agarose streptavidin  (Millipore)


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    Structured Review

    Millipore agarose streptavidin
    Agarose Streptavidin, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/agarose streptavidin/product/Millipore
    Average 85 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    agarose streptavidin - by Bioz Stars, 2020-04
    85/100 stars

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    Related Articles

    Diagnostic Assay:

    Article Title: Monoclonal Antibody 16D10 to the C-Terminal Domain of the Feto-Acinar Pancreatic Protein Binds to Membrane of Human Pancreatic Tumoral SOJ-6 Cells and Inhibits the Growth of Tumor Xenografts 1
    Article Snippet: Bovine serum albumin (BSA), streptavidin-agarose, geldanamycin, and cell dissociation solution were obtained from Sigma (St. Louis, MO). .. Bovine serum albumin (BSA), streptavidin-agarose, geldanamycin, and cell dissociation solution were obtained from Sigma (St. Louis, MO).

    Centrifugation:

    Article Title: Interaction of fascin and protein kinase C?: a novel intersection in cell adhesion and motility
    Article Snippet: Synthetic peptides corresponding to the HIV TAT protein membrane permeablization sequence ( ) fused to the sequence of aa 33–47 of fascin (identical in human and mouse fascin) were prepared in unconjugated, N-terminal biotinylated, or N-terminal FITC-conjugated forms, by Genemed Synthesis Inc. For pull-downs, biotinylated peptides were bound to streptavidin–agarose (Sigma) for 16 h at 4°C. .. Beads were collected by centrifugation, the supernatants stored separately, and the beads washed four times in buffer containing 0.1% Triton X-100.

    Article Title: Herp Regulates Hrd1-mediated Ubiquitylation in a Ubiquitin-like Domain-dependent Manner *
    Article Snippet: Cell debris was removed by centrifugation in a microcentrifuge for 10 min at 14,000 rpm. .. For precipitation of HTB-tagged proteins, supernatants were incubated with streptavidin-agarose (Novagen) for 2 h and washed in buffer B (buffer A containing 0.2% deoxyBigCHAP instead of 1%).

    Article Title: Cross-species Analysis Reveals Evolving and Conserved Features of the Nuclear Factor ?B (NF-?B) Proteins *
    Article Snippet: Proteins were overexpressed through induction with 0.2 mm isopropyl β-d -thiogalactopyranoside at 30 °C for 5 h. Pellets of cells were harvested in nickel-nitrilotriacetic acid binding buffer with added EDTA-free protease inhibitor (Roche Applied Science) and pulse-sonicated for 2 min, and debris was removed by centrifugation at 16,000 × g . .. NF-κB proteins were purified by affinity chromatography in two steps: using first the nickel-nitrilotriacetic acid His-Bind resin system (Merck) and then biotinylated DNA oligonucleotides attached to streptavidin-agarose (Sigma).

    Expressing:

    Article Title: Cross-species Analysis Reveals Evolving and Conserved Features of the Nuclear Factor ?B (NF-?B) Proteins *
    Article Snippet: Paragraph title: Protein Expression and Purification ... NF-κB proteins were purified by affinity chromatography in two steps: using first the nickel-nitrilotriacetic acid His-Bind resin system (Merck) and then biotinylated DNA oligonucleotides attached to streptavidin-agarose (Sigma).

    Stable Transfection:

    Article Title: RNA affinity tags for purification of RNAs and ribonucleoprotein complexes
    Article Snippet: For example, the S1 aptamer can stably bind to streptavidin in a solution containing up to 400 mM NaCl. .. The amount of affinity matrix used to isolate the tagged complexes should really be determined experimentally, but as a rough guide to purification of complexes from crude yeast lysates, approximately 10–20 μl Sephadex G-200 (Pharmacia) or streptavidin–agarose (Sigma) is used for each milligram of protein in the lysates.

    Construct:

    Article Title: Cross-species Analysis Reveals Evolving and Conserved Features of the Nuclear Factor ?B (NF-?B) Proteins *
    Article Snippet: Protein Expression and Purification Expression constructs for NF-κB dimers used in this study were created as described ( ). .. NF-κB proteins were purified by affinity chromatography in two steps: using first the nickel-nitrilotriacetic acid His-Bind resin system (Merck) and then biotinylated DNA oligonucleotides attached to streptavidin-agarose (Sigma).

    Electrophoresis:

    Article Title: Generation of Recombinant Polioviruses Harboring RNA Affinity Tags in the 5′ and 3′ Noncoding Regions of Genomic RNAs
    Article Snippet: In vitro transcribed RNA (11 μg) was renatured in TE buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA) by heating at 56 °C for 5 min, 37 °C for 10 min, and incubating at room temperature for 15 min. Streptavidin-agarose (SigmaAldrich,) was prepared by washing 10 times in lysis buffer (50 mM HEPES pH 7.4, 10 mM MgCl2 , 100 mM NaCl, 1 mM DTT, 0.1% Triton X-100, 10% glycerol), Complete protease inhibitors (Roche) and resuspended to 50% slurry in lysis buffer. .. RNA (1 μg) was subjected to electrophoresis on a 1% agarose gel in Tris/Borate/EDTA (TBE) buffer to confirm an intact, homogenous RNA population.

    Incubation:

    Article Title: A Unique Element in the Cytoplasmic Tail of the Type II Transforming Growth Factor-? Receptor Controls Basolateral Delivery
    Article Snippet: .. Streptavidin agarose (50 μl; Novagen, Madison, WI) was mixed with 500 μg–1 mg of protein in 1 ml total volume, and the samples were incubated for 2 h at 4°C with agitation. .. The agarose was washed four times with lysis buffer (1 ml), and the biotin-bound proteins were eluted by boiling for 5 min in Laemmli buffer.

    Article Title: The p29 and p35 Immunodominant Antigens of Neospora caninum Tachyzoites Are Homologous to the Family of Surface Antigens of Toxoplasma gondii
    Article Snippet: The soluble proteins were incubated with MAb 6C11 or 5H5 and precipitated with protein G-agarose (Sigma). .. Alternatively, the biotin-labeled proteins were precipitated with streptavidin-agarose (Sigma) and analyzed by Western blotting, as described above, using the MAb 6C11 or 5H5.

    Article Title: Two new mutations in the HIF2A gene associated with erythrocytosis
    Article Snippet: .. Ten ng of recombinant VHL was incubated without or with 1 μg of biotinylated hydroxyproline (Hyp)-564 HIF-1α (556–574) prebound to 10 μl of streptavidin-agarose (Sigma) in the absence or presence of 5 nM wild type (WT) or F540L Hyp-531 HIF-2α (527–542) peptide. .. The resins were washed, eluted, and the eluates subjected to SDS-PAGE and western blotting using anti-Flag antibodies (Sigma), essentially as described [ , ].

