating
    Article Snippet: To identify potential Fap43p or Fap44p-interacting proteins using proximity-dependent labeling, cells expressing either Fap43p–HA–BirA* or Fap44p–HA–BirA* were grown in SPP medium to a density of 2 × 105 cells/ml, starved overnight in 10 mM Tris–HCl buffer, pH 7.5, and incubated in the same buffer supplied with 50 μM biotin for 2–4 h at 30 °C. .. After washing (6 × 5 min with washing buffer: 15 mM Tris–HCl, pH 7.4 150 mM NaCl, 0.1% SDS, 0.3 mM DTT) at 4 °C, resin-bound proteins were analyzed on SDS-PAGE gel, followed by western blot with anti-streptavidin–HRP (Thermo Scientific, Rockford, IL, USA), diluted 1:40,000, or by mass spectrometry (Laboratory of Mass Spectrometry, Institute of Biochemistry and Biophysics, PAS, Warsaw, Poland).

    SDS Page:

    Article Title: Ferritin Blocks Inhibitory Effects of Two-Chain High Molecular Weight Kininogen (HKa) on Adhesion and Survival Signaling in Endothelial Cells
    Article Snippet: Proteins were separated by SDS PAGE and transferred into a polyvinylidene difluoride (PDVF) membrane. .. The membranes were blocked in 5% BSA in Tris-buffered-saline containing 0.05% Tween-20, and probed with the following antibodies: anti-phospho-MAPK 44/42, anti-total-MAPK 44/42, anti-phospho-Paxillin Y118, anti-Paxillin, anti- phospho-FAK Y397, anti-FAK, anti-phospho-Akt (Akt S473) (Cell Signaling); anti-GAPDH (Fitzgerald Industries International, Inc.); Streptavidin-HRP (Pierce); and anti-GST (Sigma).

    Article Title: Ciliary proteins Fap43 and Fap44 interact with each other and are essential for proper cilia and flagella beating
    Article Snippet: .. After washing (6 × 5 min with washing buffer: 15 mM Tris–HCl, pH 7.4 150 mM NaCl, 0.1% SDS, 0.3 mM DTT) at 4 °C, resin-bound proteins were analyzed on SDS-PAGE gel, followed by western blot with anti-streptavidin–HRP (Thermo Scientific, Rockford, IL, USA), diluted 1:40,000, or by mass spectrometry (Laboratory of Mass Spectrometry, Institute of Biochemistry and Biophysics, PAS, Warsaw, Poland). .. For the entire cilia proteome, wild-type and mutant cells were deciliated as above, the cilia were collected and washed with 10 mM Tris, pH 7.4, and an equal amount of ciliary protein was run on SDS-PAGE gel and analyzed by mass spectrometry (Laboratory of Mass Spectrometry, Institute of Biochemistry and Biophysics, PAS, Warsaw, Poland).

    Plasmid Preparation:

    Article Title: Epigallocatechin-3-gallate enhances ER stress-induced cancer cell apoptosis by directly targeting PARP16 activity
    Article Snippet: Plasmids, antibodies and other materials PARP16 was cloned into pGEX-4T-1 vector and confirmed by sequencing. .. Anti-phos-PERK (Thr 981) and anti-β -Tubulin antibodies were purchased from Santa-Cruz Biotechnologies (Dallas, TX, USA); anti-eIF2α and anti-phos-eIF2α (Ser 51) antibodies were purchased from Abcam (Cambridge, MA, USA), anti-PARP16 antibody was generated by ourselves; Streptavidin-HRP was obtained from Thermo Fisher (Waltham, MA, USA); BFA, ECG and EGCG were purchased from Sigma-Aldrich (St Louis, MO, USA); TUN was obtained from Cell Signaling Technologies (Beverly, MA, USA); Biotin-labeled NAD+ from Invitrogen (Carlsbad, CA, USA); glutathione Sepharose 4B resin was obtained from GE Healthcare (Pittsburgh, PA, USA).

