streptavidin coated 96 well plates  (Thermo Fisher)


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    Structured Review

    Thermo Fisher streptavidin coated 96 well plates
    Apical residents are internalized in mβCD-treated cells and found in early endosomes. (A) To measure internalization, WIF-B cells were continuously labeled with biotinylated antibodies diluted in LPDM in the absence or presence of 5 mM mβCD for the indicated times at 37°C. The remaining PM-associated antibodies were eluted with isoglycine for 5 min at room temperature and the cells lysed. Aliquots of the eluate and lysate (the internalized population) were assayed for amounts of biotinylated antibodies using <t>streptavidin-coated</t> 96-well plates and colorimetric detection of HRP-conjugated secondary antibodies. The total amount (in nanograms) of antibody internalized is plotted relative to the maximum observed at 60 min in control cells, which was set to 100%. Values are expressed as the mean ± SD. Measurements were done on at least three experiments each performed in duplicate. (B) WIF-B cells were pretreated for 5 min in LPDM in the absence (a, b, e, f, i, j, m, and n) or presence of 5 mM mβCD (c, d, g, h, k, l, o, and p). The indicated apical residents or recycling receptors present at the basolateral PM were continuously labeled with specific antibodies for 60 min at 37°C. The cells were fixed, permeabilized, and the trafficked antibody–antigen complexes visualized with secondary antibodies. Arrows are pointing to intracellular clusters enlarged in the insets approximately twofold. In c, d, g, and o, images were intentionally overexposed to highlight the intracellular population of the transcytosing proteins. Thus, the apparent apical labeling is exaggerated. The insets in m and n are highlighting a region where Tf-R and ASGP-R do not significantly overlap. Arrowheads are pointing to colocalized structures. Bar, 10 μm.
    Streptavidin Coated 96 Well Plates, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Transcytotic Efflux from Early Endosomes Is Dependent on Cholesterol and Glycosphingolipids in Polarized Hepatic Cells"

    Article Title: Transcytotic Efflux from Early Endosomes Is Dependent on Cholesterol and Glycosphingolipids in Polarized Hepatic Cells

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E02-12-0816

    Apical residents are internalized in mβCD-treated cells and found in early endosomes. (A) To measure internalization, WIF-B cells were continuously labeled with biotinylated antibodies diluted in LPDM in the absence or presence of 5 mM mβCD for the indicated times at 37°C. The remaining PM-associated antibodies were eluted with isoglycine for 5 min at room temperature and the cells lysed. Aliquots of the eluate and lysate (the internalized population) were assayed for amounts of biotinylated antibodies using streptavidin-coated 96-well plates and colorimetric detection of HRP-conjugated secondary antibodies. The total amount (in nanograms) of antibody internalized is plotted relative to the maximum observed at 60 min in control cells, which was set to 100%. Values are expressed as the mean ± SD. Measurements were done on at least three experiments each performed in duplicate. (B) WIF-B cells were pretreated for 5 min in LPDM in the absence (a, b, e, f, i, j, m, and n) or presence of 5 mM mβCD (c, d, g, h, k, l, o, and p). The indicated apical residents or recycling receptors present at the basolateral PM were continuously labeled with specific antibodies for 60 min at 37°C. The cells were fixed, permeabilized, and the trafficked antibody–antigen complexes visualized with secondary antibodies. Arrows are pointing to intracellular clusters enlarged in the insets approximately twofold. In c, d, g, and o, images were intentionally overexposed to highlight the intracellular population of the transcytosing proteins. Thus, the apparent apical labeling is exaggerated. The insets in m and n are highlighting a region where Tf-R and ASGP-R do not significantly overlap. Arrowheads are pointing to colocalized structures. Bar, 10 μm.
    Figure Legend Snippet: Apical residents are internalized in mβCD-treated cells and found in early endosomes. (A) To measure internalization, WIF-B cells were continuously labeled with biotinylated antibodies diluted in LPDM in the absence or presence of 5 mM mβCD for the indicated times at 37°C. The remaining PM-associated antibodies were eluted with isoglycine for 5 min at room temperature and the cells lysed. Aliquots of the eluate and lysate (the internalized population) were assayed for amounts of biotinylated antibodies using streptavidin-coated 96-well plates and colorimetric detection of HRP-conjugated secondary antibodies. The total amount (in nanograms) of antibody internalized is plotted relative to the maximum observed at 60 min in control cells, which was set to 100%. Values are expressed as the mean ± SD. Measurements were done on at least three experiments each performed in duplicate. (B) WIF-B cells were pretreated for 5 min in LPDM in the absence (a, b, e, f, i, j, m, and n) or presence of 5 mM mβCD (c, d, g, h, k, l, o, and p). The indicated apical residents or recycling receptors present at the basolateral PM were continuously labeled with specific antibodies for 60 min at 37°C. The cells were fixed, permeabilized, and the trafficked antibody–antigen complexes visualized with secondary antibodies. Arrows are pointing to intracellular clusters enlarged in the insets approximately twofold. In c, d, g, and o, images were intentionally overexposed to highlight the intracellular population of the transcytosing proteins. Thus, the apparent apical labeling is exaggerated. The insets in m and n are highlighting a region where Tf-R and ASGP-R do not significantly overlap. Arrowheads are pointing to colocalized structures. Bar, 10 μm.

    Techniques Used: Labeling

    Related Articles

    Transduction:

    Article Title: Introducing novel potent anticancer agents of 1H-benzo[f]chromene scaffolds, targeting c-Src kinase enzyme with MDA-MB-231 cell line anti-invasion effect
    Article Snippet: Streptavidin-coated plates were purchased from Pierce (Rockford, IL). .. The biotinylated substrate (catalogue number 1366) contains the residues surrounding tyrosine 160 (Tyr160) of signal transduction protein and has a sequence of EGIYDVP.

