strepavidin agarose beads  (Millipore)


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    Structured Review

    Millipore strepavidin agarose beads
    Strepavidin Agarose Beads, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/strepavidin agarose beads/product/Millipore
    Average 95 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    strepavidin agarose beads - by Bioz Stars, 2020-04
    95/100 stars

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    Related Articles

    Centrifugation:

    Article Title: TCR-dependent sensitization of human γδ T cells to non-myeloid IL-18 in cytomegalovirus and tumor stress surveillance
    Article Snippet: After centrifugation at 16,500 rpm/20 min at 4°C, cleared lysates were incubated with biotinyl-VAD-fmk (30 μmol/L) for 30 min at 37°C to precipitate the active cleaved fragment p10 of caspase-1. .. The biotinyl-VAD-fmk/caspase-1 p10 complex was recovered by using streptavidin-Sepharose beads (SIGMA), adding 30 μL of the 1:1 streptavidin-Sepharose suspension per 250 mL of IP buffer for 3 h at 4°C.

    Article Title: Nonstructural Protein Precursor NS4A/B from Hepatitis C Virus Alters Function and Ultrastructure of Host Secretory Apparatus
    Article Snippet: The MHC-I protein was collected by incubating the eluate from the streptavidin-Sepharose beads with 470 μl of lysis buffer and 2 μl of anti-MHC-I primary antibody (0.8 μg of W6/32/ml; Sigma-Aldrich, St. Louis, Mo.) at 4°C for 2 h. Subsequently, 50 μl of protein G-agarose (Life Technologies/GIBCO-BRL) was added, followed by overnight incubation at 4°C. .. Protein G-agarose beads with bound immune complexes were collected by centrifugation at 14,000 × g for 10 min and washed once with lysis buffer, once with lysis buffer containing 500 mM NaCl, and once more with lysis buffer.

    Article Title: Lectin-Induced Inhibition of Desensitization of the Kainate Receptor GluR6 Depends on the Activation State and Can Be Mediated by a Single Native or Ectopic N-Linked Carbohydrate Side Chain
    Article Snippet: .. The homogenate was kept on ice for 60 min. After centrifugation for 60 sec at 16,000 × g to remove yolk platelets and the melanin pigment granula, the supernatants were supplemented with 20 μl streptavidin–Sepharose beads (Sigma) and incubated for 3 hr at 4°C on a rotating rod. .. The streptavidin–Sepharose beads were pelleted by a 60 sec spin and washed three times with H-buffer, and the washed pellets were boiled in 40 μl/oocyte SDS polyacrylamide gel loading buffer (0.8 m β-mercaptoethanol, 6% SDS, 20% glycerol, 25 m m Tris-HCl, pH 6.8, and 0.1% bromphenol blue).

    Article Title: Expression of specific inflammasome gene modules stratifies older individuals into two extreme clinical and immunological states
    Article Snippet: The biotinyl-VAD-fmk/caspase-1 p10 complex was recovered by using streptavidin-Sepharose beads (Sigma), adding 30 µL of 1:1 streptavidin-Sepharose suspension per 250 mL of cell supernatant overnight at 4 °C. .. Beads were pelleted by centrifugation (3000 rpm/10 min/4 °C) and were washed 3 times in cold IP buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 50 mM NaF, 0.3% NP-40, 0.1 mM Na3 VO4 ) before adding SDS loading buffer on top of the beads.

    Article Title: Identification of a Novel Signaling Pathway and Its Relevance for GluA1 Recycling
    Article Snippet: .. After centrifugation of the remaining homogenate for 1 min at 16,000 g, the supernatants were supplemented with 20 µl of washed streptavidin-sepharose beads (Sigma, Munich, Germany) and incubated at 4°C for 3 hrs on a rotating rod. ..

    Article Title: Glucocorticoid adrenal steroids and glucocorticoid-inducible kinase isoforms in the regulation of GluR6 expression
    Article Snippet: .. After centrifugation of the remaining homogenate for 1 min at 16 000 g , the supernatants were supplemented with 20 μl of washed streptavidin/sepharose beads (Sigma-Aldrich) and incubated at 4°C for 3 h on a rotating rod. ..

    Amplification:

    Article Title: SAM68 is a physiological regulator of SMN2 splicing in spinal muscular atrophy
    Article Snippet: Biotin-RNA pull-down Biotin-RNA pull-down experiments were performed using HEK293T cell nuclear extracts and SMN2 RNA probes in vitro synthesized from a PCR product amplified using primers (Table S1) and the plasmid PCI-SMN2 in the presence of biotin-labeled deoxynucleotide (Roche) as described previously ( ). .. Extracts were precleared for 1 h on protein A–Sepharose beads (Sigma-Aldrich) and 1 h on streptavidin-Sepharose beads (Sigma-Aldrich).

    Positive Control:

    Article Title: TCR-dependent sensitization of human γδ T cells to non-myeloid IL-18 in cytomegalovirus and tumor stress surveillance
    Article Snippet: The biotinyl-VAD-fmk/caspase-1 p10 complex was recovered by using streptavidin-Sepharose beads (SIGMA), adding 30 μL of the 1:1 streptavidin-Sepharose suspension per 250 mL of IP buffer for 3 h at 4°C. .. For positive control of caspase-1 cleavage, THP-1 cell lines in suspension were treated with 50 ng/mL phorbol 12-myristate 13-acetate (PMA) for 12 h at 37°C.

    Synthesized:

    Article Title: Nonstructural Protein Precursor NS4A/B from Hepatitis C Virus Alters Function and Ultrastructure of Host Secretory Apparatus
    Article Snippet: Paragraph title: Cell surface expression of newly synthesized MHC-I molecules. ... The MHC-I protein was collected by incubating the eluate from the streptavidin-Sepharose beads with 470 μl of lysis buffer and 2 μl of anti-MHC-I primary antibody (0.8 μg of W6/32/ml; Sigma-Aldrich, St. Louis, Mo.) at 4°C for 2 h. Subsequently, 50 μl of protein G-agarose (Life Technologies/GIBCO-BRL) was added, followed by overnight incubation at 4°C.

    Article Title: SAM68 is a physiological regulator of SMN2 splicing in spinal muscular atrophy
    Article Snippet: Biotin-RNA pull-down Biotin-RNA pull-down experiments were performed using HEK293T cell nuclear extracts and SMN2 RNA probes in vitro synthesized from a PCR product amplified using primers (Table S1) and the plasmid PCI-SMN2 in the presence of biotin-labeled deoxynucleotide (Roche) as described previously ( ). .. Extracts were precleared for 1 h on protein A–Sepharose beads (Sigma-Aldrich) and 1 h on streptavidin-Sepharose beads (Sigma-Aldrich).

    SDS-Gel:

    Article Title: Lectin-Induced Inhibition of Desensitization of the Kainate Receptor GluR6 Depends on the Activation State and Can Be Mediated by a Single Native or Ectopic N-Linked Carbohydrate Side Chain
    Article Snippet: The homogenate was kept on ice for 60 min. After centrifugation for 60 sec at 16,000 × g to remove yolk platelets and the melanin pigment granula, the supernatants were supplemented with 20 μl streptavidin–Sepharose beads (Sigma) and incubated for 3 hr at 4°C on a rotating rod. .. SDS gel electrophoresis and Western blotting.