    Article Title: TRPC3 Activation by Erythropoietin Is Modulated by TRPC6
    Article Snippet: Blots were incubated with anti-TRPC3 (1:400, Alomone Labs, Jerusalem, Israel), anti-TRPC3 (C) targeted to the human C-terminal sequence RRRRLQDIEMGMGNSKSRLN (1:1000, Bethyl Laboratories, Inc., Montgomery, TX) , anti-TRPC3 (N) targeted to the murine N-terminal sequence LNGDLESAEPLERHGHKASL (1:1000, Bethyl Laboratories, Inc.) , anti-TRPC6 (1:250, Alomone Labs; 1:500, Abcam, Inc., Cambridge, MA), anti-V5-HRP (1:10,000, Invitrogen), anti-FLAG (1:1000, Sigma), anti-PLCγ (1:1000, SC-81, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-Epo-R (1:1000, SC697, Santa Cruz Biotechnology, Inc.), anti-actin (1:10,000, Sigma), anti-tubulin (1:10,000, Sigma) antibodies, or streptavidin-HRP (Pierce). .. To examine the interaction of TRPC3 with TRPC6, PLCγ, or Epo-R, HEK 293T cells were transfected with hTRPC3 (in pcDNA3.1/V5-His TOPO), hTRPC6 (in pCMV tag), TRPC3/TRPC6 chimeras in either vector, hEpo-R (in pcDNA3), rat PLCγ1 (in pcDNA3), or combinations of these vectors.

    Negative Control:

    Article Title: Ciliary proteins Fap43 and Fap44 interact with each other and are essential for proper cilia and flagella beating
    Article Snippet: To identify proteins that co-immunoprecipitate with GFP-tagged Fap43p, the cytoskeletal fraction [ ] or cilia from Tetrahymena cells overexpressing GFP–Fap43p or expressing Fap43p–GFP, respectively, and GFP-expressing cells (negative control) were harvested and processed as previously described [ ]. .. After washing (6 × 5 min with washing buffer: 15 mM Tris–HCl, pH 7.4 150 mM NaCl, 0.1% SDS, 0.3 mM DTT) at 4 °C, resin-bound proteins were analyzed on SDS-PAGE gel, followed by western blot with anti-streptavidin–HRP (Thermo Scientific, Rockford, IL, USA), diluted 1:40,000, or by mass spectrometry (Laboratory of Mass Spectrometry, Institute of Biochemistry and Biophysics, PAS, Warsaw, Poland).

    Recombinant:

    Article Title: Interferon-? Activates Transglutaminase 2 via a Phosphatidylinositol-3-Kinase-Dependent Pathway: Implications for Celiac Sprue Therapy S⃞
    Article Snippet: Recombinant IFN-γ was from Peprotech (Rocky Hill, NJ). .. Cell culture medium, antibiotics, trypsin-EDTA, sterile PBS, goat anti-rabbit (H-L) and goat anti-mouse (H-L) secondary antibodies, streptavidin Alexa Fluor 647, streptavidin-HRP, and goat anti-mouse HRP were from Invitrogen (Carlsbad, CA).

    Article Title: Quantitative detection of PfHRP2 in saliva of malaria patients in the Philippines
    Article Snippet: Recombinant Pf HRP2 was obtained from CTK Biotech (A3000). .. Peroxidase-labeled streptavidin (SNN2004) was obtained from Invitrogen.

    Immunoprecipitation:

    Article Title: ?-Catenin Phosphorylated at Serine 45 Is Spatially Uncoupled from ?-Catenin Phosphorylated in the GSK3 Domain: Implications for Signaling
    Article Snippet: .. Lysates were immunoprecipitated with antibodies as indicated, and Western blots were performed using streptavidin-HRP (Invitrogen). .. Phospho-peptide mapping Cytosolic β-catenin was affinity precipitated with 100 µg GST-ICAT from SW480 controls cell lysate (approximately 50 mg total protein) and subjected to SDS-PAGE.

    Article Title: Liver ubiquitome uncovers nutrient-stress-mediated trafficking and secretion of complement C3
    Article Snippet: Paragraph title: Protein immunoprecipitation and pulldown ... The following antibodies were used: anti-ubiquitin (P4D1; Santa Cruz Biotechnology, Dallas, TX, USA), anti-Iκ B (CST), anti-GAPDH (G9545; Sigma Aldrich), streptavidin-HRP (Thermo Fisher Scientific, Waltham, MA, USA), anti-C3a (BD), and anti-p-IRE1α (Thermo Fisher Scientific).