    Centrifugation:

    Article Title: Cortactin Controls Surface Expression of the Voltage-gated Potassium Channel KV10.1
    Article Snippet: The insoluble fraction was removed by centrifugation at 18,000 × g at 4 °C, and the supernatant was used for ELISA or pull-down experiments. .. After labeling, total cell lysates (30 and 150 μg of protein), were immobilized on streptavidin-coated plates (Pierce) and detected using a C-terminal monoclonal anti-KV 10.1 antibody (Ab33, 5 μg/ml) and a polyclonal anti-mouse secondary antibody (Pierce, 1:500) coupled to peroxidase.

    Article Title: PIST (GOPC) modulates the oncogenic voltage-gated potassium channel KV10.1
    Article Snippet: Insoluble fraction was removed by centrifugation at 18,000 × g at 4°C, and the supernatant used for ELISA or pull down experiments. .. After labeling, total cell lysates (30 and 150 μg protein), were immobilized on streptavidin-coated plates (Pierce) and detected using a C-terminal monoclonal anti-KV 10.1 antibody [Ab33, 5 μg/ml (Hemmerlein et al., )] and a polyclonal anti-mouse secondary antibody (Pierce, 1:500) coupled to peroxidase.

    Synthesized:

    Article Title: cAMP-dependent protein kinase phosphorylation of the acid-sensing ion channel-1 regulates its binding to the protein interacting with C-kinase-1
    Article Snippet: Streptavidin-coated plates and all developing reagents were purchased from Pierce. .. ASIC1 wild-type peptide (ASIC1/PEP) (biotin–RRGKCQKEAKRSSADKGVALSLDD), ASIC1/PEP-S478A peptide (biotin–RRGKCQKEAKRASADKGVALSLDD), ASIC1/PEP-S479A peptide (biotin–RRGKCQKEAKRSAADKGVALSLDD), and ASIC3/PEP (biotin–FWNRRSSQRRSGNTLLQE) were synthesized at the Howard Hughes Medical Institute/Keck Facility at Yale University.

    Article Title: Human single-chain antibodies that neutralize Pseudomonas aeruginosa-exotoxin A-mediated cellular apoptosis
    Article Snippet: Production of ETA-bound HuscFvs The refolded rETA-1A, rETA-3, rETA-FL, and commercially synthesized (GenScript, New Jersey, USA) biotinylated peptide containing ETA catalytic residues (biotin-6-aminohexanoic acid-ADAITGPEEEGGRLETILGW) were used as panning antigens. .. For biotinylated-ETA peptide, 2.5 µM of the peptide in 100 μl PBS were added to well of a streptavidin plate (Pierce™ Streptavidin Coated Plates, Clear Well Strips, Rockford, IL, USA) and kept at room temperature for 1 h. After blocking with Pierce™ Protein-Free (PBS) Blocking Buffer (Thermo Fisher Scientific, Rockford, IL, USA) and washed with PBS containing 0.01% Tween-20 (PBST), the previously constructed HuscFv-phage display library was added to individual antigen coated wells.

    Construct:

    Article Title: Inhibition of preS1-hepatocyte interaction by an array of recombinant human antibodies from naturally recovered individuals
    Article Snippet: Biopanning The phage rescue from the constructed library was carried out as described earlier using VCSM13 helper phage (Stratagene, 200251) . .. Three rounds of biopanning were carried out using biotinylated preS1-peptide (5 μg/ml) immobilized on high binding streptavidin coated plates (Pierce, 15501).

    Article Title: Human single-chain antibodies that neutralize Pseudomonas aeruginosa-exotoxin A-mediated cellular apoptosis
    Article Snippet: .. For biotinylated-ETA peptide, 2.5 µM of the peptide in 100 μl PBS were added to well of a streptavidin plate (Pierce™ Streptavidin Coated Plates, Clear Well Strips, Rockford, IL, USA) and kept at room temperature for 1 h. After blocking with Pierce™ Protein-Free (PBS) Blocking Buffer (Thermo Fisher Scientific, Rockford, IL, USA) and washed with PBS containing 0.01% Tween-20 (PBST), the previously constructed HuscFv-phage display library was added to individual antigen coated wells. .. Binding of HuscFv-display phages to the immobilized antigens were allowed at room temperature for 1 h on a rocking platform.

    Enzyme-linked Immunosorbent Assay:

    Article Title: DPI-ELISA: a fast and versatile method to specify the binding of plant transcription factors to DNA in vitro
    Article Snippet: .. ELISA micro well plates streptavidin-coated (5 pmol/well), pre-blocked, clear 96-well plates (Reacti-Bind™streptavidin coated clear 96-well plates with SuperBlock blocking buffer from Pierce, Thermo Fisher Scientific) .. ELISA-reader Tecan Safire plate reader

    Article Title: Cortactin Controls Surface Expression of the Voltage-gated Potassium Channel KV10.1
    Article Snippet: Quantification of the amount of labeled KV 10.1-BBS was performed by ELISA. .. After labeling, total cell lysates (30 and 150 μg of protein), were immobilized on streptavidin-coated plates (Pierce) and detected using a C-terminal monoclonal anti-KV 10.1 antibody (Ab33, 5 μg/ml) and a polyclonal anti-mouse secondary antibody (Pierce, 1:500) coupled to peroxidase.