    Article Title: Interaction of fascin and protein kinase C?: a novel intersection in cell adhesion and motility
    Article Snippet: Synthetic peptides corresponding to the HIV TAT protein membrane permeablization sequence ( ) fused to the sequence of aa 33–47 of fascin (identical in human and mouse fascin) were prepared in unconjugated, N-terminal biotinylated, or N-terminal FITC-conjugated forms, by Genemed Synthesis Inc. For pull-downs, biotinylated peptides were bound to streptavidin–agarose (Sigma) for 16 h at 4°C. .. After washing three times in 1% Triton X-100 in Tris-buffered saline, beads were incubated with 20–200 µg aliquots of cell lysates, or 10 µg of purified rabbit skeletal muscle actin (Sigma) in F-actin buffer (10 mM Tris pH 7.5, 130 mM KCl, 20 mM NaCl, 2 mM MgCl2, 1 mM β-mercaptoethanol, 0.1 mM EGTA, 0.1 mM ATP), for 4 h at 4°C.

    Article Title: RNA affinity tags for purification of RNAs and ribonucleoprotein complexes
    Article Snippet: The amount of affinity matrix used to isolate the tagged complexes should really be determined experimentally, but as a rough guide to purification of complexes from crude yeast lysates, approximately 10–20 μl Sephadex G-200 (Pharmacia) or streptavidin–agarose (Sigma) is used for each milligram of protein in the lysates. .. The binding step is usually carried out at 4 °C for 1 h. For isolation of S1-tagged complexes, incubation of the lysates with egg white avidin before the binding step is recommended, especially for yeast lysates.

    Article Title: Herp Regulates Hrd1-mediated Ubiquitylation in a Ubiquitin-like Domain-dependent Manner *
    Article Snippet: .. For precipitation of HTB-tagged proteins, supernatants were incubated with streptavidin-agarose (Novagen) for 2 h and washed in buffer B (buffer A containing 0.2% deoxyBigCHAP instead of 1%). ..

    Article Title: Interaction of Muscle and Brain Sodium Channels with Multiple Members of the Syntrophin Family of Dystrophin-Associated Proteins
    Article Snippet: Biotinylated SkM1, SkM2, and NR2B peptides (200 μg each) were coupled to 0.5 ml of streptavidin–agarose (Sigma, St. Louis, MO) in PBS, pH 7.2, for 5 hr at 4°C on a rocker platform. .. Two hundred microliters of a 50% slurry of each agarose-coupled peptide or of uncoupled streptavidin–agarose beads were added to 1 ml of detergent-solubilized cardiac muscle membranes and incubated overnight at 4°C on a rocker platform.

    Article Title: RhoA S-nitrosylation as a regulatory mechanism influencing endothelial barrier function in response to G+-bacterial toxins
    Article Snippet: To identify sites of S-nitrosylation, the biotin-switch assay was performed as described above on 1 mg of recombinant RhoA incubated with 100 μM of the S-NO donor, nitrosocysteine for 30 min. RhoA was precipitated, washed and then trypsinized overnight using 5 μg of sequencing-grade trypsin (Promega, Madison, WI) at 37 °C. .. Trypsin was inactivated by addition of 1 mM 4-(2- Aminomethyl) benzenesulfonyl fluoride hydrochloride (AEBSF, Sigma, St. Louis, MO), and biotinylated peptides of RhoA were precipitated with streptavidin–agarose (Sigma, St. Louis, MO) and washed four times with 50 mM ammonium bicarbonate.

    Mass Spectrometry:

    Article Title: RhoA S-nitrosylation as a regulatory mechanism influencing endothelial barrier function in response to G+-bacterial toxins
    Article Snippet: Paragraph title: 2.9. MS analysis ... Trypsin was inactivated by addition of 1 mM 4-(2- Aminomethyl) benzenesulfonyl fluoride hydrochloride (AEBSF, Sigma, St. Louis, MO), and biotinylated peptides of RhoA were precipitated with streptavidin–agarose (Sigma, St. Louis, MO) and washed four times with 50 mM ammonium bicarbonate.

    Western Blot:

    Article Title: XIAP upregulates expression of HIF target genes by targeting HIF1α for Lys63-linked polyubiquitination
    Article Snippet: .. Ubiquitination assays For ubiquitination assays, cells were lysed at room temperature under denaturing conditions (8 M urea, 50 mM Tris [pH 8.0], 300 mM NaCl, 50 mM Na2 HPO4 , 0.5% NP-40, 1 mM PMSF, supplemented with protease inhibitors) and ubiquitinated material was recovered by rotation with NiNTA-agarose (Invitrogen) or streptavidin-agarose (Sigma), washed 3× with lysis buffer and analysed by western blotting. .. Quantitative reverse transcription-PCR Total RNA was isolated using the Peqgold Total RNA Isolation Kit (Peqlab) according to the manufacturer's instructions.

    Article Title: A Unique Element in the Cytoplasmic Tail of the Type II Transforming Growth Factor-? Receptor Controls Basolateral Delivery
    Article Snippet: Plates were rocked at 4°C for 2 h before the biotinylation solution was replaced with fresh solution and rocked at 4°C for a further 4 h. Cells were then washed three times with ice-cold PBS, and scraped and lysed in 500 μl of RIPA buffer with protease inhibitors (as described in Western Blotting). .. Streptavidin agarose (50 μl; Novagen, Madison, WI) was mixed with 500 μg–1 mg of protein in 1 ml total volume, and the samples were incubated for 2 h at 4°C with agitation.

    Article Title: The p29 and p35 Immunodominant Antigens of Neospora caninum Tachyzoites Are Homologous to the Family of Surface Antigens of Toxoplasma gondii
    Article Snippet: .. Alternatively, the biotin-labeled proteins were precipitated with streptavidin-agarose (Sigma) and analyzed by Western blotting, as described above, using the MAb 6C11 or 5H5. ..

    Article Title: Two new mutations in the HIF2A gene associated with erythrocytosis
    Article Snippet: Ten ng of recombinant VHL was incubated without or with 1 μg of biotinylated hydroxyproline (Hyp)-564 HIF-1α (556–574) prebound to 10 μl of streptavidin-agarose (Sigma) in the absence or presence of 5 nM wild type (WT) or F540L Hyp-531 HIF-2α (527–542) peptide. .. The resins were washed, eluted, and the eluates subjected to SDS-PAGE and western blotting using anti-Flag antibodies (Sigma), essentially as described [ , ].

    Article Title: Herp Regulates Hrd1-mediated Ubiquitylation in a Ubiquitin-like Domain-dependent Manner *
    Article Snippet: Paragraph title: Preparation of Cell Extracts, Streptavidin-Agarose-based Precipitation of Tagged Proteins, Metabolic Labeling, Immunoprecipitations, and Western Blotting ... For precipitation of HTB-tagged proteins, supernatants were incubated with streptavidin-agarose (Novagen) for 2 h and washed in buffer B (buffer A containing 0.2% deoxyBigCHAP instead of 1%).

    Flow Cytometry:

    Article Title: RhoA S-nitrosylation as a regulatory mechanism influencing endothelial barrier function in response to G+-bacterial toxins
    Article Snippet: Trypsin was inactivated by addition of 1 mM 4-(2- Aminomethyl) benzenesulfonyl fluoride hydrochloride (AEBSF, Sigma, St. Louis, MO), and biotinylated peptides of RhoA were precipitated with streptavidin–agarose (Sigma, St. Louis, MO) and washed four times with 50 mM ammonium bicarbonate. .. Two microliters of reconstituted peptide was first trapped and washed on a Pepmap100 C18 trap (5 μm, 0.3 × 5 mm) at 20 μl/min using 2% acetonitrile in water (with 0.1% formic acid) for 10 min and then separated on a Pepman 100 RSLC C18 column (2.0 μm, 75-μm × 150-mm) using a gradient of 2–40% acetonitrile with 0.1% formic acid over 40 min at a flow rate of 300 nl/min and a column temperature of 40 °C.