    Article Title: TRPC3 Activation by Erythropoietin Is Modulated by TRPC6
    Article Snippet: Immunoblotting and Immunoprecipitation —For Western blotting, whole cell lysates or immunoprecipitates were separated on 8% polyacrylamide gels, followed by transfer to Hybond-C Extra membranes (Amersham Biosciences). .. Blots were incubated with anti-TRPC3 (1:400, Alomone Labs, Jerusalem, Israel), anti-TRPC3 (C) targeted to the human C-terminal sequence RRRRLQDIEMGMGNSKSRLN (1:1000, Bethyl Laboratories, Inc., Montgomery, TX) , anti-TRPC3 (N) targeted to the murine N-terminal sequence LNGDLESAEPLERHGHKASL (1:1000, Bethyl Laboratories, Inc.) , anti-TRPC6 (1:250, Alomone Labs; 1:500, Abcam, Inc., Cambridge, MA), anti-V5-HRP (1:10,000, Invitrogen), anti-FLAG (1:1000, Sigma), anti-PLCγ (1:1000, SC-81, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-Epo-R (1:1000, SC697, Santa Cruz Biotechnology, Inc.), anti-actin (1:10,000, Sigma), anti-tubulin (1:10,000, Sigma) antibodies, or streptavidin-HRP (Pierce).

    Article Title: Activity-regulated trafficking of the palmitoyl-acyl transferase DHHC5
    Article Snippet: Antibodies and complementary DNA constructs Primary antibodies used were as follows: δ-catenin (1:500 western blot (WB), 5 μg immunoprecipitation (IP); BD Transduction Laboratories 611536), N-cadherin (1:500; BD Transduction Laboratories 610921), PSD-95 for immunocytochemistry (ICC; 1:500; Abcam ab2723), PSD-95 for IP and WB (5 μg, 1:500; Calbiochem CP35), Gephyrin (1:500; Synaptic Systems 147 011), GFP for IP (10 μl; Synaptic Systems 132 002), GFP for WB (1:1,000; Roche 11814460001), DHHC5 (1:500 ICC, 1:1,000 WB, 1 μg IP; Sigma Prestige HPA014670), TfR (1:500; Millipore GR09L), VPS-35 (1:500; Abnova H000055737-M02), GluA1 (1:1,000; Millipore 05-855R), haemagglutinin (HA) for ICC (1:500; Cell Signaling Technology C29F4), HA for IP and WB (5 μg, 1:500; Covance MMS-101P), VGlut1 (1:500; Millipore AB5905), Fyn for WB and ICC (1:250, 1:500; BD Transduction Laboratories 610163), Fyn for IP (5 μg; Life Technologies MA5-13134), non-phosphorylated Y420 Fyn (1:1,000; Cell Signaling Technologies 2102S), STEP61 (1:1,000; Life Technologies MA1-16746), phospho-tyrosine (phY; 1:1,000 WB, 5 μg IP; Millipore 4G10/05-321), AP2μ (1:500; Thermo Scientific PA5-20745) and β-actin (1:1,000; Sigma A1978). .. Secondary antibodies used were as follows: IgG-horseradish peroxidase (HRP; 1:5,000; BioRad mouse 170-6516 and rabbit 170-6515), IgY-HRP (1:5,000; LifeSpan BioSciences LS-C86499), Streptavidin-HRP (1:5,000; Thermo Scientific 21126) and Alexa-Fluor 568 goat anti-mouse and 633 goat anti-rabbit (1:1,000; Life Technologies A-11004 and A-21070, respectively).

    Construct:

    Article Title: Activity-regulated trafficking of the palmitoyl-acyl transferase DHHC5
    Article Snippet: Paragraph title: Antibodies and complementary DNA constructs ... Secondary antibodies used were as follows: IgG-horseradish peroxidase (HRP; 1:5,000; BioRad mouse 170-6516 and rabbit 170-6515), IgY-HRP (1:5,000; LifeSpan BioSciences LS-C86499), Streptavidin-HRP (1:5,000; Thermo Scientific 21126) and Alexa-Fluor 568 goat anti-mouse and 633 goat anti-rabbit (1:1,000; Life Technologies A-11004 and A-21070, respectively).

    Lysis:

    Article Title: ?-Catenin Phosphorylated at Serine 45 Is Spatially Uncoupled from ?-Catenin Phosphorylated in the GSK3 Domain: Implications for Signaling
    Article Snippet: The reaction was quenched with 100 mM glycine in PBS++ , and cells were rinsed in PBS++ prior to lysis with 1% Triton buffer described above. .. Lysates were immunoprecipitated with antibodies as indicated, and Western blots were performed using streptavidin-HRP (Invitrogen).