    Article Title: Isolation of a monoclonal antibody from a phage display library binding the rhesus macaque MHC class I allomorph Mamu-A1*001
    Article Snippet: .. ELISA to detect Mamu-A1*001-specific phages MHC-coated 96-well plates were prepared by incubating pre-blocked streptavidin-coated plates (Pierce) with biotinylated rhesus macaque MHC class I monomers (20 μg/ml) and incubating for one hour at room temperature. ..

    Article Title: Mapping the Determinants of the CCR5 Amino-Terminal Sulfopeptide Interaction with Soluble Human Immunodeficiency Virus Type 1 gp120-CD4 Complexes
    Article Snippet: .. Streptavidin-coated ELISA plates (Pierce) were blocked with 5% bovine serum albumin in Dulbecco's phosphate-buffered saline for 2 h at room temperature and then were washed three times with assay buffer (Dulbecco's phosphate-buffered saline, 0.5% Tween 20, 1% fetal bovine serum, 2% bovine serum albumin). ..

    Article Title: Vaccinia virus entry/fusion complex subunit A28 is a target of neutralizing and protective antibodies
    Article Snippet: Paragraph title: ELISA ... For peptide ELISAs, streptavidin plates (Pierce) were coated with 1:1000 dilutions of peptides in PBS for 1 h. The plates were then washed in a solution of 27 g of NaCl and 3 ml of Tween 20 in 3 L of deionized water.

    Article Title: A Novel Highly Potent Therapeutic Antibody Neutralizes Multiple Human Chemokines and Mimics Viral Immune Modulation
    Article Snippet: Ten d after the final boost, serum was collected and tested for reactivity with the target chemokines by ELISA. .. Sera were screened at a range of dilutions from 1∶50 to 1∶6400 using biotinylated chemokines (0.5 µg/mL) on streptavidin-coated plates (Thermo Scientific Pierce, Rockford, IL, catalog #15124).

    Article Title: PIST (GOPC) modulates the oncogenic voltage-gated potassium channel KV10.1
    Article Snippet: Quantification of the amount of labeled KV 10.1-BBS was performed by ELISA. .. After labeling, total cell lysates (30 and 150 μg protein), were immobilized on streptavidin-coated plates (Pierce) and detected using a C-terminal monoclonal anti-KV 10.1 antibody [Ab33, 5 μg/ml (Hemmerlein et al., )] and a polyclonal anti-mouse secondary antibody (Pierce, 1:500) coupled to peroxidase.

    Article Title: Isolation of Single-Stranded DNA Aptamers That Distinguish Influenza Virus Hemagglutinin Subtype H1 from H5
    Article Snippet: Paragraph title: HA1 protein-ssDNA aptamer binding analysis by ELISA ... The 5′-biotinylated ssDNA aptamers (100 nM) were heated at 90°C for 10 min, immediately placed on ice, added to the wells of a streptavidin-coated plate (Pierce Biotechnology, Rockford, IL), and incubated for 1 h at room temperature while shaking at 100 rpm.

    Article Title: A semisynthetic Eph receptor tyrosine kinase provides insight into ligand-induced kinase activation
    Article Snippet: Paragraph title: ELISA assay and Western blotting ... Thereafter, the reaction mix was immobilized by binding to the pre-blocked streptavidin coated plates (Pierce).

    Article Title: Human single-chain antibodies that neutralize Pseudomonas aeruginosa-exotoxin A-mediated cellular apoptosis
    Article Snippet: The refolded rETA-1A, rETA-3, and rETA-FL (0.5 µg in 100 μl of coating buffer) were used to coat separate wells of EIA/RIA 8-well strips and kept at 4 °C overnight. .. For biotinylated-ETA peptide, 2.5 µM of the peptide in 100 μl PBS were added to well of a streptavidin plate (Pierce™ Streptavidin Coated Plates, Clear Well Strips, Rockford, IL, USA) and kept at room temperature for 1 h. After blocking with Pierce™ Protein-Free (PBS) Blocking Buffer (Thermo Fisher Scientific, Rockford, IL, USA) and washed with PBS containing 0.01% Tween-20 (PBST), the previously constructed HuscFv-phage display library was added to individual antigen coated wells.

    Incubation:

    Article Title: Naloxone's Pentapeptide Binding Site on Filamin A Blocks Mu Opioid Receptor-Gs Coupling and CREB Activation of Acute Morphine
    Article Snippet: VAKGL biotinated at either n- or c-terminus (0.5 µg/well) was coated onto streptavidin-coated plates (Reacti-BindTM NeutrAvidinTM High binding capacity coated 96-well plate, Pierce). .. After 3 washes with 200 µl PBS, immunoaffinity-purified FLNA (0.5 µg) from rat brain and A7 cells or MOR (0.1 µg) from rat brain or SK-N-MC cells were added into designated wells and incubation was carried out for 1 hr at 25°C with constant shaking.

    Article Title: Inhibition of preS1-hepatocyte interaction by an array of recombinant human antibodies from naturally recovered individuals
    Article Snippet: Three rounds of biopanning were carried out using biotinylated preS1-peptide (5 μg/ml) immobilized on high binding streptavidin coated plates (Pierce, 15501). .. Library phages (~1 × 1012 ) were incubated with the peptide and unbound phages were washed out with PBST (0.1%).

    Article Title: Introducing novel potent anticancer agents of 1H-benzo[f]chromene scaffolds, targeting c-Src kinase enzyme with MDA-MB-231 cell line anti-invasion effect
    Article Snippet: Streptavidin-coated plates were purchased from Pierce (Rockford, IL). .. In brief, the kinase reaction was started with the incubation of the 12.5 µL of the reaction cocktail (0.5 ng/µL of GST-Src kinase in 1.25 mM DTT) with 12.5 µL of prediluted compounds (dissolved in 1% DMSO) for 5 min at room temperature.