    Immunoprecipitation:

    Article Title: The p29 and p35 Immunodominant Antigens of Neospora caninum Tachyzoites Are Homologous to the Family of Surface Antigens of Toxoplasma gondii
    Article Snippet: The immunoprecipitated proteins were analyzed by Western blotting, as described above, using avidin-HRP conjugate (Bio-Rad Laboratories, Hercules, Calif.) and chemiluminescence to reveal biotin-labeled proteins. .. Alternatively, the biotin-labeled proteins were precipitated with streptavidin-agarose (Sigma) and analyzed by Western blotting, as described above, using the MAb 6C11 or 5H5.

    Article Title: Herp Regulates Hrd1-mediated Ubiquitylation in a Ubiquitin-like Domain-dependent Manner *
    Article Snippet: For analysis of protein complexes by tag-based precipitation or immunoprecipitation, cells were lysed in buffer A (33 m m Hepes, pH 7.3, 150 m m KOAc, 4 m m MgOAc, 1% deoxyBigCHAP (Calbiochem), 10% glycerol) supplemented with CompleteTM (Roche Applied Science) for 30 min on ice. .. For precipitation of HTB-tagged proteins, supernatants were incubated with streptavidin-agarose (Novagen) for 2 h and washed in buffer B (buffer A containing 0.2% deoxyBigCHAP instead of 1%).

    Protease Inhibitor:

    Article Title: Cross-species Analysis Reveals Evolving and Conserved Features of the Nuclear Factor ?B (NF-?B) Proteins *
    Article Snippet: Proteins were overexpressed through induction with 0.2 mm isopropyl β-d -thiogalactopyranoside at 30 °C for 5 h. Pellets of cells were harvested in nickel-nitrilotriacetic acid binding buffer with added EDTA-free protease inhibitor (Roche Applied Science) and pulse-sonicated for 2 min, and debris was removed by centrifugation at 16,000 × g . .. NF-κB proteins were purified by affinity chromatography in two steps: using first the nickel-nitrilotriacetic acid His-Bind resin system (Merck) and then biotinylated DNA oligonucleotides attached to streptavidin-agarose (Sigma).

    other:

    Article Title: INTRACELLULAR GLUTATHIONE MEDIATES THE DENITROSYLATION OF PROTEIN NITROSOTHIOLS IN THE RAT SPINAL CORD
    Article Snippet: Ascorbic acid, aurothioglucose (ATG), caffeic acid (CA), diethyl maleate (DEM), 5,5′-dithiobis(2-nitrobenzoic) acid (DTNB), L-glutathione (GSH), L-glutathione monoethyl ester (GSH-EE), mefenamic acid (MEF), mercaptosuccinic acid (MSA), methyl methanethiol sulfonate, 4-methylpyrazole (4-MP), phenylarsine oxide (PAO) and streptavidin-agarose were purchased from Sigma-Aldrich (St. Louis, MO).

    Article Title: Lasiodin Inhibits Proliferation of Human Nasopharyngeal Carcinoma Cells by Simultaneous Modulation of the Apaf-1/Caspase, AKT/MAPK and COX-2/NF-?B Signaling Pathways
    Article Snippet: Reagents LY294002, SB203580, SP600125, U0126, celecoxib and streptavidin-agarose were purchased from Sigma (St. Louis, MO).

    Tandem Mass Spectroscopy:

    Article Title: RhoA S-nitrosylation as a regulatory mechanism influencing endothelial barrier function in response to G+-bacterial toxins
    Article Snippet: Trypsin was inactivated by addition of 1 mM 4-(2- Aminomethyl) benzenesulfonyl fluoride hydrochloride (AEBSF, Sigma, St. Louis, MO), and biotinylated peptides of RhoA were precipitated with streptavidin–agarose (Sigma, St. Louis, MO) and washed four times with 50 mM ammonium bicarbonate. .. Samples were analyzed by data-dependent acquisition in positive mode using Orbitrap MS analyzer for precursor scan at 120,000 FWHM from 300 to 1500 m / z and ion-trap MS analyzer for MS/MS scans at top speed mode (3-s cycle time).

    Sequencing:

    Article Title: Interaction of fascin and protein kinase C?: a novel intersection in cell adhesion and motility
    Article Snippet: .. Synthetic peptides corresponding to the HIV TAT protein membrane permeablization sequence ( ) fused to the sequence of aa 33–47 of fascin (identical in human and mouse fascin) were prepared in unconjugated, N-terminal biotinylated, or N-terminal FITC-conjugated forms, by Genemed Synthesis Inc. For pull-downs, biotinylated peptides were bound to streptavidin–agarose (Sigma) for 16 h at 4°C. .. After washing three times in 1% Triton X-100 in Tris-buffered saline, beads were incubated with 20–200 µg aliquots of cell lysates, or 10 µg of purified rabbit skeletal muscle actin (Sigma) in F-actin buffer (10 mM Tris pH 7.5, 130 mM KCl, 20 mM NaCl, 2 mM MgCl2, 1 mM β-mercaptoethanol, 0.1 mM EGTA, 0.1 mM ATP), for 4 h at 4°C.

    Article Title: RhoA S-nitrosylation as a regulatory mechanism influencing endothelial barrier function in response to G+-bacterial toxins
    Article Snippet: To identify sites of S-nitrosylation, the biotin-switch assay was performed as described above on 1 mg of recombinant RhoA incubated with 100 μM of the S-NO donor, nitrosocysteine for 30 min. RhoA was precipitated, washed and then trypsinized overnight using 5 μg of sequencing-grade trypsin (Promega, Madison, WI) at 37 °C. .. Trypsin was inactivated by addition of 1 mM 4-(2- Aminomethyl) benzenesulfonyl fluoride hydrochloride (AEBSF, Sigma, St. Louis, MO), and biotinylated peptides of RhoA were precipitated with streptavidin–agarose (Sigma, St. Louis, MO) and washed four times with 50 mM ammonium bicarbonate.

    Affinity Purification:

    Article Title: Generation of Recombinant Polioviruses Harboring RNA Affinity Tags in the 5′ and 3′ Noncoding Regions of Genomic RNAs
    Article Snippet: Paragraph title: 2.8. S1 and D8 Aptamer Affinity Purification ... In vitro transcribed RNA (11 μg) was renatured in TE buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA) by heating at 56 °C for 5 min, 37 °C for 10 min, and incubating at room temperature for 15 min. Streptavidin-agarose (SigmaAldrich,) was prepared by washing 10 times in lysis buffer (50 mM HEPES pH 7.4, 10 mM MgCl2 , 100 mM NaCl, 1 mM DTT, 0.1% Triton X-100, 10% glycerol), Complete protease inhibitors (Roche) and resuspended to 50% slurry in lysis buffer.