    Article Title: Ferritin Blocks Inhibitory Effects of Two-Chain High Molecular Weight Kininogen (HKa) on Adhesion and Survival Signaling in Endothelial Cells
    Article Snippet: Cells were lysed in Triton X-100 (TX-100) lysis buffer (50 mM Tris, pH 7.5, 150 mM sodium chloride, 0.5% TX-100) supplemented with protease and phosphatase inhibitor cocktails (Roche) and protein concentration of the clarified samples determined using BCA protein assay kit (Pierce). .. The membranes were blocked in 5% BSA in Tris-buffered-saline containing 0.05% Tween-20, and probed with the following antibodies: anti-phospho-MAPK 44/42, anti-total-MAPK 44/42, anti-phospho-Paxillin Y118, anti-Paxillin, anti- phospho-FAK Y397, anti-FAK, anti-phospho-Akt (Akt S473) (Cell Signaling); anti-GAPDH (Fitzgerald Industries International, Inc.); Streptavidin-HRP (Pierce); and anti-GST (Sigma).

    Article Title: Liver ubiquitome uncovers nutrient-stress-mediated trafficking and secretion of complement C3
    Article Snippet: FLAG-tagged proteins were immunoprecipitated with FLAG M2 affinity gel (Sigma Aldrich, St Louis, MO, USA) and eluted two times with lysis buffer+0.05 mg of FLAG peptide at 4 °C. .. The following antibodies were used: anti-ubiquitin (P4D1; Santa Cruz Biotechnology, Dallas, TX, USA), anti-Iκ B (CST), anti-GAPDH (G9545; Sigma Aldrich), streptavidin-HRP (Thermo Fisher Scientific, Waltham, MA, USA), anti-C3a (BD), and anti-p-IRE1α (Thermo Fisher Scientific).

    Article Title: Ciliary proteins Fap43 and Fap44 interact with each other and are essential for proper cilia and flagella beating
    Article Snippet: After centrifugation (10 min at 21,100× g at 4 °C), the axonemes were resuspended in a lysis buffer (50 mM Tris–HCl, pH 7.4, 500 mM NaCl, 0.4% SDS, 1 mM DTT), incubated at RT for 1 h, and then centrifuged at 8000× g at 4 °C. .. After washing (6 × 5 min with washing buffer: 15 mM Tris–HCl, pH 7.4 150 mM NaCl, 0.1% SDS, 0.3 mM DTT) at 4 °C, resin-bound proteins were analyzed on SDS-PAGE gel, followed by western blot with anti-streptavidin–HRP (Thermo Scientific, Rockford, IL, USA), diluted 1:40,000, or by mass spectrometry (Laboratory of Mass Spectrometry, Institute of Biochemistry and Biophysics, PAS, Warsaw, Poland).