    Incubation:

    Article Title: TCR-dependent sensitization of human γδ T cells to non-myeloid IL-18 in cytomegalovirus and tumor stress surveillance
    Article Snippet: After centrifugation at 16,500 rpm/20 min at 4°C, cleared lysates were incubated with biotinyl-VAD-fmk (30 μmol/L) for 30 min at 37°C to precipitate the active cleaved fragment p10 of caspase-1. .. The biotinyl-VAD-fmk/caspase-1 p10 complex was recovered by using streptavidin-Sepharose beads (SIGMA), adding 30 μL of the 1:1 streptavidin-Sepharose suspension per 250 mL of IP buffer for 3 h at 4°C.

    Article Title: Nonstructural Protein Precursor NS4A/B from Hepatitis C Virus Alters Function and Ultrastructure of Host Secretory Apparatus
    Article Snippet: .. The MHC-I protein was collected by incubating the eluate from the streptavidin-Sepharose beads with 470 μl of lysis buffer and 2 μl of anti-MHC-I primary antibody (0.8 μg of W6/32/ml; Sigma-Aldrich, St. Louis, Mo.) at 4°C for 2 h. Subsequently, 50 μl of protein G-agarose (Life Technologies/GIBCO-BRL) was added, followed by overnight incubation at 4°C. .. Protein G-agarose beads with bound immune complexes were collected by centrifugation at 14,000 × g for 10 min and washed once with lysis buffer, once with lysis buffer containing 500 mM NaCl, and once more with lysis buffer.

    Article Title: Osp/Claudin-11 Forms a Complex with a Novel Member of the Tetraspanin Super Family and ?1 Integrin and Regulates Proliferation and Migration of Oligodendrocytes
    Article Snippet: In brief, primary oligodendrocyte monolayers were washed and then incubated for 30 min at room temperature in the presence of 1 mM sulfosuccinimydyl-6 (biotinamido)-hexanoate (Sulpho-NHS-Biotin; Pierce Chemical Co.) in PBS. .. Cells were solubilized after washing with PBS and 0.5 M Tris-Cl, centrifuged, and Strepavidin-agarose beads (Sigma-Aldrich) were added to the supernatant.

    Article Title: SAM68 is a physiological regulator of SMN2 splicing in spinal muscular atrophy
    Article Snippet: Extracts were precleared for 1 h on protein A–Sepharose beads (Sigma-Aldrich) and 1 h on streptavidin-Sepharose beads (Sigma-Aldrich). .. Precleared extracts were then incubated with streptavidin-Sepharose beads in the presence of 0.1% BSA and biotinylated RNA probe for 2 h at 4°C under rotation.

    Article Title: Kif26b controls endothelial cell polarity through the Dishevelled/Daam1-dependent planar cell polarity–signaling pathway
    Article Snippet: In brief, for immunoprecipitation, lysates were incubated with primary antibody at +4°C, followed by incubation with protein G–Sepharose slurry. .. Active Cdc42–CRIB complexes were precipitated using streptavidin–Sepharose beads (Sigma-Aldrich).

    Article Title: Lectin-Induced Inhibition of Desensitization of the Kainate Receptor GluR6 Depends on the Activation State and Can Be Mediated by a Single Native or Ectopic N-Linked Carbohydrate Side Chain
    Article Snippet: .. The homogenate was kept on ice for 60 min. After centrifugation for 60 sec at 16,000 × g to remove yolk platelets and the melanin pigment granula, the supernatants were supplemented with 20 μl streptavidin–Sepharose beads (Sigma) and incubated for 3 hr at 4°C on a rotating rod. .. The streptavidin–Sepharose beads were pelleted by a 60 sec spin and washed three times with H-buffer, and the washed pellets were boiled in 40 μl/oocyte SDS polyacrylamide gel loading buffer (0.8 m β-mercaptoethanol, 6% SDS, 20% glycerol, 25 m m Tris-HCl, pH 6.8, and 0.1% bromphenol blue).

    Article Title: Expression of specific inflammasome gene modules stratifies older individuals into two extreme clinical and immunological states
    Article Snippet: For recovery of active caspase-1, cell culture supernatants of adherent THP-1 cells that were treated with one of the four compounds of interest were incubated with biotinyl-VAD-fmk (30 µM) overnight at 4 °C in order to precipitate the active, cleaved fragment p10 of caspase-1. .. The biotinyl-VAD-fmk/caspase-1 p10 complex was recovered by using streptavidin-Sepharose beads (Sigma), adding 30 µL of 1:1 streptavidin-Sepharose suspension per 250 mL of cell supernatant overnight at 4 °C.

    Article Title: Identification of a Novel Signaling Pathway and Its Relevance for GluA1 Recycling
    Article Snippet: .. After centrifugation of the remaining homogenate for 1 min at 16,000 g, the supernatants were supplemented with 20 µl of washed streptavidin-sepharose beads (Sigma, Munich, Germany) and incubated at 4°C for 3 hrs on a rotating rod. ..

    Article Title: Regulation of mRNA Abundance by Polypyrimidine Tract-Binding Protein-Controlled Alternate 5′ Splice Site Choice
    Article Snippet: .. RNA-protein complexes were then incubated with 20 µl of Streptavidin Sepharose Beads (Sigma) pre-washed in buffer D for 1 hour at 4°C. .. The beads were then washed thrice with buffer D and the RNA-associated proteins were eluted by boiling the beads for 10 min in 30 µl of 1× SDS PAGE sample buffer (0.0625 M Tris-HCl pH 6.8, 2% SDS, 5% β-mercapthoethanol, 10% glycerol and 0.01% bromophenol blue) and subsequently analyzed by immunoblotting.

    Article Title: Glucocorticoid adrenal steroids and glucocorticoid-inducible kinase isoforms in the regulation of GluR6 expression
    Article Snippet: .. After centrifugation of the remaining homogenate for 1 min at 16 000 g , the supernatants were supplemented with 20 μl of washed streptavidin/sepharose beads (Sigma-Aldrich) and incubated at 4°C for 3 h on a rotating rod. ..

    Proliferation Assay:

    Article Title: A small-molecule inhibitor of isoprenylcysteine carboxyl methyltransferase with antitumor activity in cancer cells
    Article Snippet: Farnesyl pyrophosphate was from Biomol, the chemical library was from PPD Discovery (Research Triangle Park, NC), streptavidin-Sepharose beads were from Amersham Pharmacia, puromycin and S -adenosylmethionine (AdoMet) were from Sigma, and S -(5′-adenosyl)- l -homocysteine was from Fluka. .. CellTiter 96 Aqueous One solution cell proliferation assay was from Promega. pEGFP and pLPCX were from Clontech.