    Binding Assay:

    Article Title: Two new mutations in the HIF2A gene associated with erythrocytosis
    Article Snippet: Paragraph title: VHL binding ... Ten ng of recombinant VHL was incubated without or with 1 μg of biotinylated hydroxyproline (Hyp)-564 HIF-1α (556–574) prebound to 10 μl of streptavidin-agarose (Sigma) in the absence or presence of 5 nM wild type (WT) or F540L Hyp-531 HIF-2α (527–542) peptide.

    Article Title: Interaction of fascin and protein kinase C?: a novel intersection in cell adhesion and motility
    Article Snippet: Synthetic peptides corresponding to the HIV TAT protein membrane permeablization sequence ( ) fused to the sequence of aa 33–47 of fascin (identical in human and mouse fascin) were prepared in unconjugated, N-terminal biotinylated, or N-terminal FITC-conjugated forms, by Genemed Synthesis Inc. For pull-downs, biotinylated peptides were bound to streptavidin–agarose (Sigma) for 16 h at 4°C. .. To examine direct binding between fascin and PKCα, 400 ng of the purified fascin was bound to Ni-NTA beads and mixed with 100 ng recombinant PKCα (PanVera), or with cell extracts, in buffer containing 20 mM HEPES, pH 7.4, 5 mM MgCl2 , 1.25 mM CaCl2 , 1 mM dithiothreitol, 1 mM ATP, 0.03% Triton X-100 in the presence or absence of activating lipid mixture, with or without TAT-FAS peptides, for 30 min at 30°C.

    Article Title: RNA affinity tags for purification of RNAs and ribonucleoprotein complexes
    Article Snippet: Therefore, different buffer compositions (e.g., lower or higher salt concentration, addition of glycerol or detergents) should be tested and might be used successfully as binding buffer with the aptamer tags to suit specific requirements of various systems. .. The amount of affinity matrix used to isolate the tagged complexes should really be determined experimentally, but as a rough guide to purification of complexes from crude yeast lysates, approximately 10–20 μl Sephadex G-200 (Pharmacia) or streptavidin–agarose (Sigma) is used for each milligram of protein in the lysates.

    Article Title: Cross-species Analysis Reveals Evolving and Conserved Features of the Nuclear Factor ?B (NF-?B) Proteins *
    Article Snippet: Proteins were overexpressed through induction with 0.2 mm isopropyl β-d -thiogalactopyranoside at 30 °C for 5 h. Pellets of cells were harvested in nickel-nitrilotriacetic acid binding buffer with added EDTA-free protease inhibitor (Roche Applied Science) and pulse-sonicated for 2 min, and debris was removed by centrifugation at 16,000 × g . .. NF-κB proteins were purified by affinity chromatography in two steps: using first the nickel-nitrilotriacetic acid His-Bind resin system (Merck) and then biotinylated DNA oligonucleotides attached to streptavidin-agarose (Sigma).

    Isolation:

    Article Title: RNA affinity tags for purification of RNAs and ribonucleoprotein complexes
    Article Snippet: Paragraph title: 2.3. Isolation of tagged complexes ... The amount of affinity matrix used to isolate the tagged complexes should really be determined experimentally, but as a rough guide to purification of complexes from crude yeast lysates, approximately 10–20 μl Sephadex G-200 (Pharmacia) or streptavidin–agarose (Sigma) is used for each milligram of protein in the lysates.

    Avidin-Biotin Assay:

    Article Title: The p29 and p35 Immunodominant Antigens of Neospora caninum Tachyzoites Are Homologous to the Family of Surface Antigens of Toxoplasma gondii
    Article Snippet: The immunoprecipitated proteins were analyzed by Western blotting, as described above, using avidin-HRP conjugate (Bio-Rad Laboratories, Hercules, Calif.) and chemiluminescence to reveal biotin-labeled proteins. .. Alternatively, the biotin-labeled proteins were precipitated with streptavidin-agarose (Sigma) and analyzed by Western blotting, as described above, using the MAb 6C11 or 5H5.

    Article Title: RNA affinity tags for purification of RNAs and ribonucleoprotein complexes
    Article Snippet: The amount of affinity matrix used to isolate the tagged complexes should really be determined experimentally, but as a rough guide to purification of complexes from crude yeast lysates, approximately 10–20 μl Sephadex G-200 (Pharmacia) or streptavidin–agarose (Sigma) is used for each milligram of protein in the lysates. .. The binding step is usually carried out at 4 °C for 1 h. For isolation of S1-tagged complexes, incubation of the lysates with egg white avidin before the binding step is recommended, especially for yeast lysates.

    Labeling:

    Article Title: The p29 and p35 Immunodominant Antigens of Neospora caninum Tachyzoites Are Homologous to the Family of Surface Antigens of Toxoplasma gondii
    Article Snippet: The labeled parasite pellet was lysed in 1 ml of radioimmunoprecipitation assay buffer (50 mM Tris [pH 7.5], 1% Triton X-100, 0.5% sodium deoxycholate, 0.2% sodium dodecyl sulfate, 100 mM NaCl, 5 mM EDTA) supplemented with RNase, DNase, and protease inhibitors E64, APMSF, leupeptin, and TLCK, and the sample was centrifuged at 16,000 × g to remove the insoluble fraction. .. Alternatively, the biotin-labeled proteins were precipitated with streptavidin-agarose (Sigma) and analyzed by Western blotting, as described above, using the MAb 6C11 or 5H5.

    Article Title: Herp Regulates Hrd1-mediated Ubiquitylation in a Ubiquitin-like Domain-dependent Manner *
    Article Snippet: Paragraph title: Preparation of Cell Extracts, Streptavidin-Agarose-based Precipitation of Tagged Proteins, Metabolic Labeling, Immunoprecipitations, and Western Blotting ... For precipitation of HTB-tagged proteins, supernatants were incubated with streptavidin-agarose (Novagen) for 2 h and washed in buffer B (buffer A containing 0.2% deoxyBigCHAP instead of 1%).

    Purification:

    Article Title: Interaction of fascin and protein kinase C?: a novel intersection in cell adhesion and motility
    Article Snippet: Synthetic peptides corresponding to the HIV TAT protein membrane permeablization sequence ( ) fused to the sequence of aa 33–47 of fascin (identical in human and mouse fascin) were prepared in unconjugated, N-terminal biotinylated, or N-terminal FITC-conjugated forms, by Genemed Synthesis Inc. For pull-downs, biotinylated peptides were bound to streptavidin–agarose (Sigma) for 16 h at 4°C. .. After washing three times in 1% Triton X-100 in Tris-buffered saline, beads were incubated with 20–200 µg aliquots of cell lysates, or 10 µg of purified rabbit skeletal muscle actin (Sigma) in F-actin buffer (10 mM Tris pH 7.5, 130 mM KCl, 20 mM NaCl, 2 mM MgCl2, 1 mM β-mercaptoethanol, 0.1 mM EGTA, 0.1 mM ATP), for 4 h at 4°C.