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    Thermo Fisher high sensitivity streptavidin hrp antibody
    Multiple types of analyses show that GDF11 does not cross the BBB. ( a ) GDF11 levels in the serum of 24-month-old mice following acute GDF11 treatment. Full-length blot is presented in Supplementary Fig. 7a . ( b ) ELISA of SMAD2/3 phosphorylation in whole tissue lysates of 24-month-old mice following acute GDF11 treatment (1 mg/kg). n = 6 for each experimental group. Data plotted as the background-subtracted absorbance and shown as mean ± s.e.m., statistical analysis by unpaired, two-tailed Student’s t- test, *p = 0.03 (heart), ***p = 0.0003 (kidney), ***p = 0.0006 (liver), **p = 0.01 (spleen), # p = 0.06 (muscle), not significant (ns) (brain) compared to vehicle controls of each tissue type. ( c ) ELISA of SMAD2/3 phosphorylation in primary mouse cortical neurons and primary mouse astrocytes following 1-hour treatment with GDF11 (50 ng/ml) or vehicle. n = 6 for each neuron condition, n = 4 for each astrocyte condition. Data calculated as fold change from vehicle controls and shown as mean ± s.e.m., statistical analysis by unpaired, two-tailed Student’s t- test, ***p = 0.0001 (neuron), # p = 0.06 (astrocyte), compared to vehicle controls of each cell type. ( d ) Number of pSMAD2/3 + nuclei in SVZ-derived, dissociated neurospheres following 90-minute treatment with GDF11 (50 ng/ml) or vehicle. n = 3 for each condition. Data shown as mean ± s.e.m., statistical analysis by unpaired, two-tailed Student’s t -test, *p = 0.01 compared to vehicle control. ( e ) Average colony size (number of cells per sphere) of young and old SVZ-derived neurospheres following 10-day treatment with GDF11 (50 ng/ml) or vehicle, in a clonal assay. n = 11 for young, n = 3 for old condition. Data shown as mean ± s.e.m., statistical analysis by unpaired, two-tailed Student’s t -test, *p = 0.02 compared to vehicle control. ( f ) Number of Tuj1+ cells differentiated from young and old SVZ-derived neurospheres following 7-day treatment with GDF11 (50 ng/ml) or vehicle, in a differentiation assay. n = 4 for each condition. Data shown as mean ± s.e.m., statistical analysis by unpaired, two-tailed Student’s t -test, *p = 0.04, **p = 0.006 compared to vehicle control. ( g ) Detection of biotinylated recombinant GDF11 with <t>streptavidin-HRP</t> (left) or Coomassie staining (right). Full-length blot and gel are presented in Supplementary Fig. 7b. ( h ) Biotinylated GDF11 levels in the brain parenchyma (left) and the spleen (right) of 3–4-month-old mice following acute GDF11 treatment (8 mg/kg). Biotinylated recombinant GDF11 protein was loaded to help detect the biotinylated protein in tissue samples. Tubulin was used as a loading control. Full-length blots are presented in Supplementary Fig. 7c.
    High Sensitivity Streptavidin Hrp Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher streptavidin hrp
    Characterization of APEX2 fusion constructs. HEK 293 T cells stably expressing the indicated constructs ( right ) were labeled and crosslinked via Protocol II ( Figure 1—figure supplement 1A ). Cell lysates were analyzed by SDS-PAGE, blotting with <t>streptavidin-HRP,</t> anti-V5 and anti-FLAG. L: ladder; U: untransfected HEK 293T cells. Anti-V5 and anti-FLAG blots ( bottom left ) measure fusion construct expression.
    Streptavidin Hrp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 162 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher streptavidin biotinylated horseradish peroxidase conjugated antibody
    Characterization of APEX2 fusion constructs. HEK 293 T cells stably expressing the indicated constructs ( right ) were labeled and crosslinked via Protocol II ( Figure 1—figure supplement 1A ). Cell lysates were analyzed by SDS-PAGE, blotting with <t>streptavidin-HRP,</t> anti-V5 and anti-FLAG. L: ladder; U: untransfected HEK 293T cells. Anti-V5 and anti-FLAG blots ( bottom left ) measure fusion construct expression.
    Streptavidin Biotinylated Horseradish Peroxidase Conjugated Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Multiple types of analyses show that GDF11 does not cross the BBB. ( a ) GDF11 levels in the serum of 24-month-old mice following acute GDF11 treatment. Full-length blot is presented in Supplementary Fig. 7a . ( b ) ELISA of SMAD2/3 phosphorylation in whole tissue lysates of 24-month-old mice following acute GDF11 treatment (1 mg/kg). n = 6 for each experimental group. Data plotted as the background-subtracted absorbance and shown as mean ± s.e.m., statistical analysis by unpaired, two-tailed Student’s t- test, *p = 0.03 (heart), ***p = 0.0003 (kidney), ***p = 0.0006 (liver), **p = 0.01 (spleen), # p = 0.06 (muscle), not significant (ns) (brain) compared to vehicle controls of each tissue type. ( c ) ELISA of SMAD2/3 phosphorylation in primary mouse cortical neurons and primary mouse astrocytes following 1-hour treatment with GDF11 (50 ng/ml) or vehicle. n = 6 for each neuron condition, n = 4 for each astrocyte condition. Data calculated as fold change from vehicle controls and shown as mean ± s.e.m., statistical analysis by unpaired, two-tailed Student’s t- test, ***p = 0.0001 (neuron), # p = 0.06 (astrocyte), compared to vehicle controls of each cell type. ( d ) Number of pSMAD2/3 + nuclei in SVZ-derived, dissociated neurospheres following 90-minute treatment with GDF11 (50 ng/ml) or vehicle. n = 3 for each condition. Data shown as mean ± s.e.m., statistical analysis by unpaired, two-tailed Student’s t -test, *p = 0.01 compared to vehicle control. ( e ) Average colony size (number of cells per sphere) of young and old SVZ-derived neurospheres following 10-day treatment with GDF11 (50 ng/ml) or vehicle, in a clonal assay. n = 11 for young, n = 3 for old condition. Data shown as mean ± s.e.m., statistical analysis by unpaired, two-tailed Student’s t -test, *p = 0.02 compared to vehicle control. ( f ) Number of Tuj1+ cells differentiated from young and old SVZ-derived neurospheres following 7-day treatment with GDF11 (50 ng/ml) or vehicle, in a differentiation assay. n = 4 for each condition. Data shown as mean ± s.e.m., statistical analysis by unpaired, two-tailed Student’s t -test, *p = 0.04, **p = 0.006 compared to vehicle control. ( g ) Detection of biotinylated recombinant GDF11 with streptavidin-HRP (left) or Coomassie staining (right). Full-length blot and gel are presented in Supplementary Fig. 7b. ( h ) Biotinylated GDF11 levels in the brain parenchyma (left) and the spleen (right) of 3–4-month-old mice following acute GDF11 treatment (8 mg/kg). Biotinylated recombinant GDF11 protein was loaded to help detect the biotinylated protein in tissue samples. Tubulin was used as a loading control. Full-length blots are presented in Supplementary Fig. 7c.