    Infection:

    Article Title: TCR-dependent sensitization of human γδ T cells to non-myeloid IL-18 in cytomegalovirus and tumor stress surveillance
    Article Snippet: For recovery of active caspase-1, pellets of HUVECs uninfected or infected with HCMV were suspended in IP buffer (50 mmol/L Tris–HCl, pH 7.4, 150 mmol/L NaCl, 50 mmol/L NaF, 0.3% NP-40, 0.1 mmol/L Na3 VO4 ), 20 μg/mL leupeptin, 20 μg/mL aprotinin, 1 mmol/L phenylmethyl sulfonyl fluoride [PMSF]). .. The biotinyl-VAD-fmk/caspase-1 p10 complex was recovered by using streptavidin-Sepharose beads (SIGMA), adding 30 μL of the 1:1 streptavidin-Sepharose suspension per 250 mL of IP buffer for 3 h at 4°C.

    Expressing:

    Article Title: Nonstructural Protein Precursor NS4A/B from Hepatitis C Virus Alters Function and Ultrastructure of Host Secretory Apparatus
    Article Snippet: Paragraph title: Cell surface expression of newly synthesized MHC-I molecules. ... The MHC-I protein was collected by incubating the eluate from the streptavidin-Sepharose beads with 470 μl of lysis buffer and 2 μl of anti-MHC-I primary antibody (0.8 μg of W6/32/ml; Sigma-Aldrich, St. Louis, Mo.) at 4°C for 2 h. Subsequently, 50 μl of protein G-agarose (Life Technologies/GIBCO-BRL) was added, followed by overnight incubation at 4°C.

    BIA-KA:

    Article Title: Spatial oxidation of L-plastin downmodulates actin-based functions of tumor cells
    Article Snippet: .. A site-directed mutagenesis kit, (Invitrogen, A13282) and LPL-specific AcceII siRNAs (A-011716-13 and custom-designed control siRNA and siTRX1, Dharmacon), Vivaspin 6 (VS0601, Sartorius); ZebaSpin desalting columns (89893), BCA protein assay kit (23225), protein concentrators (88513), Pierce centrifuge columns (89868) and Ni-NTA beads (88221), Thermo Fischer Scientific; CAT (C9322), NEM (E3876), mmPEG24 (22713), poly-D-lysine (P6407), Streptavidin sepharose beads (71-5004-40AE), gelatin (G1393), Imidazole (I0250), Biotin (B4501), anti-FLAG M2 affinity gel (A2220), and Duolink In Situ Orange Starter Kit Mouse/Rabbit (PLA, DUO92102) were purchased from Sigma–Aldrich. .. Additionally, DTT (6908.1, Carl Roth), d5-NEM (D-6141, EQ Laboratories), Polypropylene columns (35964, Qiagen), recombinant actin (AKL99, Tebu-Bio), an actin-bundling assay kit (BK001, Tebu-Bio), a H2 O2 detection kit (ICT-9132, Biomol), 35 mm μ-Dishes (81156, Ibidi), 8-μm pore-sized Transwell inserts (3422, Corning), Matrigel invasion chambers (354480, Corning), a gelatin matrix degradation assay kit (ECM670, Merck), recombinant human TRX1 (ab51064, Abcam), an MMP activity assay kit (ab112146, Abcam), and an MMP antibody array (ab134004, Abcam) were purchased.

    Modification:

    Article Title: Osp/Claudin-11 Forms a Complex with a Novel Member of the Tetraspanin Super Family and ?1 Integrin and Regulates Proliferation and Migration of Oligodendrocytes
    Article Snippet: Cell Surface Biotinylation Cell surface proteins of primary oligodendrocytes were biotinylated by using a method modified from . .. Cells were solubilized after washing with PBS and 0.5 M Tris-Cl, centrifuged, and Strepavidin-agarose beads (Sigma-Aldrich) were added to the supernatant.

    Article Title: Identification of a Novel Signaling Pathway and Its Relevance for GluA1 Recycling
    Article Snippet: Paragraph title: Isolation of cell surface proteins after biotinyl-ConA modification ... After centrifugation of the remaining homogenate for 1 min at 16,000 g, the supernatants were supplemented with 20 µl of washed streptavidin-sepharose beads (Sigma, Munich, Germany) and incubated at 4°C for 3 hrs on a rotating rod.

    Western Blot:

    Article Title: SAM68 is a physiological regulator of SMN2 splicing in spinal muscular atrophy
    Article Snippet: Extracts were precleared for 1 h on protein A–Sepharose beads (Sigma-Aldrich) and 1 h on streptavidin-Sepharose beads (Sigma-Aldrich). .. Beads were washed three times with washing buffer, and proteins were eluted in SDS sample buffer for Western blot analysis ( ).

    Article Title: Kif26b controls endothelial cell polarity through the Dishevelled/Daam1-dependent planar cell polarity–signaling pathway
    Article Snippet: Paragraph title: Western blot, cdc42-pull-down activation, and immunoprecipitation assays ... Active Cdc42–CRIB complexes were precipitated using streptavidin–Sepharose beads (Sigma-Aldrich).

    Article Title: Lectin-Induced Inhibition of Desensitization of the Kainate Receptor GluR6 Depends on the Activation State and Can Be Mediated by a Single Native or Ectopic N-Linked Carbohydrate Side Chain
    Article Snippet: The homogenate was kept on ice for 60 min. After centrifugation for 60 sec at 16,000 × g to remove yolk platelets and the melanin pigment granula, the supernatants were supplemented with 20 μl streptavidin–Sepharose beads (Sigma) and incubated for 3 hr at 4°C on a rotating rod. .. SDS gel electrophoresis and Western blotting.

    Activation Assay:

    Article Title: Kif26b controls endothelial cell polarity through the Dishevelled/Daam1-dependent planar cell polarity–signaling pathway
    Article Snippet: Paragraph title: Western blot, cdc42-pull-down activation, and immunoprecipitation assays ... Active Cdc42–CRIB complexes were precipitated using streptavidin–Sepharose beads (Sigma-Aldrich).

    Cell Culture:

    Article Title: Spatial oxidation of L-plastin downmodulates actin-based functions of tumor cells
    Article Snippet: Materials The following cell culture reagents were purchased: RPMI 1640 (11875093, Thermo Fischer Scientific), DMEM (M-L2624-I, Cell Concepts), fetal calf serum (FCS, Pan Biotech), puromycin (540411, Calbiochem), and L-glutamine (25030081, Life Technologies). .. A site-directed mutagenesis kit, (Invitrogen, A13282) and LPL-specific AcceII siRNAs (A-011716-13 and custom-designed control siRNA and siTRX1, Dharmacon), Vivaspin 6 (VS0601, Sartorius); ZebaSpin desalting columns (89893), BCA protein assay kit (23225), protein concentrators (88513), Pierce centrifuge columns (89868) and Ni-NTA beads (88221), Thermo Fischer Scientific; CAT (C9322), NEM (E3876), mmPEG24 (22713), poly-D-lysine (P6407), Streptavidin sepharose beads (71-5004-40AE), gelatin (G1393), Imidazole (I0250), Biotin (B4501), anti-FLAG M2 affinity gel (A2220), and Duolink In Situ Orange Starter Kit Mouse/Rabbit (PLA, DUO92102) were purchased from Sigma–Aldrich.