    Article Title: RNA affinity tags for purification of RNAs and ribonucleoprotein complexes
    Article Snippet: .. The amount of affinity matrix used to isolate the tagged complexes should really be determined experimentally, but as a rough guide to purification of complexes from crude yeast lysates, approximately 10–20 μl Sephadex G-200 (Pharmacia) or streptavidin–agarose (Sigma) is used for each milligram of protein in the lysates. .. The binding step is usually carried out at 4 °C for 1 h. For isolation of S1-tagged complexes, incubation of the lysates with egg white avidin before the binding step is recommended, especially for yeast lysates.

    Article Title: Cross-species Analysis Reveals Evolving and Conserved Features of the Nuclear Factor ?B (NF-?B) Proteins *
    Article Snippet: .. NF-κB proteins were purified by affinity chromatography in two steps: using first the nickel-nitrilotriacetic acid His-Bind resin system (Merck) and then biotinylated DNA oligonucleotides attached to streptavidin-agarose (Sigma). ..

    Positron Emission Tomography:

    Article Title: Cross-species Analysis Reveals Evolving and Conserved Features of the Nuclear Factor ?B (NF-?B) Proteins *
    Article Snippet: Briefly, pET vectors for expression in BL21(DE3) Escherichia coli (Merck) were used to produce His-tagged recombinant proteins. .. NF-κB proteins were purified by affinity chromatography in two steps: using first the nickel-nitrilotriacetic acid His-Bind resin system (Merck) and then biotinylated DNA oligonucleotides attached to streptavidin-agarose (Sigma).

    Biotin Switch Assay:

    Article Title: RhoA S-nitrosylation as a regulatory mechanism influencing endothelial barrier function in response to G+-bacterial toxins
    Article Snippet: To identify sites of S-nitrosylation, the biotin-switch assay was performed as described above on 1 mg of recombinant RhoA incubated with 100 μM of the S-NO donor, nitrosocysteine for 30 min. RhoA was precipitated, washed and then trypsinized overnight using 5 μg of sequencing-grade trypsin (Promega, Madison, WI) at 37 °C. .. Trypsin was inactivated by addition of 1 mM 4-(2- Aminomethyl) benzenesulfonyl fluoride hydrochloride (AEBSF, Sigma, St. Louis, MO), and biotinylated peptides of RhoA were precipitated with streptavidin–agarose (Sigma, St. Louis, MO) and washed four times with 50 mM ammonium bicarbonate.

    Mouse Assay:

    Article Title: Interaction of Muscle and Brain Sodium Channels with Multiple Members of the Syntrophin Family of Dystrophin-Associated Proteins
    Article Snippet: Tissues were dissected from C57BL6 mice or Sprague Dawley rats and were frozen in liquid nitrogen or used fresh as needed. .. Biotinylated SkM1, SkM2, and NR2B peptides (200 μg each) were coupled to 0.5 ml of streptavidin–agarose (Sigma, St. Louis, MO) in PBS, pH 7.2, for 5 hr at 4°C on a rocker platform.

    SDS Page:

    Article Title: A Unique Element in the Cytoplasmic Tail of the Type II Transforming Growth Factor-? Receptor Controls Basolateral Delivery
    Article Snippet: Streptavidin agarose (50 μl; Novagen, Madison, WI) was mixed with 500 μg–1 mg of protein in 1 ml total volume, and the samples were incubated for 2 h at 4°C with agitation. .. Proteins resolved on 8% SDS-PAGE gel were probed with a rabbit anti-GM-CSF β chain antibody (sc-676; Santa Cruz Biotechnology) at 1:800 dilution, and a goat anti-rabbit horseradish peroxidase secondary (sc2004) at 1:2500.

    Article Title: Two new mutations in the HIF2A gene associated with erythrocytosis
    Article Snippet: Ten ng of recombinant VHL was incubated without or with 1 μg of biotinylated hydroxyproline (Hyp)-564 HIF-1α (556–574) prebound to 10 μl of streptavidin-agarose (Sigma) in the absence or presence of 5 nM wild type (WT) or F540L Hyp-531 HIF-2α (527–542) peptide. .. The resins were washed, eluted, and the eluates subjected to SDS-PAGE and western blotting using anti-Flag antibodies (Sigma), essentially as described [ , ].

    Article Title: Interaction of Muscle and Brain Sodium Channels with Multiple Members of the Syntrophin Family of Dystrophin-Associated Proteins
    Article Snippet: Biotinylated SkM1, SkM2, and NR2B peptides (200 μg each) were coupled to 0.5 ml of streptavidin–agarose (Sigma, St. Louis, MO) in PBS, pH 7.2, for 5 hr at 4°C on a rocker platform. .. After extensive washing with the above buffer, the beads were aspirated to near dryness and heated to 80°C in 100 μl of SDS-PAGE loading buffer.

    Affinity Chromatography:

    Article Title: Interaction of Muscle and Brain Sodium Channels with Multiple Members of the Syntrophin Family of Dystrophin-Associated Proteins
    Article Snippet: Peptide affinity chromatography. .. Biotinylated SkM1, SkM2, and NR2B peptides (200 μg each) were coupled to 0.5 ml of streptavidin–agarose (Sigma, St. Louis, MO) in PBS, pH 7.2, for 5 hr at 4°C on a rocker platform.

    Article Title: Cross-species Analysis Reveals Evolving and Conserved Features of the Nuclear Factor ?B (NF-?B) Proteins *
    Article Snippet: .. NF-κB proteins were purified by affinity chromatography in two steps: using first the nickel-nitrilotriacetic acid His-Bind resin system (Merck) and then biotinylated DNA oligonucleotides attached to streptavidin-agarose (Sigma). ..

    Agarose Gel Electrophoresis:

    Article Title: Generation of Recombinant Polioviruses Harboring RNA Affinity Tags in the 5′ and 3′ Noncoding Regions of Genomic RNAs
    Article Snippet: In vitro transcribed RNA (11 μg) was renatured in TE buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA) by heating at 56 °C for 5 min, 37 °C for 10 min, and incubating at room temperature for 15 min. Streptavidin-agarose (SigmaAldrich,) was prepared by washing 10 times in lysis buffer (50 mM HEPES pH 7.4, 10 mM MgCl2 , 100 mM NaCl, 1 mM DTT, 0.1% Triton X-100, 10% glycerol), Complete protease inhibitors (Roche) and resuspended to 50% slurry in lysis buffer. .. RNA (1 μg) was subjected to electrophoresis on a 1% agarose gel in Tris/Borate/EDTA (TBE) buffer to confirm an intact, homogenous RNA population.

    In Vitro:

    Article Title: Generation of Recombinant Polioviruses Harboring RNA Affinity Tags in the 5′ and 3′ Noncoding Regions of Genomic RNAs
    Article Snippet: .. In vitro transcribed RNA (11 μg) was renatured in TE buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA) by heating at 56 °C for 5 min, 37 °C for 10 min, and incubating at room temperature for 15 min. Streptavidin-agarose (SigmaAldrich,) was prepared by washing 10 times in lysis buffer (50 mM HEPES pH 7.4, 10 mM MgCl2 , 100 mM NaCl, 1 mM DTT, 0.1% Triton X-100, 10% glycerol), Complete protease inhibitors (Roche) and resuspended to 50% slurry in lysis buffer. .. Sephadex matrix was prepared by swelling 0.5 g Sephadex G-200 (Sigma-Aldrich) in 40 mL lysis buffer overnight at room temperature.