    Article Title: Vaccinia virus entry/fusion complex subunit A28 is a target of neutralizing and protective antibodies
    Article Snippet: Polystyrene plates with 96-well round bottoms (Corning, Inc., Acton, MA) were coated by overnight incubation with either 60 ng/well of recombinant protein at 4°C or 1×106 PFU/well of purified VACV WR at 37°C. .. For peptide ELISAs, streptavidin plates (Pierce) were coated with 1:1000 dilutions of peptides in PBS for 1 h. The plates were then washed in a solution of 27 g of NaCl and 3 ml of Tween 20 in 3 L of deionized water.

    Article Title: A Novel Highly Potent Therapeutic Antibody Neutralizes Multiple Human Chemokines and Mimics Viral Immune Modulation
    Article Snippet: Sera were screened at a range of dilutions from 1∶50 to 1∶6400 using biotinylated chemokines (0.5 µg/mL) on streptavidin-coated plates (Thermo Scientific Pierce, Rockford, IL, catalog #15124). .. Sera incubations were for 90 min at 37°C, plates were blocked using 1% BSA in PBS, and bound antibodies were detected using goat anti-mouse IgG Fc-HRP (Jackson Immuno Research, West Grove, PA, #115-035-071) incubated for 90 min at 37°C.

    Article Title: Isolation of Single-Stranded DNA Aptamers That Distinguish Influenza Virus Hemagglutinin Subtype H1 from H5
    Article Snippet: .. The 5′-biotinylated ssDNA aptamers (100 nM) were heated at 90°C for 10 min, immediately placed on ice, added to the wells of a streptavidin-coated plate (Pierce Biotechnology, Rockford, IL), and incubated for 1 h at room temperature while shaking at 100 rpm. .. The wells were washed four times with PBST (0.1% Tween 20 in PBS; pH 7.4), blocked with 5% BSA in PBST at room temperature for 1 h, re-washed four times, and incubated with various concentrations of purified GST-H1-HA1 in PBS at room temperature for 1 h. After washing four times with PBST, incubation with GST antibody-conjugated horseradish peroxidase (HRP; 1:1,000 in PBST, Santa Cruz Biotechnology, Dallas, TX) at room temperature for 1 h, and four additional washes, bound GST-tagged HA1 protein was detected by adding 3,3′,5,5′-Tetramethylbenzidine (TMB) solution (Merck, Darmstadt, Germany) and terminating with 0.5 N H2 SO4 .

    Article Title: A semisynthetic Eph receptor tyrosine kinase provides insight into ligand-induced kinase activation
    Article Snippet: The enzyme reactions were incubated for 5 minutes at 30°C and then quenched with 100 mM EDTA. .. Thereafter, the reaction mix was immobilized by binding to the pre-blocked streptavidin coated plates (Pierce).

    Article Title: Longitudinal Analysis of the Human Antibody Response to Chikungunya Virus Infection: Implications for Serodiagnosis and Vaccine Development
    Article Snippet: .. Briefly, streptavidin-coated plates (Pierce) were first blocked with 1% sodium caseinate (Sigma-Aldrich) diluted in 0.1% PBST (0.1% Tween 20 in PBS) before coating with peptides diluted at 1:1,000 in 0.1% PBST and incubated at room temperature for 1 h on a rotating platform. .. Plates were then rinsed with 0.1% PBST before incubation with patient plasma samples diluted at 1:2,000 with 0.1% PBST for 1 h. Plates were rinsed, followed by incubation with anti-human IgG antibodies conjugated to HRP diluted in 0.1% blocking buffer for 1 h at room temperature to detect peptide-bound antibodies.

    Activity Assay:

    Article Title: Introducing novel potent anticancer agents of 1H-benzo[f]chromene scaffolds, targeting c-Src kinase enzyme with MDA-MB-231 cell line anti-invasion effect
    Article Snippet: 2.3.4. c-Src kinase assay The effect of the synthesised compounds on the activity of c-Src kinase was determined by HTScan Src Kinase Assay Kit, catalogue number 7776 from Cell Signaling Technology (Danvers, MA); according to the manufacturer’s protocol. .. Streptavidin-coated plates were purchased from Pierce (Rockford, IL).

    Article Title: A semisynthetic Eph receptor tyrosine kinase provides insight into ligand-induced kinase activation
    Article Snippet: The kinase activity of both was monitored over a period of time using the ELISA assay described above. .. Thereafter, the reaction mix was immobilized by binding to the pre-blocked streptavidin coated plates (Pierce).

    Western Blot:

    Article Title: PIST (GOPC) modulates the oncogenic voltage-gated potassium channel KV10.1
    Article Snippet: Bound protein was eluted at 70°C for 10 min using LDS sample buffer containing reducing agent (Invitrogen) and analyzed by SDS PAGE (Invitrogen) and western blotting. .. After labeling, total cell lysates (30 and 150 μg protein), were immobilized on streptavidin-coated plates (Pierce) and detected using a C-terminal monoclonal anti-KV 10.1 antibody [Ab33, 5 μg/ml (Hemmerlein et al., )] and a polyclonal anti-mouse secondary antibody (Pierce, 1:500) coupled to peroxidase.

    Article Title: A semisynthetic Eph receptor tyrosine kinase provides insight into ligand-induced kinase activation
    Article Snippet: Paragraph title: ELISA assay and Western blotting ... Thereafter, the reaction mix was immobilized by binding to the pre-blocked streptavidin coated plates (Pierce).