    Article Title: Expression of specific inflammasome gene modules stratifies older individuals into two extreme clinical and immunological states
    Article Snippet: For recovery of active caspase-1, cell culture supernatants of adherent THP-1 cells that were treated with one of the four compounds of interest were incubated with biotinyl-VAD-fmk (30 µM) overnight at 4 °C in order to precipitate the active, cleaved fragment p10 of caspase-1. .. The biotinyl-VAD-fmk/caspase-1 p10 complex was recovered by using streptavidin-Sepharose beads (Sigma), adding 30 µL of 1:1 streptavidin-Sepharose suspension per 250 mL of cell supernatant overnight at 4 °C.

    Generated:

    Article Title: Regulation of mRNA Abundance by Polypyrimidine Tract-Binding Protein-Controlled Alternate 5′ Splice Site Choice
    Article Snippet: Biotinylated RNA pull-down assays RNA probes were generated by transcribing linearized plasmid DNA in vitro with T7 polymerase (Promega) and biotin RNA labeling mix (Roche) for 2 hours at 37°C. .. RNA-protein complexes were then incubated with 20 µl of Streptavidin Sepharose Beads (Sigma) pre-washed in buffer D for 1 hour at 4°C.

    Imaging:

    Article Title: Kif26b controls endothelial cell polarity through the Dishevelled/Daam1-dependent planar cell polarity–signaling pathway
    Article Snippet: Signal was detected using an Odyssey infrared imaging system (LI-COR Biosciences) or an enhanced chemiluminescence detection system (Amersham). .. Active Cdc42–CRIB complexes were precipitated using streptavidin–Sepharose beads (Sigma-Aldrich).

    Polymerase Chain Reaction:

    Article Title: SAM68 is a physiological regulator of SMN2 splicing in spinal muscular atrophy
    Article Snippet: Biotin-RNA pull-down Biotin-RNA pull-down experiments were performed using HEK293T cell nuclear extracts and SMN2 RNA probes in vitro synthesized from a PCR product amplified using primers (Table S1) and the plasmid PCI-SMN2 in the presence of biotin-labeled deoxynucleotide (Roche) as described previously ( ). .. Extracts were precleared for 1 h on protein A–Sepharose beads (Sigma-Aldrich) and 1 h on streptavidin-Sepharose beads (Sigma-Aldrich).

    Injection:

    Article Title: Lectin-Induced Inhibition of Desensitization of the Kainate Receptor GluR6 Depends on the Activation State and Can Be Mediated by a Single Native or Ectopic N-Linked Carbohydrate Side Chain
    Article Snippet: To identify only that fraction of receptor protein that is inserted in the plasma membrane of the oocytes, surface proteins were tagged with biotin and isolated by streptavidin–Sepharose-mediated precipitation of the labeled protein 4–6 d after RNA injection. .. The homogenate was kept on ice for 60 min. After centrifugation for 60 sec at 16,000 × g to remove yolk platelets and the melanin pigment granula, the supernatants were supplemented with 20 μl streptavidin–Sepharose beads (Sigma) and incubated for 3 hr at 4°C on a rotating rod.

    Recombinant:

    Article Title: Kif26b controls endothelial cell polarity through the Dishevelled/Daam1-dependent planar cell polarity–signaling pathway
    Article Snippet: The activation status of cdc42 was assayed using Wnt5a recombinant protein (400 ng/ml) and the biotinylated peptide corresponding to the CRIB domain of PAK (Proteogenix). .. Active Cdc42–CRIB complexes were precipitated using streptavidin–Sepharose beads (Sigma-Aldrich).

    Article Title: Spatial oxidation of L-plastin downmodulates actin-based functions of tumor cells
    Article Snippet: A site-directed mutagenesis kit, (Invitrogen, A13282) and LPL-specific AcceII siRNAs (A-011716-13 and custom-designed control siRNA and siTRX1, Dharmacon), Vivaspin 6 (VS0601, Sartorius); ZebaSpin desalting columns (89893), BCA protein assay kit (23225), protein concentrators (88513), Pierce centrifuge columns (89868) and Ni-NTA beads (88221), Thermo Fischer Scientific; CAT (C9322), NEM (E3876), mmPEG24 (22713), poly-D-lysine (P6407), Streptavidin sepharose beads (71-5004-40AE), gelatin (G1393), Imidazole (I0250), Biotin (B4501), anti-FLAG M2 affinity gel (A2220), and Duolink In Situ Orange Starter Kit Mouse/Rabbit (PLA, DUO92102) were purchased from Sigma–Aldrich. .. Additionally, DTT (6908.1, Carl Roth), d5-NEM (D-6141, EQ Laboratories), Polypropylene columns (35964, Qiagen), recombinant actin (AKL99, Tebu-Bio), an actin-bundling assay kit (BK001, Tebu-Bio), a H2 O2 detection kit (ICT-9132, Biomol), 35 mm μ-Dishes (81156, Ibidi), 8-μm pore-sized Transwell inserts (3422, Corning), Matrigel invasion chambers (354480, Corning), a gelatin matrix degradation assay kit (ECM670, Merck), recombinant human TRX1 (ab51064, Abcam), an MMP activity assay kit (ab112146, Abcam), and an MMP antibody array (ab134004, Abcam) were purchased.

    Mutagenesis:

    Article Title: Spatial oxidation of L-plastin downmodulates actin-based functions of tumor cells
    Article Snippet: .. A site-directed mutagenesis kit, (Invitrogen, A13282) and LPL-specific AcceII siRNAs (A-011716-13 and custom-designed control siRNA and siTRX1, Dharmacon), Vivaspin 6 (VS0601, Sartorius); ZebaSpin desalting columns (89893), BCA protein assay kit (23225), protein concentrators (88513), Pierce centrifuge columns (89868) and Ni-NTA beads (88221), Thermo Fischer Scientific; CAT (C9322), NEM (E3876), mmPEG24 (22713), poly-D-lysine (P6407), Streptavidin sepharose beads (71-5004-40AE), gelatin (G1393), Imidazole (I0250), Biotin (B4501), anti-FLAG M2 affinity gel (A2220), and Duolink In Situ Orange Starter Kit Mouse/Rabbit (PLA, DUO92102) were purchased from Sigma–Aldrich. .. Additionally, DTT (6908.1, Carl Roth), d5-NEM (D-6141, EQ Laboratories), Polypropylene columns (35964, Qiagen), recombinant actin (AKL99, Tebu-Bio), an actin-bundling assay kit (BK001, Tebu-Bio), a H2 O2 detection kit (ICT-9132, Biomol), 35 mm μ-Dishes (81156, Ibidi), 8-μm pore-sized Transwell inserts (3422, Corning), Matrigel invasion chambers (354480, Corning), a gelatin matrix degradation assay kit (ECM670, Merck), recombinant human TRX1 (ab51064, Abcam), an MMP activity assay kit (ab112146, Abcam), and an MMP antibody array (ab134004, Abcam) were purchased.

    Isolation:

    Article Title: Lectin-Induced Inhibition of Desensitization of the Kainate Receptor GluR6 Depends on the Activation State and Can Be Mediated by a Single Native or Ectopic N-Linked Carbohydrate Side Chain
    Article Snippet: To identify only that fraction of receptor protein that is inserted in the plasma membrane of the oocytes, surface proteins were tagged with biotin and isolated by streptavidin–Sepharose-mediated precipitation of the labeled protein 4–6 d after RNA injection. .. The homogenate was kept on ice for 60 min. After centrifugation for 60 sec at 16,000 × g to remove yolk platelets and the melanin pigment granula, the supernatants were supplemented with 20 μl streptavidin–Sepharose beads (Sigma) and incubated for 3 hr at 4°C on a rotating rod.