    Radio Immunoprecipitation:

    Article Title: The p29 and p35 Immunodominant Antigens of Neospora caninum Tachyzoites Are Homologous to the Family of Surface Antigens of Toxoplasma gondii
    Article Snippet: The labeled parasite pellet was lysed in 1 ml of radioimmunoprecipitation assay buffer (50 mM Tris [pH 7.5], 1% Triton X-100, 0.5% sodium deoxycholate, 0.2% sodium dodecyl sulfate, 100 mM NaCl, 5 mM EDTA) supplemented with RNase, DNase, and protease inhibitors E64, APMSF, leupeptin, and TLCK, and the sample was centrifuged at 16,000 × g to remove the insoluble fraction. .. Alternatively, the biotin-labeled proteins were precipitated with streptavidin-agarose (Sigma) and analyzed by Western blotting, as described above, using the MAb 6C11 or 5H5.

    Homogenization:

    Article Title: Interaction of Muscle and Brain Sodium Channels with Multiple Members of the Syntrophin Family of Dystrophin-Associated Proteins
    Article Snippet: The following protease inhibitors were added immediately before homogenization: 1 m m phenylmethylsulfonyl fluoride and 1 μg/ml each leupeptin, pepstatin, aprotinin, and antipain. .. Biotinylated SkM1, SkM2, and NR2B peptides (200 μg each) were coupled to 0.5 ml of streptavidin–agarose (Sigma, St. Louis, MO) in PBS, pH 7.2, for 5 hr at 4°C on a rocker platform.

    Concentration Assay:

    Article Title: A Unique Element in the Cytoplasmic Tail of the Type II Transforming Growth Factor-? Receptor Controls Basolateral Delivery
    Article Snippet: EZ-Link Sulfo-NHS-LC-Biotin (Pierce Chemical, Rockford, IL) was dissolved in ice-cold KLH buffer at a concentration of 1 mg/ml, and it was applied in triplicate to the six-well plate wells (1 ml), apical (0.5 ml) or basolateral (1.0 ml) surfaces. .. Streptavidin agarose (50 μl; Novagen, Madison, WI) was mixed with 500 μg–1 mg of protein in 1 ml total volume, and the samples were incubated for 2 h at 4°C with agitation.

    Article Title: The p29 and p35 Immunodominant Antigens of Neospora caninum Tachyzoites Are Homologous to the Family of Surface Antigens of Toxoplasma gondii
    Article Snippet: Sulfo– N -hydroxysuccinimide–biotin (50 mg/ml in dimethyl sulfoxide; Pierce) was added to a concentration of 0.5 mg/ml and incubated at room temperature for 30 min. .. Alternatively, the biotin-labeled proteins were precipitated with streptavidin-agarose (Sigma) and analyzed by Western blotting, as described above, using the MAb 6C11 or 5H5.

    Article Title: RNA affinity tags for purification of RNAs and ribonucleoprotein complexes
    Article Snippet: Therefore, different buffer compositions (e.g., lower or higher salt concentration, addition of glycerol or detergents) should be tested and might be used successfully as binding buffer with the aptamer tags to suit specific requirements of various systems. .. The amount of affinity matrix used to isolate the tagged complexes should really be determined experimentally, but as a rough guide to purification of complexes from crude yeast lysates, approximately 10–20 μl Sephadex G-200 (Pharmacia) or streptavidin–agarose (Sigma) is used for each milligram of protein in the lysates.

    Lysis:

    Article Title: XIAP upregulates expression of HIF target genes by targeting HIF1α for Lys63-linked polyubiquitination
    Article Snippet: .. Ubiquitination assays For ubiquitination assays, cells were lysed at room temperature under denaturing conditions (8 M urea, 50 mM Tris [pH 8.0], 300 mM NaCl, 50 mM Na2 HPO4 , 0.5% NP-40, 1 mM PMSF, supplemented with protease inhibitors) and ubiquitinated material was recovered by rotation with NiNTA-agarose (Invitrogen) or streptavidin-agarose (Sigma), washed 3× with lysis buffer and analysed by western blotting. .. Quantitative reverse transcription-PCR Total RNA was isolated using the Peqgold Total RNA Isolation Kit (Peqlab) according to the manufacturer's instructions.

    Article Title: A Unique Element in the Cytoplasmic Tail of the Type II Transforming Growth Factor-? Receptor Controls Basolateral Delivery
    Article Snippet: Streptavidin agarose (50 μl; Novagen, Madison, WI) was mixed with 500 μg–1 mg of protein in 1 ml total volume, and the samples were incubated for 2 h at 4°C with agitation. .. The agarose was washed four times with lysis buffer (1 ml), and the biotin-bound proteins were eluted by boiling for 5 min in Laemmli buffer.

    Article Title: Generation of Recombinant Polioviruses Harboring RNA Affinity Tags in the 5′ and 3′ Noncoding Regions of Genomic RNAs
    Article Snippet: .. In vitro transcribed RNA (11 μg) was renatured in TE buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA) by heating at 56 °C for 5 min, 37 °C for 10 min, and incubating at room temperature for 15 min. Streptavidin-agarose (SigmaAldrich,) was prepared by washing 10 times in lysis buffer (50 mM HEPES pH 7.4, 10 mM MgCl2 , 100 mM NaCl, 1 mM DTT, 0.1% Triton X-100, 10% glycerol), Complete protease inhibitors (Roche) and resuspended to 50% slurry in lysis buffer. .. Sephadex matrix was prepared by swelling 0.5 g Sephadex G-200 (Sigma-Aldrich) in 40 mL lysis buffer overnight at room temperature.

    Recombinant:

    Article Title: Peptide EphB2/CTF2 Generated by the ?-Secretase Processing of EphB2 Receptor Promotes Tyrosine Phosphorylation and Cell Surface Localization of N-Methyl-d-aspartate Receptors *
    Article Snippet: .. Magnesium/ATP mixture and recombinant active EphB2R C-terminal kinase were acquired from Upstate Biotechnology (catalog number 14-553) and [γ-32 P]ATP was from PerkinElmer Life Sciences, and streptavidin-agarose was from Sigma. .. Retroviral EphB2R expression constructs were described previously ( ).

    Article Title: Two new mutations in the HIF2A gene associated with erythrocytosis
    Article Snippet: .. Ten ng of recombinant VHL was incubated without or with 1 μg of biotinylated hydroxyproline (Hyp)-564 HIF-1α (556–574) prebound to 10 μl of streptavidin-agarose (Sigma) in the absence or presence of 5 nM wild type (WT) or F540L Hyp-531 HIF-2α (527–542) peptide. .. The resins were washed, eluted, and the eluates subjected to SDS-PAGE and western blotting using anti-Flag antibodies (Sigma), essentially as described [ , ].