    Kinase Assay:

    Article Title: Introducing novel potent anticancer agents of 1H-benzo[f]chromene scaffolds, targeting c-Src kinase enzyme with MDA-MB-231 cell line anti-invasion effect
    Article Snippet: Paragraph title: c-Src kinase assay ... Streptavidin-coated plates were purchased from Pierce (Rockford, IL).

    Infection:

    Article Title: Inhibition of preS1-hepatocyte interaction by an array of recombinant human antibodies from naturally recovered individuals
    Article Snippet: Three rounds of biopanning were carried out using biotinylated preS1-peptide (5 μg/ml) immobilized on high binding streptavidin coated plates (Pierce, 15501). .. The eluted phages were infected into mid-exponential phase (OD600 ~ 0.4) TG1 cells and grown for 1 hour at 37 °C and plated on 2 × YT/ Chloramphenicol/2%Glc/ Agar plates and incubated overnight at 37 °C.

    Article Title: Isolation of a monoclonal antibody from a phage display library binding the rhesus macaque MHC class I allomorph Mamu-A1*001
    Article Snippet: ELISA to detect Mamu-A1*001-specific phages MHC-coated 96-well plates were prepared by incubating pre-blocked streptavidin-coated plates (Pierce) with biotinylated rhesus macaque MHC class I monomers (20 μg/ml) and incubating for one hour at room temperature. .. E .coli TG1 was infected with phages, grown overnight at 37°C on TYE plates containing 100 μg/ml ampicillin and 1% glucose.

    Generated:

    Article Title: Longitudinal Analysis of the Human Antibody Response to Chikungunya Virus Infection: Implications for Serodiagnosis and Vaccine Development
    Article Snippet: A biotinylated peptide library consisting of 18-mer overlapping peptides (Mimotopes) was generated from sequence alignments of different CHIKV amino acid sequences as described previously ( ). .. Briefly, streptavidin-coated plates (Pierce) were first blocked with 1% sodium caseinate (Sigma-Aldrich) diluted in 0.1% PBST (0.1% Tween 20 in PBS) before coating with peptides diluted at 1:1,000 in 0.1% PBST and incubated at room temperature for 1 h on a rotating platform.

    Sequencing:

    Article Title: Introducing novel potent anticancer agents of 1H-benzo[f]chromene scaffolds, targeting c-Src kinase enzyme with MDA-MB-231 cell line anti-invasion effect
    Article Snippet: Streptavidin-coated plates were purchased from Pierce (Rockford, IL). .. The biotinylated substrate (catalogue number 1366) contains the residues surrounding tyrosine 160 (Tyr160) of signal transduction protein and has a sequence of EGIYDVP.

    Article Title: Longitudinal Analysis of the Human Antibody Response to Chikungunya Virus Infection: Implications for Serodiagnosis and Vaccine Development
    Article Snippet: A biotinylated peptide library consisting of 18-mer overlapping peptides (Mimotopes) was generated from sequence alignments of different CHIKV amino acid sequences as described previously ( ). .. Briefly, streptavidin-coated plates (Pierce) were first blocked with 1% sodium caseinate (Sigma-Aldrich) diluted in 0.1% PBST (0.1% Tween 20 in PBS) before coating with peptides diluted at 1:1,000 in 0.1% PBST and incubated at room temperature for 1 h on a rotating platform.

    Injection:

    Article Title: Cortactin Controls Surface Expression of the Voltage-gated Potassium Channel KV10.1
    Article Snippet: KV 10.1-BBS expressed in Xenopus oocytes injected with the corresponding cRNA was processed in the same way as that from HEK-BBS cells. .. After labeling, total cell lysates (30 and 150 μg of protein), were immobilized on streptavidin-coated plates (Pierce) and detected using a C-terminal monoclonal anti-KV 10.1 antibody (Ab33, 5 μg/ml) and a polyclonal anti-mouse secondary antibody (Pierce, 1:500) coupled to peroxidase.

    Recombinant:

    Article Title: Vaccinia virus entry/fusion complex subunit A28 is a target of neutralizing and protective antibodies
    Article Snippet: Polystyrene plates with 96-well round bottoms (Corning, Inc., Acton, MA) were coated by overnight incubation with either 60 ng/well of recombinant protein at 4°C or 1×106 PFU/well of purified VACV WR at 37°C. .. For peptide ELISAs, streptavidin plates (Pierce) were coated with 1:1000 dilutions of peptides in PBS for 1 h. The plates were then washed in a solution of 27 g of NaCl and 3 ml of Tween 20 in 3 L of deionized water.

    Magnetic Beads:

    Article Title: Cortactin Controls Surface Expression of the Voltage-gated Potassium Channel KV10.1
    Article Snippet: For pulldown approaches, labeled KV 10.1-BBS was bound to streptavidin-coated magnetic beads (T1, Invitrogen) for at least 30 min at 4 °C. .. After labeling, total cell lysates (30 and 150 μg of protein), were immobilized on streptavidin-coated plates (Pierce) and detected using a C-terminal monoclonal anti-KV 10.1 antibody (Ab33, 5 μg/ml) and a polyclonal anti-mouse secondary antibody (Pierce, 1:500) coupled to peroxidase.

    Article Title: PIST (GOPC) modulates the oncogenic voltage-gated potassium channel KV10.1
    Article Snippet: For pull down approaches, labeled KV 10.1-BBS was bound on streptavidin-coated magnetic beads (T1, Invitrogen) for 2 h at 4°C. .. After labeling, total cell lysates (30 and 150 μg protein), were immobilized on streptavidin-coated plates (Pierce) and detected using a C-terminal monoclonal anti-KV 10.1 antibody [Ab33, 5 μg/ml (Hemmerlein et al., )] and a polyclonal anti-mouse secondary antibody (Pierce, 1:500) coupled to peroxidase.