    Article Title: Identification of a Novel Signaling Pathway and Its Relevance for GluA1 Recycling
    Article Snippet: Paragraph title: Isolation of cell surface proteins after biotinyl-ConA modification ... After centrifugation of the remaining homogenate for 1 min at 16,000 g, the supernatants were supplemented with 20 µl of washed streptavidin-sepharose beads (Sigma, Munich, Germany) and incubated at 4°C for 3 hrs on a rotating rod.

    Article Title: Glucocorticoid adrenal steroids and glucocorticoid-inducible kinase isoforms in the regulation of GluR6 expression
    Article Snippet: To identify the fraction of receptor protein inserted in the plasma membrane, surface proteins were tagged with biotinylated ConA (Sigma-Aldrich), and isolated by streptavidin/sepharose-mediated precipitation of the biotinyl-ConA/protein complex, as described elsewere ( ). .. After centrifugation of the remaining homogenate for 1 min at 16 000 g , the supernatants were supplemented with 20 μl of washed streptavidin/sepharose beads (Sigma-Aldrich) and incubated at 4°C for 3 h on a rotating rod.

    Size-exclusion Chromatography:

    Article Title: Lectin-Induced Inhibition of Desensitization of the Kainate Receptor GluR6 Depends on the Activation State and Can Be Mediated by a Single Native or Ectopic N-Linked Carbohydrate Side Chain
    Article Snippet: .. The homogenate was kept on ice for 60 min. After centrifugation for 60 sec at 16,000 × g to remove yolk platelets and the melanin pigment granula, the supernatants were supplemented with 20 μl streptavidin–Sepharose beads (Sigma) and incubated for 3 hr at 4°C on a rotating rod. .. The streptavidin–Sepharose beads were pelleted by a 60 sec spin and washed three times with H-buffer, and the washed pellets were boiled in 40 μl/oocyte SDS polyacrylamide gel loading buffer (0.8 m β-mercaptoethanol, 6% SDS, 20% glycerol, 25 m m Tris-HCl, pH 6.8, and 0.1% bromphenol blue).

    Labeling:

    Article Title: Lectin-Induced Inhibition of Desensitization of the Kainate Receptor GluR6 Depends on the Activation State and Can Be Mediated by a Single Native or Ectopic N-Linked Carbohydrate Side Chain
    Article Snippet: To identify only that fraction of receptor protein that is inserted in the plasma membrane of the oocytes, surface proteins were tagged with biotin and isolated by streptavidin–Sepharose-mediated precipitation of the labeled protein 4–6 d after RNA injection. .. The homogenate was kept on ice for 60 min. After centrifugation for 60 sec at 16,000 × g to remove yolk platelets and the melanin pigment granula, the supernatants were supplemented with 20 μl streptavidin–Sepharose beads (Sigma) and incubated for 3 hr at 4°C on a rotating rod.

    Article Title: Regulation of mRNA Abundance by Polypyrimidine Tract-Binding Protein-Controlled Alternate 5′ Splice Site Choice
    Article Snippet: Biotinylated RNA pull-down assays RNA probes were generated by transcribing linearized plasmid DNA in vitro with T7 polymerase (Promega) and biotin RNA labeling mix (Roche) for 2 hours at 37°C. .. RNA-protein complexes were then incubated with 20 µl of Streptavidin Sepharose Beads (Sigma) pre-washed in buffer D for 1 hour at 4°C.

    Purification:

    Article Title: Regulation of mRNA Abundance by Polypyrimidine Tract-Binding Protein-Controlled Alternate 5′ Splice Site Choice
    Article Snippet: Pull-down assays were carried out by incubating 2 µg of purified RNA probes in 20 µl of buffer D (20 mM HEPES, pH 7.9, 100 mM KCl, 20% Glycerol, 0.5 mM DTT and 0.2 mM EDTA) supplemented with 80 ng yeast tRNA, 2.5 µg heparin, 40 units of rRNAsin (Promega) and 50% HeLa S3 NE (vol/vol; dialyzed against buffer D; ∼100 µg protein in total) for 30 min at room temperature. .. RNA-protein complexes were then incubated with 20 µl of Streptavidin Sepharose Beads (Sigma) pre-washed in buffer D for 1 hour at 4°C.

    Sequencing:

    Article Title: Lectin-Induced Inhibition of Desensitization of the Kainate Receptor GluR6 Depends on the Activation State and Can Be Mediated by a Single Native or Ectopic N-Linked Carbohydrate Side Chain
    Article Snippet: All mutations were verified by chain-termination method sequencing using the Sequenase kit from USB. .. The homogenate was kept on ice for 60 min. After centrifugation for 60 sec at 16,000 × g to remove yolk platelets and the melanin pigment granula, the supernatants were supplemented with 20 μl streptavidin–Sepharose beads (Sigma) and incubated for 3 hr at 4°C on a rotating rod.

    Degradation Assay:

    Article Title: Spatial oxidation of L-plastin downmodulates actin-based functions of tumor cells
    Article Snippet: A site-directed mutagenesis kit, (Invitrogen, A13282) and LPL-specific AcceII siRNAs (A-011716-13 and custom-designed control siRNA and siTRX1, Dharmacon), Vivaspin 6 (VS0601, Sartorius); ZebaSpin desalting columns (89893), BCA protein assay kit (23225), protein concentrators (88513), Pierce centrifuge columns (89868) and Ni-NTA beads (88221), Thermo Fischer Scientific; CAT (C9322), NEM (E3876), mmPEG24 (22713), poly-D-lysine (P6407), Streptavidin sepharose beads (71-5004-40AE), gelatin (G1393), Imidazole (I0250), Biotin (B4501), anti-FLAG M2 affinity gel (A2220), and Duolink In Situ Orange Starter Kit Mouse/Rabbit (PLA, DUO92102) were purchased from Sigma–Aldrich. .. Additionally, DTT (6908.1, Carl Roth), d5-NEM (D-6141, EQ Laboratories), Polypropylene columns (35964, Qiagen), recombinant actin (AKL99, Tebu-Bio), an actin-bundling assay kit (BK001, Tebu-Bio), a H2 O2 detection kit (ICT-9132, Biomol), 35 mm μ-Dishes (81156, Ibidi), 8-μm pore-sized Transwell inserts (3422, Corning), Matrigel invasion chambers (354480, Corning), a gelatin matrix degradation assay kit (ECM670, Merck), recombinant human TRX1 (ab51064, Abcam), an MMP activity assay kit (ab112146, Abcam), and an MMP antibody array (ab134004, Abcam) were purchased.