    Article Title: Interaction of fascin and protein kinase C?: a novel intersection in cell adhesion and motility
    Article Snippet: Synthetic peptides corresponding to the HIV TAT protein membrane permeablization sequence ( ) fused to the sequence of aa 33–47 of fascin (identical in human and mouse fascin) were prepared in unconjugated, N-terminal biotinylated, or N-terminal FITC-conjugated forms, by Genemed Synthesis Inc. For pull-downs, biotinylated peptides were bound to streptavidin–agarose (Sigma) for 16 h at 4°C. .. To examine direct binding between fascin and PKCα, 400 ng of the purified fascin was bound to Ni-NTA beads and mixed with 100 ng recombinant PKCα (PanVera), or with cell extracts, in buffer containing 20 mM HEPES, pH 7.4, 5 mM MgCl2 , 1.25 mM CaCl2 , 1 mM dithiothreitol, 1 mM ATP, 0.03% Triton X-100 in the presence or absence of activating lipid mixture, with or without TAT-FAS peptides, for 30 min at 30°C.

    Article Title: RhoA S-nitrosylation as a regulatory mechanism influencing endothelial barrier function in response to G+-bacterial toxins
    Article Snippet: To identify sites of S-nitrosylation, the biotin-switch assay was performed as described above on 1 mg of recombinant RhoA incubated with 100 μM of the S-NO donor, nitrosocysteine for 30 min. RhoA was precipitated, washed and then trypsinized overnight using 5 μg of sequencing-grade trypsin (Promega, Madison, WI) at 37 °C. .. Trypsin was inactivated by addition of 1 mM 4-(2- Aminomethyl) benzenesulfonyl fluoride hydrochloride (AEBSF, Sigma, St. Louis, MO), and biotinylated peptides of RhoA were precipitated with streptavidin–agarose (Sigma, St. Louis, MO) and washed four times with 50 mM ammonium bicarbonate.

    Article Title: Cross-species Analysis Reveals Evolving and Conserved Features of the Nuclear Factor ?B (NF-?B) Proteins *
    Article Snippet: Briefly, pET vectors for expression in BL21(DE3) Escherichia coli (Merck) were used to produce His-tagged recombinant proteins. .. NF-κB proteins were purified by affinity chromatography in two steps: using first the nickel-nitrilotriacetic acid His-Bind resin system (Merck) and then biotinylated DNA oligonucleotides attached to streptavidin-agarose (Sigma).

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  • 99
    Millipore streptavidin
    ( A ) Binding analyses of Csn2 in the presence and absence of EGTA and free DNA ends on 2% Tris-acetate agarose gel. In each lane 168 ng linear DNA and 7.2 mM CaCl 2 were employed. The numbers above the lanes indicate the order of addition of <t>streptavidin</t> (2 µg), Csn2 (4.7 µg), or EGTA (14 mM) in a total volume of 14.4 µl. Lanes 2–5: Influence of EGTA on Csn2-DNA interaction is shown. Lanes 6–9: 168 ng of the end-biotinylated DNA fragment were incubated first with streptavidin to block the DNA ends. Lanes 10 and 11: Streptavidin was added after binding of Csn2. After separation of the complexes the agarose gel was stained with ethidium bromide. ( B ) Schematic presentation of the binding analysis, shown in (A).
    Streptavidin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Millipore streptavidin sepharose affinity columns
    <t>Streptavidin-affinity-enriched</t> PBI686-tagged protein extracts. Total protein extract, photo-cross-linked with PBI686 was enriched using <t>streptavidin—Sepharose</t> affinity chromatography. Eluted proteins were desalted, concentrated and analysed using far-Western blot analysis with a streptavidin—HRP conjugate (lane 1) and silver-staining (lane 2) techniques. Band regions that were excised are indicated with letters A-C. The molecular mass in kDa is indicated.
    Streptavidin Sepharose Affinity Columns, supplied by Millipore, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Millipore streptavidin agarose beads
    Proline-rich reading array screen and peptide pulldown. (A) Use of biotinylated eVP40 WT (MRRVILPTAPPEYMEAI[Lys-biotin]) peptide (50 μg) to screen a proline-rich reading array. The GST-WW domain fusion proteins are arrayed in duplicate and at different angles, as indicated in enlarged box C. Box C shows duplicate samples of all four WW domains from WWP1, WWP2, and ITCH as indicated. Additional positive interactions are indicated in the highlighted red boxes and ovals (A to H). The eVP40 mutant peptide (MRRVILPTAAAEAMEAI[Lys-biotin]) did not interact with any GST-WW domain fusion protein (data not shown). (B) Exogenously expressed FLAG-tagged WWP1-WT was pulled down with <t>streptavidin</t> beads bound to either eVP40 WT (WT) or PPXY mutant (mut) peptides and detected by Western blotting using anti-Flag antiserum (top). Expression controls for WWP1 and actin are shown (bottom).
    Streptavidin Agarose Beads, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 293 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    ( A ) Binding analyses of Csn2 in the presence and absence of EGTA and free DNA ends on 2% Tris-acetate agarose gel. In each lane 168 ng linear DNA and 7.2 mM CaCl 2 were employed. The numbers above the lanes indicate the order of addition of streptavidin (2 µg), Csn2 (4.7 µg), or EGTA (14 mM) in a total volume of 14.4 µl. Lanes 2–5: Influence of EGTA on Csn2-DNA interaction is shown. Lanes 6–9: 168 ng of the end-biotinylated DNA fragment were incubated first with streptavidin to block the DNA ends. Lanes 10 and 11: Streptavidin was added after binding of Csn2. After separation of the complexes the agarose gel was stained with ethidium bromide. ( B ) Schematic presentation of the binding analysis, shown in (A).

    Journal: Nucleic Acids Research

    Article Title: Double-strand DNA end-binding and sliding of the toroidal CRISPR-associated protein Csn2

    doi: 10.1093/nar/gkt315

    Figure Lengend Snippet: ( A ) Binding analyses of Csn2 in the presence and absence of EGTA and free DNA ends on 2% Tris-acetate agarose gel. In each lane 168 ng linear DNA and 7.2 mM CaCl 2 were employed. The numbers above the lanes indicate the order of addition of streptavidin (2 µg), Csn2 (4.7 µg), or EGTA (14 mM) in a total volume of 14.4 µl. Lanes 2–5: Influence of EGTA on Csn2-DNA interaction is shown. Lanes 6–9: 168 ng of the end-biotinylated DNA fragment were incubated first with streptavidin to block the DNA ends. Lanes 10 and 11: Streptavidin was added after binding of Csn2. After separation of the complexes the agarose gel was stained with ethidium bromide. ( B ) Schematic presentation of the binding analysis, shown in (A).

    Article Snippet: The volumes of the binding reaction without EGTA or streptavidin were adjusted by addition of deionized water (Millipore).

    Techniques: Binding Assay, Agarose Gel Electrophoresis, Incubation, Blocking Assay, Staining

    Streptavidin-affinity-enriched PBI686-tagged protein extracts. Total protein extract, photo-cross-linked with PBI686 was enriched using streptavidin—Sepharose affinity chromatography. Eluted proteins were desalted, concentrated and analysed using far-Western blot analysis with a streptavidin—HRP conjugate (lane 1) and silver-staining (lane 2) techniques. Band regions that were excised are indicated with letters A-C. The molecular mass in kDa is indicated.