    Isolation:

    Article Title: Isolation of a monoclonal antibody from a phage display library binding the rhesus macaque MHC class I allomorph Mamu-A1*001
    Article Snippet: ELISA to detect Mamu-A1*001-specific phages MHC-coated 96-well plates were prepared by incubating pre-blocked streptavidin-coated plates (Pierce) with biotinylated rhesus macaque MHC class I monomers (20 μg/ml) and incubating for one hour at room temperature. .. Isolated bacterial colonies were grown overnight in 96-deep-well plates (Thermo Fisher Scientific) in 2xTY + 1% glu at 37°C while shaking.

    Labeling:

    Article Title: Cortactin Controls Surface Expression of the Voltage-gated Potassium Channel KV10.1
    Article Snippet: .. After labeling, total cell lysates (30 and 150 μg of protein), were immobilized on streptavidin-coated plates (Pierce) and detected using a C-terminal monoclonal anti-KV 10.1 antibody (Ab33, 5 μg/ml) and a polyclonal anti-mouse secondary antibody (Pierce, 1:500) coupled to peroxidase. .. ABTS (Invitrogen) was used as a substrate for development and detected in a Wallac Victor2 reader at 405 nm (reference 490 nm).

    Article Title: PIST (GOPC) modulates the oncogenic voltage-gated potassium channel KV10.1
    Article Snippet: .. After labeling, total cell lysates (30 and 150 μg protein), were immobilized on streptavidin-coated plates (Pierce) and detected using a C-terminal monoclonal anti-KV 10.1 antibody [Ab33, 5 μg/ml (Hemmerlein et al., )] and a polyclonal anti-mouse secondary antibody (Pierce, 1:500) coupled to peroxidase. .. ABTS (Invitrogen) was used as a substrate for development and detected in a Wallac Victor2 reader at 405 nm (reference 490 nm).

    Purification:

    Article Title: Cortactin Controls Surface Expression of the Voltage-gated Potassium Channel KV10.1
    Article Snippet: Paragraph title: Fractional Labeling, Quantification, and Purification of KV 10.1-BBS ... After labeling, total cell lysates (30 and 150 μg of protein), were immobilized on streptavidin-coated plates (Pierce) and detected using a C-terminal monoclonal anti-KV 10.1 antibody (Ab33, 5 μg/ml) and a polyclonal anti-mouse secondary antibody (Pierce, 1:500) coupled to peroxidase.

    Article Title: Vaccinia virus entry/fusion complex subunit A28 is a target of neutralizing and protective antibodies
    Article Snippet: Polystyrene plates with 96-well round bottoms (Corning, Inc., Acton, MA) were coated by overnight incubation with either 60 ng/well of recombinant protein at 4°C or 1×106 PFU/well of purified VACV WR at 37°C. .. For peptide ELISAs, streptavidin plates (Pierce) were coated with 1:1000 dilutions of peptides in PBS for 1 h. The plates were then washed in a solution of 27 g of NaCl and 3 ml of Tween 20 in 3 L of deionized water.

    Article Title: PIST (GOPC) modulates the oncogenic voltage-gated potassium channel KV10.1
    Article Snippet: Paragraph title: Fractional labeling, quantification and purification of KV 10.1-BBS ... After labeling, total cell lysates (30 and 150 μg protein), were immobilized on streptavidin-coated plates (Pierce) and detected using a C-terminal monoclonal anti-KV 10.1 antibody [Ab33, 5 μg/ml (Hemmerlein et al., )] and a polyclonal anti-mouse secondary antibody (Pierce, 1:500) coupled to peroxidase.

    Article Title: Isolation of Single-Stranded DNA Aptamers That Distinguish Influenza Virus Hemagglutinin Subtype H1 from H5
    Article Snippet: The 5′-biotinylated ssDNA aptamers (100 nM) were heated at 90°C for 10 min, immediately placed on ice, added to the wells of a streptavidin-coated plate (Pierce Biotechnology, Rockford, IL), and incubated for 1 h at room temperature while shaking at 100 rpm. .. The wells were washed four times with PBST (0.1% Tween 20 in PBS; pH 7.4), blocked with 5% BSA in PBST at room temperature for 1 h, re-washed four times, and incubated with various concentrations of purified GST-H1-HA1 in PBS at room temperature for 1 h. After washing four times with PBST, incubation with GST antibody-conjugated horseradish peroxidase (HRP; 1:1,000 in PBST, Santa Cruz Biotechnology, Dallas, TX) at room temperature for 1 h, and four additional washes, bound GST-tagged HA1 protein was detected by adding 3,3′,5,5′-Tetramethylbenzidine (TMB) solution (Merck, Darmstadt, Germany) and terminating with 0.5 N H2 SO4 .

    Polymerase Chain Reaction:

    Article Title: Isolation of Single-Stranded DNA Aptamers That Distinguish Influenza Virus Hemagglutinin Subtype H1 from H5
    Article Snippet: HA1 protein-ssDNA aptamer binding analysis by ELISA Aptamers were 5′-biotinylated by asymmetric PCR using the forward primer 5′-Biotin-GCAATGTACGGTACTTCC-3′ followed by lambda exonuclease digestion, as previously described [ , ]. .. The 5′-biotinylated ssDNA aptamers (100 nM) were heated at 90°C for 10 min, immediately placed on ice, added to the wells of a streptavidin-coated plate (Pierce Biotechnology, Rockford, IL), and incubated for 1 h at room temperature while shaking at 100 rpm.