    Journal: Scientific Reports

    Article Title: Growth Differentiation Factor 11 treatment leads to neuronal and vascular improvements in the hippocampus of aged mice

    doi: 10.1038/s41598-018-35716-6

    Figure Lengend Snippet: Multiple types of analyses show that GDF11 does not cross the BBB. ( a ) GDF11 levels in the serum of 24-month-old mice following acute GDF11 treatment. Full-length blot is presented in Supplementary Fig. 7a . ( b ) ELISA of SMAD2/3 phosphorylation in whole tissue lysates of 24-month-old mice following acute GDF11 treatment (1 mg/kg). n = 6 for each experimental group. Data plotted as the background-subtracted absorbance and shown as mean ± s.e.m., statistical analysis by unpaired, two-tailed Student’s t- test, *p = 0.03 (heart), ***p = 0.0003 (kidney), ***p = 0.0006 (liver), **p = 0.01 (spleen), # p = 0.06 (muscle), not significant (ns) (brain) compared to vehicle controls of each tissue type. ( c ) ELISA of SMAD2/3 phosphorylation in primary mouse cortical neurons and primary mouse astrocytes following 1-hour treatment with GDF11 (50 ng/ml) or vehicle. n = 6 for each neuron condition, n = 4 for each astrocyte condition. Data calculated as fold change from vehicle controls and shown as mean ± s.e.m., statistical analysis by unpaired, two-tailed Student’s t- test, ***p = 0.0001 (neuron), # p = 0.06 (astrocyte), compared to vehicle controls of each cell type. ( d ) Number of pSMAD2/3 + nuclei in SVZ-derived, dissociated neurospheres following 90-minute treatment with GDF11 (50 ng/ml) or vehicle. n = 3 for each condition. Data shown as mean ± s.e.m., statistical analysis by unpaired, two-tailed Student’s t -test, *p = 0.01 compared to vehicle control. ( e ) Average colony size (number of cells per sphere) of young and old SVZ-derived neurospheres following 10-day treatment with GDF11 (50 ng/ml) or vehicle, in a clonal assay. n = 11 for young, n = 3 for old condition. Data shown as mean ± s.e.m., statistical analysis by unpaired, two-tailed Student’s t -test, *p = 0.02 compared to vehicle control. ( f ) Number of Tuj1+ cells differentiated from young and old SVZ-derived neurospheres following 7-day treatment with GDF11 (50 ng/ml) or vehicle, in a differentiation assay. n = 4 for each condition. Data shown as mean ± s.e.m., statistical analysis by unpaired, two-tailed Student’s t -test, *p = 0.04, **p = 0.006 compared to vehicle control. ( g ) Detection of biotinylated recombinant GDF11 with streptavidin-HRP (left) or Coomassie staining (right). Full-length blot and gel are presented in Supplementary Fig. 7b. ( h ) Biotinylated GDF11 levels in the brain parenchyma (left) and the spleen (right) of 3–4-month-old mice following acute GDF11 treatment (8 mg/kg). Biotinylated recombinant GDF11 protein was loaded to help detect the biotinylated protein in tissue samples. Tubulin was used as a loading control. Full-length blots are presented in Supplementary Fig. 7c.