    Activity Assay:

    Article Title: Spatial oxidation of L-plastin downmodulates actin-based functions of tumor cells
    Article Snippet: A site-directed mutagenesis kit, (Invitrogen, A13282) and LPL-specific AcceII siRNAs (A-011716-13 and custom-designed control siRNA and siTRX1, Dharmacon), Vivaspin 6 (VS0601, Sartorius); ZebaSpin desalting columns (89893), BCA protein assay kit (23225), protein concentrators (88513), Pierce centrifuge columns (89868) and Ni-NTA beads (88221), Thermo Fischer Scientific; CAT (C9322), NEM (E3876), mmPEG24 (22713), poly-D-lysine (P6407), Streptavidin sepharose beads (71-5004-40AE), gelatin (G1393), Imidazole (I0250), Biotin (B4501), anti-FLAG M2 affinity gel (A2220), and Duolink In Situ Orange Starter Kit Mouse/Rabbit (PLA, DUO92102) were purchased from Sigma–Aldrich. .. Additionally, DTT (6908.1, Carl Roth), d5-NEM (D-6141, EQ Laboratories), Polypropylene columns (35964, Qiagen), recombinant actin (AKL99, Tebu-Bio), an actin-bundling assay kit (BK001, Tebu-Bio), a H2 O2 detection kit (ICT-9132, Biomol), 35 mm μ-Dishes (81156, Ibidi), 8-μm pore-sized Transwell inserts (3422, Corning), Matrigel invasion chambers (354480, Corning), a gelatin matrix degradation assay kit (ECM670, Merck), recombinant human TRX1 (ab51064, Abcam), an MMP activity assay kit (ab112146, Abcam), and an MMP antibody array (ab134004, Abcam) were purchased.

    Chloramphenicol Acetyltransferase Assay:

    Article Title: Spatial oxidation of L-plastin downmodulates actin-based functions of tumor cells
    Article Snippet: .. A site-directed mutagenesis kit, (Invitrogen, A13282) and LPL-specific AcceII siRNAs (A-011716-13 and custom-designed control siRNA and siTRX1, Dharmacon), Vivaspin 6 (VS0601, Sartorius); ZebaSpin desalting columns (89893), BCA protein assay kit (23225), protein concentrators (88513), Pierce centrifuge columns (89868) and Ni-NTA beads (88221), Thermo Fischer Scientific; CAT (C9322), NEM (E3876), mmPEG24 (22713), poly-D-lysine (P6407), Streptavidin sepharose beads (71-5004-40AE), gelatin (G1393), Imidazole (I0250), Biotin (B4501), anti-FLAG M2 affinity gel (A2220), and Duolink In Situ Orange Starter Kit Mouse/Rabbit (PLA, DUO92102) were purchased from Sigma–Aldrich. .. Additionally, DTT (6908.1, Carl Roth), d5-NEM (D-6141, EQ Laboratories), Polypropylene columns (35964, Qiagen), recombinant actin (AKL99, Tebu-Bio), an actin-bundling assay kit (BK001, Tebu-Bio), a H2 O2 detection kit (ICT-9132, Biomol), 35 mm μ-Dishes (81156, Ibidi), 8-μm pore-sized Transwell inserts (3422, Corning), Matrigel invasion chambers (354480, Corning), a gelatin matrix degradation assay kit (ECM670, Merck), recombinant human TRX1 (ab51064, Abcam), an MMP activity assay kit (ab112146, Abcam), and an MMP antibody array (ab134004, Abcam) were purchased.

    SDS Page:

    Article Title: Osp/Claudin-11 Forms a Complex with a Novel Member of the Tetraspanin Super Family and ?1 Integrin and Regulates Proliferation and Migration of Oligodendrocytes
    Article Snippet: Cells were solubilized after washing with PBS and 0.5 M Tris-Cl, centrifuged, and Strepavidin-agarose beads (Sigma-Aldrich) were added to the supernatant. .. The sample was subjected through 12% SDS-PAGE, and electroblotted protein was probed with anti–OSP/claudin-11 and anti–OAP-1 antibodies.

    Article Title: Kif26b controls endothelial cell polarity through the Dishevelled/Daam1-dependent planar cell polarity–signaling pathway
    Article Snippet: Proteins were then resolved by SDS–PAGE and blotted with the following antibodies: anti Dvl3 from Santa Cruz Biotechnology, anti-Myc and active β-catenin (anti-ABC, clone 8E7) from Millipore, anti-Daam1 from Peprotech, anti–α-tubulin, anti–acetylated tubulin, anti–β-catenin, and anti-Kif26b from Sigma-Aldrich, and anti-V5 from Life Technologies. .. Active Cdc42–CRIB complexes were precipitated using streptavidin–Sepharose beads (Sigma-Aldrich).

    Article Title: Identification of a Novel Signaling Pathway and Its Relevance for GluA1 Recycling
    Article Snippet: After centrifugation of the remaining homogenate for 1 min at 16,000 g, the supernatants were supplemented with 20 µl of washed streptavidin-sepharose beads (Sigma, Munich, Germany) and incubated at 4°C for 3 hrs on a rotating rod. .. The final pellets, containing plasma membrane receptors, were boiled in 20 µl of SDS-PAGE loading buffer (0.8 M ß-mercaptoethanol, 6% SDS, 20% glycerol, 25 mM Tris-HCl, pH 6.8, and 0.1% bromphenol blue).

    Article Title: Regulation of mRNA Abundance by Polypyrimidine Tract-Binding Protein-Controlled Alternate 5′ Splice Site Choice
    Article Snippet: RNA-protein complexes were then incubated with 20 µl of Streptavidin Sepharose Beads (Sigma) pre-washed in buffer D for 1 hour at 4°C. .. The beads were then washed thrice with buffer D and the RNA-associated proteins were eluted by boiling the beads for 10 min in 30 µl of 1× SDS PAGE sample buffer (0.0625 M Tris-HCl pH 6.8, 2% SDS, 5% β-mercapthoethanol, 10% glycerol and 0.01% bromophenol blue) and subsequently analyzed by immunoblotting.

    Article Title: Glucocorticoid adrenal steroids and glucocorticoid-inducible kinase isoforms in the regulation of GluR6 expression
    Article Snippet: After centrifugation of the remaining homogenate for 1 min at 16 000 g , the supernatants were supplemented with 20 μl of washed streptavidin/sepharose beads (Sigma-Aldrich) and incubated at 4°C for 3 h on a rotating rod. .. The final pellets (P) containing plasma membrane receptors were boiled in 20 μl of SDS-PAGE loading buffer (0.8 m β–mercaptoethanol, 6% SDS, 20% glycerol, 25 m m Tris-HCl, pH 6.8, and 0.1% bromophenol blue).

    Plasmid Preparation:

    Article Title: SAM68 is a physiological regulator of SMN2 splicing in spinal muscular atrophy
    Article Snippet: Biotin-RNA pull-down Biotin-RNA pull-down experiments were performed using HEK293T cell nuclear extracts and SMN2 RNA probes in vitro synthesized from a PCR product amplified using primers (Table S1) and the plasmid PCI-SMN2 in the presence of biotin-labeled deoxynucleotide (Roche) as described previously ( ). .. Extracts were precleared for 1 h on protein A–Sepharose beads (Sigma-Aldrich) and 1 h on streptavidin-Sepharose beads (Sigma-Aldrich).