    Blocking Assay:

    Article Title: DPI-ELISA: a fast and versatile method to specify the binding of plant transcription factors to DNA in vitro
    Article Snippet: .. ELISA micro well plates streptavidin-coated (5 pmol/well), pre-blocked, clear 96-well plates (Reacti-Bind™streptavidin coated clear 96-well plates with SuperBlock blocking buffer from Pierce, Thermo Fisher Scientific) .. ELISA-reader Tecan Safire plate reader

    Article Title: Vaccinia virus entry/fusion complex subunit A28 is a target of neutralizing and protective antibodies
    Article Snippet: For peptide ELISAs, streptavidin plates (Pierce) were coated with 1:1000 dilutions of peptides in PBS for 1 h. The plates were then washed in a solution of 27 g of NaCl and 3 ml of Tween 20 in 3 L of deionized water. .. Plates were incubated for 1 h at 37°C with blocking buffer (5% nonfat dry milk in PBS with 0.2% Tween 20) and washed.

    Article Title: Human single-chain antibodies that neutralize Pseudomonas aeruginosa-exotoxin A-mediated cellular apoptosis
    Article Snippet: .. For biotinylated-ETA peptide, 2.5 µM of the peptide in 100 μl PBS were added to well of a streptavidin plate (Pierce™ Streptavidin Coated Plates, Clear Well Strips, Rockford, IL, USA) and kept at room temperature for 1 h. After blocking with Pierce™ Protein-Free (PBS) Blocking Buffer (Thermo Fisher Scientific, Rockford, IL, USA) and washed with PBS containing 0.01% Tween-20 (PBST), the previously constructed HuscFv-phage display library was added to individual antigen coated wells. .. Binding of HuscFv-display phages to the immobilized antigens were allowed at room temperature for 1 h on a rocking platform.

    Article Title: Longitudinal Analysis of the Human Antibody Response to Chikungunya Virus Infection: Implications for Serodiagnosis and Vaccine Development
    Article Snippet: Briefly, streptavidin-coated plates (Pierce) were first blocked with 1% sodium caseinate (Sigma-Aldrich) diluted in 0.1% PBST (0.1% Tween 20 in PBS) before coating with peptides diluted at 1:1,000 in 0.1% PBST and incubated at room temperature for 1 h on a rotating platform. .. Plates were then rinsed with 0.1% PBST before incubation with patient plasma samples diluted at 1:2,000 with 0.1% PBST for 1 h. Plates were rinsed, followed by incubation with anti-human IgG antibodies conjugated to HRP diluted in 0.1% blocking buffer for 1 h at room temperature to detect peptide-bound antibodies.

    Peptide ELISA:

    Article Title: Longitudinal Analysis of the Human Antibody Response to Chikungunya Virus Infection: Implications for Serodiagnosis and Vaccine Development
    Article Snippet: Paragraph title: Peptide-based ELISA. ... Briefly, streptavidin-coated plates (Pierce) were first blocked with 1% sodium caseinate (Sigma-Aldrich) diluted in 0.1% PBST (0.1% Tween 20 in PBS) before coating with peptides diluted at 1:1,000 in 0.1% PBST and incubated at room temperature for 1 h on a rotating platform.

    Mouse Assay:

    Article Title: A Novel Highly Potent Therapeutic Antibody Neutralizes Multiple Human Chemokines and Mimics Viral Immune Modulation
    Article Snippet: 18V4F Hybridoma Generation Ten-to 12-wk-old female BALB/c mice were immunized sequentially with three CC-chemokines in random order: CCL3, CCL4, and CCL5 (PeproTech, Rocky Hill, NJ). .. Sera were screened at a range of dilutions from 1∶50 to 1∶6400 using biotinylated chemokines (0.5 µg/mL) on streptavidin-coated plates (Thermo Scientific Pierce, Rockford, IL, catalog #15124).

    SDS Page:

    Article Title: PIST (GOPC) modulates the oncogenic voltage-gated potassium channel KV10.1
    Article Snippet: Bound protein was eluted at 70°C for 10 min using LDS sample buffer containing reducing agent (Invitrogen) and analyzed by SDS PAGE (Invitrogen) and western blotting. .. After labeling, total cell lysates (30 and 150 μg protein), were immobilized on streptavidin-coated plates (Pierce) and detected using a C-terminal monoclonal anti-KV 10.1 antibody [Ab33, 5 μg/ml (Hemmerlein et al., )] and a polyclonal anti-mouse secondary antibody (Pierce, 1:500) coupled to peroxidase.

    Binding Assay:

    Article Title: Naloxone's Pentapeptide Binding Site on Filamin A Blocks Mu Opioid Receptor-Gs Coupling and CREB Activation of Acute Morphine
    Article Snippet: .. VAKGL biotinated at either n- or c-terminus (0.5 µg/well) was coated onto streptavidin-coated plates (Reacti-BindTM NeutrAvidinTM High binding capacity coated 96-well plate, Pierce). .. After 3 washes with 200 µl PBS, immunoaffinity-purified FLNA (0.5 µg) from rat brain and A7 cells or MOR (0.1 µg) from rat brain or SK-N-MC cells were added into designated wells and incubation was carried out for 1 hr at 25°C with constant shaking.

    Article Title: Inhibition of preS1-hepatocyte interaction by an array of recombinant human antibodies from naturally recovered individuals
    Article Snippet: .. Three rounds of biopanning were carried out using biotinylated preS1-peptide (5 μg/ml) immobilized on high binding streptavidin coated plates (Pierce, 15501). .. Library phages (~1 × 1012 ) were incubated with the peptide and unbound phages were washed out with PBST (0.1%).