    Journal: PLoS ONE

    Article Title: Identification of Interactions between Abscisic Acid and Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase

    doi: 10.1371/journal.pone.0133033

    Figure Lengend Snippet: Streptavidin-affinity-enriched PBI686-tagged protein extracts. Total protein extract, photo-cross-linked with PBI686 was enriched using streptavidin—Sepharose affinity chromatography. Eluted proteins were desalted, concentrated and analysed using far-Western blot analysis with a streptavidin—HRP conjugate (lane 1) and silver-staining (lane 2) techniques. Band regions that were excised are indicated with letters A-C. The molecular mass in kDa is indicated.

    Article Snippet: Protein fractions were eluted by streptavidin—Sepharose affinity columns, desalted, concentrated using AmiconTM Ultrafree centrifugal filters (Millipore), and visualized using a FOCUS-FAST silver-stain kit.

    Techniques: Affinity Chromatography, Far Western Blot, Silver Staining

    Proline-rich reading array screen and peptide pulldown. (A) Use of biotinylated eVP40 WT (MRRVILPTAPPEYMEAI[Lys-biotin]) peptide (50 μg) to screen a proline-rich reading array. The GST-WW domain fusion proteins are arrayed in duplicate and at different angles, as indicated in enlarged box C. Box C shows duplicate samples of all four WW domains from WWP1, WWP2, and ITCH as indicated. Additional positive interactions are indicated in the highlighted red boxes and ovals (A to H). The eVP40 mutant peptide (MRRVILPTAAAEAMEAI[Lys-biotin]) did not interact with any GST-WW domain fusion protein (data not shown). (B) Exogenously expressed FLAG-tagged WWP1-WT was pulled down with streptavidin beads bound to either eVP40 WT (WT) or PPXY mutant (mut) peptides and detected by Western blotting using anti-Flag antiserum (top). Expression controls for WWP1 and actin are shown (bottom).

    Journal: Journal of Virology

    Article Title: Ubiquitin Ligase WWP1 Interacts with Ebola Virus VP40 To Regulate Egress

    doi: 10.1128/JVI.00812-17

    Figure Lengend Snippet: Proline-rich reading array screen and peptide pulldown. (A) Use of biotinylated eVP40 WT (MRRVILPTAPPEYMEAI[Lys-biotin]) peptide (50 μg) to screen a proline-rich reading array. The GST-WW domain fusion proteins are arrayed in duplicate and at different angles, as indicated in enlarged box C. Box C shows duplicate samples of all four WW domains from WWP1, WWP2, and ITCH as indicated. Additional positive interactions are indicated in the highlighted red boxes and ovals (A to H). The eVP40 mutant peptide (MRRVILPTAAAEAMEAI[Lys-biotin]) did not interact with any GST-WW domain fusion protein (data not shown). (B) Exogenously expressed FLAG-tagged WWP1-WT was pulled down with streptavidin beads bound to either eVP40 WT (WT) or PPXY mutant (mut) peptides and detected by Western blotting using anti-Flag antiserum (top). Expression controls for WWP1 and actin are shown (bottom).

    Article Snippet: Streptavidin agarose beads (Millipore) were prewashed once with 1× mild buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% NP-40, 5 mM EDTA, 5 mM EGTA, 15 mM MgCl2 ), and 15 μg of the WT or PPXY mutant eVP40 peptide was incubated with the prewashed streptavidin beads in 500 μl of 1× mild buffer for 1 h at 4°C with rocking.

    Techniques: Mutagenesis, Western Blot, Expressing

    The rate of cell surface expression/appearance/transport of BRI2 is reduced in the absence of N-glycosylation. Wild-type mycBRI2 or mycBRI2/N170A was expressed in HEK293 cells. The newly synthesized proteins were labeled with 35 S in radiolabeling medium for 2 h (pulse) at 16°C and then were incubated in non-radiolabeling medium for 0′, 20′, 40′ and 60′ (chase). ( A ) Cell surface proteins were labeled with biotin and precipitated with streptavidin beads. Precipitated cell surface proteins were eluted from the beads and immunoprecipitated with 9B11 antibody against the myc epitope before electrophoresis and autoradiography. ( B ) Immunoprecipitation of cell extracts with 9B11, electrophoresis and autoradiography were performed to verify the expression levels of BRI2.

    Journal: Glycobiology

    Article Title: Glycosylation of BRI2 on asparagine 170 is involved in its trafficking to the cell surface but not in its processing by furin or ADAM10

    doi: 10.1093/glycob/cwr097

    Figure Lengend Snippet: The rate of cell surface expression/appearance/transport of BRI2 is reduced in the absence of N-glycosylation. Wild-type mycBRI2 or mycBRI2/N170A was expressed in HEK293 cells. The newly synthesized proteins were labeled with 35 S in radiolabeling medium for 2 h (pulse) at 16°C and then were incubated in non-radiolabeling medium for 0′, 20′, 40′ and 60′ (chase). ( A ) Cell surface proteins were labeled with biotin and precipitated with streptavidin beads. Precipitated cell surface proteins were eluted from the beads and immunoprecipitated with 9B11 antibody against the myc epitope before electrophoresis and autoradiography. ( B ) Immunoprecipitation of cell extracts with 9B11, electrophoresis and autoradiography were performed to verify the expression levels of BRI2.

    Article Snippet: The cell extracts were centrifuged at 15,000 × g for 30 min and supernatants were incubated with 50 μL of streptavidin–agarose beads (Millipore) for 1 h at 4°C.

    Techniques: Expressing, Synthesized, Labeling, Radioactivity, Incubation, Immunoprecipitation, Electrophoresis, Autoradiography

    Inhibition of N-glycosylation of BRI2 inhibits its expression at the cell surface. Wild-type mycBRI2 or mycBRI2/N170A was expressed in HEK293 cells. Cell surface proteins were labeled with biotin (lanes 1 and 2) or were not labeled (lanes 3 and 4), as a control for biotinylation specificity. ( A ) Cell extracts were precipitated with streptavidin beads and analyzed with western blot against myc with 9B11 antibody. ( B ) Cell extracts were directly analyzed with western blot as a control for protein expression. The two immunoreactive bands of BRI2 proteins correspond to the furin-cleaved and the non-cleaved wild-type mycBRI2 or mycBRI2/N170A.

    Journal: Glycobiology

    Article Title: Glycosylation of BRI2 on asparagine 170 is involved in its trafficking to the cell surface but not in its processing by furin or ADAM10

    doi: 10.1093/glycob/cwr097

    Figure Lengend Snippet: Inhibition of N-glycosylation of BRI2 inhibits its expression at the cell surface. Wild-type mycBRI2 or mycBRI2/N170A was expressed in HEK293 cells. Cell surface proteins were labeled with biotin (lanes 1 and 2) or were not labeled (lanes 3 and 4), as a control for biotinylation specificity. ( A ) Cell extracts were precipitated with streptavidin beads and analyzed with western blot against myc with 9B11 antibody. ( B ) Cell extracts were directly analyzed with western blot as a control for protein expression. The two immunoreactive bands of BRI2 proteins correspond to the furin-cleaved and the non-cleaved wild-type mycBRI2 or mycBRI2/N170A.

    Article Snippet: The cell extracts were centrifuged at 15,000 × g for 30 min and supernatants were incubated with 50 μL of streptavidin–agarose beads (Millipore) for 1 h at 4°C.

    Techniques: Inhibition, Expressing, Labeling, Western Blot