    Article Title: Regulation of mRNA Abundance by Polypyrimidine Tract-Binding Protein-Controlled Alternate 5′ Splice Site Choice
    Article Snippet: Biotinylated RNA pull-down assays RNA probes were generated by transcribing linearized plasmid DNA in vitro with T7 polymerase (Promega) and biotin RNA labeling mix (Roche) for 2 hours at 37°C. .. RNA-protein complexes were then incubated with 20 µl of Streptavidin Sepharose Beads (Sigma) pre-washed in buffer D for 1 hour at 4°C.

    In Vitro:

    Article Title: SAM68 is a physiological regulator of SMN2 splicing in spinal muscular atrophy
    Article Snippet: Biotin-RNA pull-down Biotin-RNA pull-down experiments were performed using HEK293T cell nuclear extracts and SMN2 RNA probes in vitro synthesized from a PCR product amplified using primers (Table S1) and the plasmid PCI-SMN2 in the presence of biotin-labeled deoxynucleotide (Roche) as described previously ( ). .. Extracts were precleared for 1 h on protein A–Sepharose beads (Sigma-Aldrich) and 1 h on streptavidin-Sepharose beads (Sigma-Aldrich).

    Article Title: Regulation of mRNA Abundance by Polypyrimidine Tract-Binding Protein-Controlled Alternate 5′ Splice Site Choice
    Article Snippet: Biotinylated RNA pull-down assays RNA probes were generated by transcribing linearized plasmid DNA in vitro with T7 polymerase (Promega) and biotin RNA labeling mix (Roche) for 2 hours at 37°C. .. RNA-protein complexes were then incubated with 20 µl of Streptavidin Sepharose Beads (Sigma) pre-washed in buffer D for 1 hour at 4°C.

    Electrophoresis:

    Article Title: Lectin-Induced Inhibition of Desensitization of the Kainate Receptor GluR6 Depends on the Activation State and Can Be Mediated by a Single Native or Ectopic N-Linked Carbohydrate Side Chain
    Article Snippet: The homogenate was kept on ice for 60 min. After centrifugation for 60 sec at 16,000 × g to remove yolk platelets and the melanin pigment granula, the supernatants were supplemented with 20 μl streptavidin–Sepharose beads (Sigma) and incubated for 3 hr at 4°C on a rotating rod. .. SDS gel electrophoresis and Western blotting.

    In Situ:

    Article Title: Spatial oxidation of L-plastin downmodulates actin-based functions of tumor cells
    Article Snippet: .. A site-directed mutagenesis kit, (Invitrogen, A13282) and LPL-specific AcceII siRNAs (A-011716-13 and custom-designed control siRNA and siTRX1, Dharmacon), Vivaspin 6 (VS0601, Sartorius); ZebaSpin desalting columns (89893), BCA protein assay kit (23225), protein concentrators (88513), Pierce centrifuge columns (89868) and Ni-NTA beads (88221), Thermo Fischer Scientific; CAT (C9322), NEM (E3876), mmPEG24 (22713), poly-D-lysine (P6407), Streptavidin sepharose beads (71-5004-40AE), gelatin (G1393), Imidazole (I0250), Biotin (B4501), anti-FLAG M2 affinity gel (A2220), and Duolink In Situ Orange Starter Kit Mouse/Rabbit (PLA, DUO92102) were purchased from Sigma–Aldrich. .. Additionally, DTT (6908.1, Carl Roth), d5-NEM (D-6141, EQ Laboratories), Polypropylene columns (35964, Qiagen), recombinant actin (AKL99, Tebu-Bio), an actin-bundling assay kit (BK001, Tebu-Bio), a H2 O2 detection kit (ICT-9132, Biomol), 35 mm μ-Dishes (81156, Ibidi), 8-μm pore-sized Transwell inserts (3422, Corning), Matrigel invasion chambers (354480, Corning), a gelatin matrix degradation assay kit (ECM670, Merck), recombinant human TRX1 (ab51064, Abcam), an MMP activity assay kit (ab112146, Abcam), and an MMP antibody array (ab134004, Abcam) were purchased.

    Proximity Ligation Assay:

    Article Title: Spatial oxidation of L-plastin downmodulates actin-based functions of tumor cells
    Article Snippet: .. A site-directed mutagenesis kit, (Invitrogen, A13282) and LPL-specific AcceII siRNAs (A-011716-13 and custom-designed control siRNA and siTRX1, Dharmacon), Vivaspin 6 (VS0601, Sartorius); ZebaSpin desalting columns (89893), BCA protein assay kit (23225), protein concentrators (88513), Pierce centrifuge columns (89868) and Ni-NTA beads (88221), Thermo Fischer Scientific; CAT (C9322), NEM (E3876), mmPEG24 (22713), poly-D-lysine (P6407), Streptavidin sepharose beads (71-5004-40AE), gelatin (G1393), Imidazole (I0250), Biotin (B4501), anti-FLAG M2 affinity gel (A2220), and Duolink In Situ Orange Starter Kit Mouse/Rabbit (PLA, DUO92102) were purchased from Sigma–Aldrich. .. Additionally, DTT (6908.1, Carl Roth), d5-NEM (D-6141, EQ Laboratories), Polypropylene columns (35964, Qiagen), recombinant actin (AKL99, Tebu-Bio), an actin-bundling assay kit (BK001, Tebu-Bio), a H2 O2 detection kit (ICT-9132, Biomol), 35 mm μ-Dishes (81156, Ibidi), 8-μm pore-sized Transwell inserts (3422, Corning), Matrigel invasion chambers (354480, Corning), a gelatin matrix degradation assay kit (ECM670, Merck), recombinant human TRX1 (ab51064, Abcam), an MMP activity assay kit (ab112146, Abcam), and an MMP antibody array (ab134004, Abcam) were purchased.

    Homogenization:

    Article Title: Identification of a Novel Signaling Pathway and Its Relevance for GluA1 Recycling
    Article Snippet: Since exclusively intact oocytes were used for homogenization, only plasma membrane proteins, not proteins of internal membranes, were labelled. .. After centrifugation of the remaining homogenate for 1 min at 16,000 g, the supernatants were supplemented with 20 µl of washed streptavidin-sepharose beads (Sigma, Munich, Germany) and incubated at 4°C for 3 hrs on a rotating rod.

    Immunoprecipitation:

    Article Title: Osp/Claudin-11 Forms a Complex with a Novel Member of the Tetraspanin Super Family and ?1 Integrin and Regulates Proliferation and Migration of Oligodendrocytes
    Article Snippet: For immunoprecipitation, flasks were washed with PBS and 0.5 M Tris-Cl, and cells were solubilized in buffer A with 1% Triton X-100. .. Cells were solubilized after washing with PBS and 0.5 M Tris-Cl, centrifuged, and Strepavidin-agarose beads (Sigma-Aldrich) were added to the supernatant.

    Article Title: Kif26b controls endothelial cell polarity through the Dishevelled/Daam1-dependent planar cell polarity–signaling pathway
    Article Snippet: Paragraph title: Western blot, cdc42-pull-down activation, and immunoprecipitation assays ... Active Cdc42–CRIB complexes were precipitated using streptavidin–Sepharose beads (Sigma-Aldrich).