    Article Title: Isolation of Single-Stranded DNA Aptamers That Distinguish Influenza Virus Hemagglutinin Subtype H1 from H5
    Article Snippet: Paragraph title: HA1 protein-ssDNA aptamer binding analysis by ELISA ... The 5′-biotinylated ssDNA aptamers (100 nM) were heated at 90°C for 10 min, immediately placed on ice, added to the wells of a streptavidin-coated plate (Pierce Biotechnology, Rockford, IL), and incubated for 1 h at room temperature while shaking at 100 rpm.

    Article Title: A semisynthetic Eph receptor tyrosine kinase provides insight into ligand-induced kinase activation
    Article Snippet: .. Thereafter, the reaction mix was immobilized by binding to the pre-blocked streptavidin coated plates (Pierce). .. The phosphorylation of the biotinylated peptide substrate was visualized by ELISA using a phosphotyrosine monoclonal antibody conjugated to HRP and an ensuing chromogenic substrate reaction as described above.

    Article Title: Human single-chain antibodies that neutralize Pseudomonas aeruginosa-exotoxin A-mediated cellular apoptosis
    Article Snippet: For biotinylated-ETA peptide, 2.5 µM of the peptide in 100 μl PBS were added to well of a streptavidin plate (Pierce™ Streptavidin Coated Plates, Clear Well Strips, Rockford, IL, USA) and kept at room temperature for 1 h. After blocking with Pierce™ Protein-Free (PBS) Blocking Buffer (Thermo Fisher Scientific, Rockford, IL, USA) and washed with PBS containing 0.01% Tween-20 (PBST), the previously constructed HuscFv-phage display library was added to individual antigen coated wells. .. Binding of HuscFv-display phages to the immobilized antigens were allowed at room temperature for 1 h on a rocking platform.

    Concentration Assay:

    Article Title: A semisynthetic Eph receptor tyrosine kinase provides insight into ligand-induced kinase activation
    Article Snippet: To measure the Km for ATP, the concentrations of the biotinylated peptide substrate, the unligated Eph ICD, and the semisynthetic Eph receptor proteins, were kept constant at 5 µM, 11 nM and 10.4 nM, respectively, whereas the ATP concentration was varied from 0 to 500 µM. .. Thereafter, the reaction mix was immobilized by binding to the pre-blocked streptavidin coated plates (Pierce).

    Article Title: Longitudinal Analysis of the Human Antibody Response to Chikungunya Virus Infection: Implications for Serodiagnosis and Vaccine Development
    Article Snippet: Peptides were dissolved in dimethyl sulfoxide (DMSO) to obtain a stock concentration of approximately 15 μg/ml. .. Briefly, streptavidin-coated plates (Pierce) were first blocked with 1% sodium caseinate (Sigma-Aldrich) diluted in 0.1% PBST (0.1% Tween 20 in PBS) before coating with peptides diluted at 1:1,000 in 0.1% PBST and incubated at room temperature for 1 h on a rotating platform.

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    Thermo Fisher streptavidin coated 96 well plate
    Time course of epitope-specific antibody production in Patient H. Biotin-linked peptides containing the 1H8 binding site and the specific mutation of the binding site were incubated with <t>streptavidin-coated</t> 96-well plates at 200 ng/well. The plasma samples obtained from Patient H were diluted 1∶800 for the ELISA. An unrelated peptide was used as a negative control for the assay. The data were obtained from at least three independent experiments. The x-axis indicates the peptide used in the ELISA and the time when the sample was collected. The y-axis indicates the absorbance at 450 nm, representing the binding activity of the “1H8-like” antibodies in the samples from Patient H.
    Streptavidin Coated 96 Well Plate, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Time course of epitope-specific antibody production in Patient H. Biotin-linked peptides containing the 1H8 binding site and the specific mutation of the binding site were incubated with streptavidin-coated 96-well plates at 200 ng/well. The plasma samples obtained from Patient H were diluted 1∶800 for the ELISA. An unrelated peptide was used as a negative control for the assay. The data were obtained from at least three independent experiments. The x-axis indicates the peptide used in the ELISA and the time when the sample was collected. The y-axis indicates the absorbance at 450 nm, representing the binding activity of the “1H8-like” antibodies in the samples from Patient H.

    Journal: PLoS ONE

    Article Title: A Neutralization Epitope in the Hepatitis C Virus E2 Glycoprotein Interacts with Host Entry Factor CD81

    doi: 10.1371/journal.pone.0084346

    Figure Lengend Snippet: Time course of epitope-specific antibody production in Patient H. Biotin-linked peptides containing the 1H8 binding site and the specific mutation of the binding site were incubated with streptavidin-coated 96-well plates at 200 ng/well. The plasma samples obtained from Patient H were diluted 1∶800 for the ELISA. An unrelated peptide was used as a negative control for the assay. The data were obtained from at least three independent experiments. The x-axis indicates the peptide used in the ELISA and the time when the sample was collected. The y-axis indicates the absorbance at 450 nm, representing the binding activity of the “1H8-like” antibodies in the samples from Patient H.

    Article Snippet: ELISA A biotinylated peptide (200 ng) was prepared in 100 µL SuperBlock blocking buffer (Thermo Fisher Scientific, Rockford, IL) and added to each well of a streptavidin-coated 96-well plate (Thermo Fisher Scientific).

    Techniques: Binding Assay, Mutagenesis, Incubation, Enzyme-linked Immunosorbent Assay, Negative Control, Activity Assay