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Horseradish peroxidase hrp conjugated anti m13 antibody

Manufactured by GE Healthcare
Sourced in United States

The Horseradish peroxidase (HRP)-conjugated anti-M13 antibody is a laboratory reagent used in various immunoassay techniques. It consists of an antibody directed against the M13 phage protein, which is conjugated to the enzyme horseradish peroxidase. This conjugated antibody can be used to detect the presence of the M13 phage protein in samples through colorimetric or chemiluminescent signal generation.

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6 protocols using horseradish peroxidase hrp conjugated anti m13 antibody

1

Screening Phage Library for TeNT-Hc Binders

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After three rounds of panning, the phage clones were screened by an enzyme-linked immunosorbent assay (ELISA). The 96-well ELISA plate (Costar, Corning, NY, USA) was coated with 40 nM TeNT-Hc, and also coated with 130 nM Yersinia pestis capsular F1, 54 nM Yersinia pestis capsular V protein (F1 and V, purified in our lab) and 91 nM interferon-omega (IFN-ω, purified in our lab) individually as negative antigens.50 μL of phage particles at a concentration of approximately 8 μM in 3% Marvel™ PBS (pH 7.4) were added, and the binding of the phages to the immobilized antigen was performed at 37 °C for 2 h. The unbound phages were removed by washing extensively with PBST. The plate wells were detected by horseradish peroxidase (HRP)-conjugated anti-M13 antibody (GE Healthcare), followed by the addition of peroxidase substrate, o-phenylenediamine dihydrochloride (OPD). 2 M H2SO4 was used to stop the reaction, and the results were monitored at OD492/630 with a microplate reader (Multiskin MK3, Shanghai Labsystems, Shanghai, China). The particular clones that were bound to TeNT-Hc were selected, and the corresponding phage plasmids were sequenced.
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2

Enzyme-Linked Immunosorbent Assay for Peptide Binding

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Maxisorp 96-well microtiter plates were coated overnight with 5 µg/mL streptavidin (50 µL/well) in NaHCO3 buffer (50 mM, pH 9.6), then washed and blocked with PBST buffer (0.05% Tween 20, 2% BSA in PBS) for one hour. Peptides (2 µg/mL) were then loaded on the plate for 30 min. Rabbit sera, individual phage clones or purified antibodies were added to the plates for a one-hour incubation. The plates were then washed with PBST and incubated with the detecting antibody: alkaline phosphatase (AP)-conjugated goat anti rabbit for rabbit serum (Sigma-A8025), horseradish peroxidase (HRP)-conjugated anti-M13 antibody (GE healthcare, Little Chalfont, UK) for phage clones and AP-conjugated anti-human IgG (Sigma-A3187) for scFv-Fc antibodies. Detection of HRP conjugates was carried out with 3,3′,5,5′-tetramethybenzidine (TMB/E, Millipore, Billerica, MA, USA). Detection of AP-conjugated antibodies was carried out with SIGMAFAST p-nitrophenyl phosphate tablets (Sigma-N2770).
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3

Phage Display Peptide Binding Assay

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The phage displayed peptide clones with different amino acid sequences were subjected to ELISA. Briefly, ELISA plates were coated with the G1 protein at a concentration of 10 μg/well diluted in 0.1 mol/L NaHCO3 (pH 8.6) overnight at 4°C. Meanwhile, the purified pET32a(+) vector control protein coated with the same concentration was used as control group. All ELISA plates were blocked with 200 μL blocking buffer (5% BSA) for 2 h at room temperature. Ten-fold serial dilutions of the selected phages were added to both coated plates starting with 1012 phages in the first well. Plates were incubated for 2 h at room temperature and then washed 6 times with TBST (TBS containing 0.5% Tween-20). Bound phage was then detected with 200 μL 1:1000 diluted horseradish peroxidase (HRP)-conjugated anti-M13 antibody (GE Healthcare, 27–9421-01, USA) in blocking buffer. Finally, the peroxidase activity was rapidly detected with substrate solution TMB upon addition of a sulfuric acid stop solution. The absorbance of the reaction was determined at 450 nm with an ELISA microplate reader (Shanghai Utrao Medical Instrument Co., Ltd., Shanghai, China).
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4

Phage Display Screening of Eosinophil Protein

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Ph.D-12mer phage peptide library was purchased from New England Biolabs (New England Biolabs, Beverly, MA, USA). This contains a structurally constrained 12-mer random peptide library with complexity of 1.2 × 109 and E. coli ER 2738 as a host cell. Recombinant Human Eosinophil Cationic protein was purchased from Abcam (Cambridge, Massachusetts, USA). ELISA microplate 96-well, hingh binding PS, F-bottom, (Chimney Well), clear, Microlon®, were purchased from Greiner Bio one (Kremsmunster, Austria), bovine serum albumin (BSA) was from Bovogen (East Keilor, Australia), horseradish peroxidase (HRP)-conjugated anti-M13 antibody was from GE Healthcare (New Jersey, USA), 3,3’,5,5’-tetramethylbenzidine (TMB) substrate was from BD OptEIA™ (California, USA) and 5-Bromo-4-Chloro-3-Indolyl β-D-Galactopyranoside (X-Gal) was from Ludwig Biotecnology (Rio Grande do Sul, Brazil). Absorbance was measured using the Sunrise Basic microplate reader (Tecan group Ltd, Männedorf, Switzerland).
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5

Screening Nanobody Clones from Phage Display

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Blocked recombinant phage supernatants (100 μl) were added to 96-well clear flat-bottom polystyrene High Bind microplates (Corning, USA) coated with 3 μg/ml Ptedn (to screen nanobody clones) or with 3 μg/ml BSA (negative control) for 1 h at 37 ℃. Microplates were rinsed 10 times with PBS/0.1% Tween 20 (PBST), incubated with 100 μl/ well of horseradish peroxidase (HRP)-conjugated anti-M13 antibody (1:5000; GE Healthcare, USA) for 1 h at 37 ℃, rinsed 10 times with PBST, and 100 μl/well of TMB singlecomponent substrate solution (Solarbio Science & Technology) was added in the dark for 10 min at room temperature. After the reaction was terminated with equal volume of 1 M H 2 SO 4 , optical density (OD) was measured at 450 nm and the positive clones with the highest OD values were selected for sequencing.
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6

Monoclonal Antibody Production and Characterization

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The anti-Cry1Ab MAb used was previously produced in our laboratory.32 (link) Cry1Ab, Cry1Ac, Cry1F, Cry2A, and Cry3B toxins were purchased from You Long Bio. Co. Ltd. (Shanghai, China). SuperScript III First-Strand Synthesis SuperMix was purchased from Invitrogen (Carlsbad, CA, USA). Restriction enzymes and T4 DNA ligase were purchased from NEB (Ipswich, MA, USA). Escherichia coli strain TG1 and helper phage M13K07 were obtained from MRC (Cambridge, England). Horseradish peroxidase (HRP) conjugated anti-M13 antibody was purchased from GE Healthcare (Beijing, China). Tetramethylbenzidine was purchased from Sigma (St. Louis, MO, USA). SuperSignal ELISA Pico chemiluminescent substrate was obtained from Thermo Scientific (Waltham, MA, USA). All other reagents used were of analytical grade.
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