    Article Snippet: Goat anti-rabbit HRP secondary antibody or Pierce™ high sensitivity streptavidin-HRP antibody (21130, ThermoFisher Scientific) was added at 1:10,000 dilution in blocking solution and membranes were incubated for 1 hour at room temperature.

    Techniques: Mouse Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Derivative Assay, Clone Assay, Differentiation Assay, Recombinant, Staining

    Multiple types of analyses show that GDF11 does not cross the BBB. ( a . ( b ) ELISA of SMAD2/3 phosphorylation in whole tissue lysates of 24-month-old mice following acute GDF11 treatment (1 mg/kg). n = 6 for each experimental group. Data plotted as the background-subtracted absorbance and shown as mean ± s.e.m., statistical analysis by unpaired, two-tailed Student’s t- test, *p = 0.03 (heart), ***p = 0.0003 (kidney), ***p = 0.0006 (liver), **p = 0.01 (spleen), # p = 0.06 (muscle), not significant (ns) (brain) compared to vehicle controls of each tissue type. ( c ) ELISA of SMAD2/3 phosphorylation in primary mouse cortical neurons and primary mouse astrocytes following 1-hour treatment with GDF11 (50 ng/ml) or vehicle. n = 6 for each neuron condition, n = 4 for each astrocyte condition. Data calculated as fold change from vehicle controls and shown as mean ± s.e.m., statistical analysis by unpaired, two-tailed Student’s t- test, ***p = 0.0001 (neuron), # p = 0.06 (astrocyte), compared to vehicle controls of each cell type. ( d ) Number of pSMAD2/3 + nuclei in SVZ-derived, dissociated neurospheres following 90-minute treatment with GDF11 (50 ng/ml) or vehicle. n = 3 for each condition. Data shown as mean ± s.e.m., statistical analysis by unpaired, two-tailed Student’s t -test, *p = 0.01 compared to vehicle control. ( e ) Average colony size (number of cells per sphere) of young and old SVZ-derived neurospheres following 10-day treatment with GDF11 (50 ng/ml) or vehicle, in a clonal assay. n = 11 for young, n = 3 for old condition. Data shown as mean ± s.e.m., statistical analysis by unpaired, two-tailed Student’s t -test, *p = 0.02 compared to vehicle control. ( f ) Number of Tuj1+ cells differentiated from young and old SVZ-derived neurospheres following 7-day treatment with GDF11 (50 ng/ml) or vehicle, in a differentiation assay. n = 4 for each condition. Data shown as mean ± s.e.m., statistical analysis by unpaired, two-tailed Student’s t -test, *p = 0.04, **p = 0.006 compared to vehicle control. ( g ) Detection of biotinylated recombinant GDF11 with streptavidin-HRP (left) or Coomassie staining (right). Full-length blot and gel are presented in Supplementary Fig. 7b. ( h ) Biotinylated GDF11 levels in the brain parenchyma (left) and the spleen (right) of 3–4-month-old mice following acute GDF11 treatment (8 mg/kg). Biotinylated recombinant GDF11 protein was loaded to help detect the biotinylated protein in tissue samples. Tubulin was used as a loading control. Full-length blots are presented in Supplementary Fig. 7c.

    Journal: Scientific Reports

    Article Title: Growth Differentiation Factor 11 treatment leads to neuronal and vascular improvements in the hippocampus of aged mice