    Lysis:

    Article Title: Nonstructural Protein Precursor NS4A/B from Hepatitis C Virus Alters Function and Ultrastructure of Host Secretory Apparatus
    Article Snippet: .. The MHC-I protein was collected by incubating the eluate from the streptavidin-Sepharose beads with 470 μl of lysis buffer and 2 μl of anti-MHC-I primary antibody (0.8 μg of W6/32/ml; Sigma-Aldrich, St. Louis, Mo.) at 4°C for 2 h. Subsequently, 50 μl of protein G-agarose (Life Technologies/GIBCO-BRL) was added, followed by overnight incubation at 4°C. .. Protein G-agarose beads with bound immune complexes were collected by centrifugation at 14,000 × g for 10 min and washed once with lysis buffer, once with lysis buffer containing 500 mM NaCl, and once more with lysis buffer.

    Article Title: Kif26b controls endothelial cell polarity through the Dishevelled/Daam1-dependent planar cell polarity–signaling pathway
    Article Snippet: Briefly, cells were washed with ice-cold phosphate-buffered saline (containing 1 mM MgCl2 and 1 mM CaCl2 ) and lysed for 5 min in cell lysis buffer (1% Nonidet P40, 2% glycerol, 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 7.4, 5 mM MgCl2 , and 150 mM NaCl) containing protease inhibitors and 2 μg of biotin–CRIB peptide. .. Active Cdc42–CRIB complexes were precipitated using streptavidin–Sepharose beads (Sigma-Aldrich).

    Ab Array:

    Article Title: Spatial oxidation of L-plastin downmodulates actin-based functions of tumor cells
    Article Snippet: A site-directed mutagenesis kit, (Invitrogen, A13282) and LPL-specific AcceII siRNAs (A-011716-13 and custom-designed control siRNA and siTRX1, Dharmacon), Vivaspin 6 (VS0601, Sartorius); ZebaSpin desalting columns (89893), BCA protein assay kit (23225), protein concentrators (88513), Pierce centrifuge columns (89868) and Ni-NTA beads (88221), Thermo Fischer Scientific; CAT (C9322), NEM (E3876), mmPEG24 (22713), poly-D-lysine (P6407), Streptavidin sepharose beads (71-5004-40AE), gelatin (G1393), Imidazole (I0250), Biotin (B4501), anti-FLAG M2 affinity gel (A2220), and Duolink In Situ Orange Starter Kit Mouse/Rabbit (PLA, DUO92102) were purchased from Sigma–Aldrich. .. Additionally, DTT (6908.1, Carl Roth), d5-NEM (D-6141, EQ Laboratories), Polypropylene columns (35964, Qiagen), recombinant actin (AKL99, Tebu-Bio), an actin-bundling assay kit (BK001, Tebu-Bio), a H2 O2 detection kit (ICT-9132, Biomol), 35 mm μ-Dishes (81156, Ibidi), 8-μm pore-sized Transwell inserts (3422, Corning), Matrigel invasion chambers (354480, Corning), a gelatin matrix degradation assay kit (ECM670, Merck), recombinant human TRX1 (ab51064, Abcam), an MMP activity assay kit (ab112146, Abcam), and an MMP antibody array (ab134004, Abcam) were purchased.

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    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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    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

    A19 phosphorylation. (A) Western blot analysis. BS-C-1 cells were infected with vFS-A11 and vFS-A19 in the presence of 100 μCi/ml 32 P i . After 18 h, the cells were lysed, and the soluble extract was bound to streptavidin-agarose beads. Bound proteins

    Journal: Journal of Virology

    Article Title: Vaccinia Virus A19 Protein Participates in the Transformation of Spherical Immature Particles to Barrel-Shaped Infectious Virions

    doi: 10.1128/JVI.01258-13

    Figure Lengend Snippet: A19 phosphorylation. (A) Western blot analysis. BS-C-1 cells were infected with vFS-A11 and vFS-A19 in the presence of 100 μCi/ml 32 P i . After 18 h, the cells were lysed, and the soluble extract was bound to streptavidin-agarose beads. Bound proteins

    Article Snippet: Soluble extracts obtained by low-speed centrifugation were allowed to bind to streptavidin-agarose beads (Millipore, Billerica, MA) for 3 h at 4°C.

    Techniques: Western Blot, Infection

    Expression and characterization of the A19 protein. (A) Schematic representation of the DNA construct used for generating recombinant vFS-A19. The FLAG- and streptavidin-binding peptide tag fused at the N terminus of the A19 ORF (FS) is indicated. The

    Journal: Journal of Virology

    Article Title: Vaccinia Virus A19 Protein Participates in the Transformation of Spherical Immature Particles to Barrel-Shaped Infectious Virions

    doi: 10.1128/JVI.01258-13

    Figure Lengend Snippet: Expression and characterization of the A19 protein. (A) Schematic representation of the DNA construct used for generating recombinant vFS-A19. The FLAG- and streptavidin-binding peptide tag fused at the N terminus of the A19 ORF (FS) is indicated. The

    Article Snippet: Soluble extracts obtained by low-speed centrifugation were allowed to bind to streptavidin-agarose beads (Millipore, Billerica, MA) for 3 h at 4°C.

    Techniques: Expressing, Construct, Recombinant, Binding Assay

    Analysis of viral PPxY-host WW-domain interactions between BAG3 and LFV-Z by peptide pull-down assays. ( A ) Flow chart of the peptide pull-down assay using LFV-Z peptides and cell lysates expressing BAG3-WT; ( B ) Schematic diagram of BAG3-WT, BAG3-ΔN, and BAG3-ΔC mutants with the various domains highlighted in color and amino acid positions indicated; ( C ) Western blot of peptide pull-down assay using streptavidin agarose beads conjugated with either the LFV-Z WT or LFV-Z PY mutant peptide. BAG3 proteins were detected using anti-c-myc antibody (top blot). Expression controls for BAG3 and actin are shown in the bottom blot. These results are from 1 of 2 independent experiments.

    Journal: Diseases

    Article Title: Host Protein BAG3 is a Negative Regulator of Lassa VLP Egress

    doi: 10.3390/diseases6030064

    Figure Lengend Snippet: Analysis of viral PPxY-host WW-domain interactions between BAG3 and LFV-Z by peptide pull-down assays. ( A ) Flow chart of the peptide pull-down assay using LFV-Z peptides and cell lysates expressing BAG3-WT; ( B ) Schematic diagram of BAG3-WT, BAG3-ΔN, and BAG3-ΔC mutants with the various domains highlighted in color and amino acid positions indicated; ( C ) Western blot of peptide pull-down assay using streptavidin agarose beads conjugated with either the LFV-Z WT or LFV-Z PY mutant peptide. BAG3 proteins were detected using anti-c-myc antibody (top blot). Expression controls for BAG3 and actin are shown in the bottom blot. These results are from 1 of 2 independent experiments.

    Article Snippet: Briefly, extracts from HEK293T cells expressing either BAG3-WT, BAG3-ΔN, or BAG3-ΔC ( B) were incubated with streptavidin agarose beads bound with either the LFV-Z-WT or LFV-Z-mutant peptides.

    Techniques: Flow Cytometry, Pull Down Assay, Expressing, Western Blot, Mutagenesis