    doi: 10.1038/s41598-018-35716-6

    Figure Lengend Snippet: Multiple types of analyses show that GDF11 does not cross the BBB. ( a . ( b ) ELISA of SMAD2/3 phosphorylation in whole tissue lysates of 24-month-old mice following acute GDF11 treatment (1 mg/kg). n = 6 for each experimental group. Data plotted as the background-subtracted absorbance and shown as mean ± s.e.m., statistical analysis by unpaired, two-tailed Student’s t- test, *p = 0.03 (heart), ***p = 0.0003 (kidney), ***p = 0.0006 (liver), **p = 0.01 (spleen), # p = 0.06 (muscle), not significant (ns) (brain) compared to vehicle controls of each tissue type. ( c ) ELISA of SMAD2/3 phosphorylation in primary mouse cortical neurons and primary mouse astrocytes following 1-hour treatment with GDF11 (50 ng/ml) or vehicle. n = 6 for each neuron condition, n = 4 for each astrocyte condition. Data calculated as fold change from vehicle controls and shown as mean ± s.e.m., statistical analysis by unpaired, two-tailed Student’s t- test, ***p = 0.0001 (neuron), # p = 0.06 (astrocyte), compared to vehicle controls of each cell type. ( d ) Number of pSMAD2/3 + nuclei in SVZ-derived, dissociated neurospheres following 90-minute treatment with GDF11 (50 ng/ml) or vehicle. n = 3 for each condition. Data shown as mean ± s.e.m., statistical analysis by unpaired, two-tailed Student’s t -test, *p = 0.01 compared to vehicle control. ( e ) Average colony size (number of cells per sphere) of young and old SVZ-derived neurospheres following 10-day treatment with GDF11 (50 ng/ml) or vehicle, in a clonal assay. n = 11 for young, n = 3 for old condition. Data shown as mean ± s.e.m., statistical analysis by unpaired, two-tailed Student’s t -test, *p = 0.02 compared to vehicle control. ( f ) Number of Tuj1+ cells differentiated from young and old SVZ-derived neurospheres following 7-day treatment with GDF11 (50 ng/ml) or vehicle, in a differentiation assay. n = 4 for each condition. Data shown as mean ± s.e.m., statistical analysis by unpaired, two-tailed Student’s t -test, *p = 0.04, **p = 0.006 compared to vehicle control. ( g ) Detection of biotinylated recombinant GDF11 with streptavidin-HRP (left) or Coomassie staining (right). Full-length blot and gel are presented in Supplementary Fig. 7b. ( h ) Biotinylated GDF11 levels in the brain parenchyma (left) and the spleen (right) of 3–4-month-old mice following acute GDF11 treatment (8 mg/kg). Biotinylated recombinant GDF11 protein was loaded to help detect the biotinylated protein in tissue samples. Tubulin was used as a loading control. Full-length blots are presented in Supplementary Fig. 7c.

    Article Snippet: Goat anti-rabbit HRP secondary antibody or Pierce™ high sensitivity streptavidin-HRP antibody (21130, ThermoFisher Scientific) was added at 1:10,000 dilution in blocking solution and membranes were incubated for 1 hour at room temperature.

    Techniques: Enzyme-linked Immunosorbent Assay, Mouse Assay, Two Tailed Test, Derivative Assay, Clone Assay, Differentiation Assay, Recombinant, Staining

    Characterization of APEX2 fusion constructs. HEK 293 T cells stably expressing the indicated constructs ( right ) were labeled and crosslinked via Protocol II ( Figure 1—figure supplement 1A ). Cell lysates were analyzed by SDS-PAGE, blotting with streptavidin-HRP, anti-V5 and anti-FLAG. L: ladder; U: untransfected HEK 293T cells. Anti-V5 and anti-FLAG blots ( bottom left ) measure fusion construct expression.

    Journal: eLife

    Article Title: Live-cell mapping of organelle-associated RNAs via proximity biotinylation combined with protein-RNA crosslinking

    doi: 10.7554/eLife.29224

    Figure Lengend Snippet: Characterization of APEX2 fusion constructs. HEK 293 T cells stably expressing the indicated constructs ( right ) were labeled and crosslinked via Protocol II ( Figure 1—figure supplement 1A ). Cell lysates were analyzed by SDS-PAGE, blotting with streptavidin-HRP, anti-V5 and anti-FLAG. L: ladder; U: untransfected HEK 293T cells. Anti-V5 and anti-FLAG blots ( bottom left ) measure fusion construct expression.

    Article Snippet: Following Ponceau imaging, blots were blocked in blocking buffer for 30 min at room temperature, immersed in blocking buffer supplemented with streptavidin-HRP (1:3000 dilution, ThermoFisher Scientific, RRID: AB_2619743 ) at room temperature for 15 min, rinsed with blocking buffer five times for 5 min each, developed and imaged using the Clarity reagent and an Alpha Innotech gel imaging system.

    Techniques: Construct, Stable Transfection, Expressing, Labeling, SDS Page