The gene-transfected DO-11.10 cells were lysed with RIPA buffer (50 mM Tris-HCL pH 7.6, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, and protease inhibitor cocktail; Nacalai Tesque) on ice for 1 h. Cell lysates were centrifuged at 10,000 × g for 10 min at 4 °C and the supernatants used for ELISA. The stock silk solutions (80 mg/mL in 9 M LiBr) were diluted with 1 mM Tris-HCl (pH 8.0) to a concentration of 0.25 mg/mL. One hundred microlitres of the cell lysate, culture supernatants from hybridoma cells, and diluted silk solutions were applied to 96-well plates and incubated overnight at 4 °C. After three washes with PBS, each well was blocked with ELISA Assay Diluent (BioLegend, San Diego, CA, USA) at room temperature for 1 h. After five washes with PBS and Tween 20, CEA (Abcam, code no. ab742, Cambridge, UK) was added to the wells and incubated at room temperature for 2 h. Binding was detected with anti-CEA polyclonal antibody (Abcam, code no. ab15987), then HRP-conjugated anti-rabbit Igs (Dako), finally by incubation with ELISA POD Substrate TMB solution (Nacalai Tesque). After colour development, the reaction was stopped with 2 N H2SO4 and the absorbance read at 450 nm using a microplate reader (iMarkTM Microplate Reader; Bio-Rad).
Elisa pod substrate tmb solution
Manufactured by Nacalai Tesque
Sourced in Japan
ELISA POD substrate TMB solution is a reagent used in enzyme-linked immunosorbent assay (ELISA) protocols. It serves as a substrate for the detection of enzyme activity, producing a colored product that can be measured spectrophotometrically.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Nacalai Tesque (2 mentions)
Elisa assay diluent, by BioLegend (2 mentions)
Imark microplate reader, by Bio-Rad (2 mentions)
Hrp conjugated anti rabbit ig, by Agilent Technologies (1 mentions)
Nunc maxisorp flat bottom plate, by Thermo Fisher Scientific (1 mentions)
Recombinant human tnf α, by BioLegend (1 mentions)
Multiskan fc microplate reader, by Thermo Fisher Scientific (1 mentions)
Eia plate, by Thermo Fisher Scientific (1 mentions)
Elisa diluent, by BioLegend (1 mentions)
Hrp conjugated protein a, by BioLegend (1 mentions)
Fc tag, by ACROBiosystems (1 mentions)
Block ace, by Sumitomo Pharma (1 mentions)
Varioskan, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Equitech-Bio (1 mentions)
6 protocols using elisa pod substrate tmb solution
1
ELISA-based Silk Protein Quantification
2
Measuring Antibody-Antigen Binding Activity
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The binding activity of antibody to its antigen, recombinant human TNFα (BioLegend, San Diego, CA, USA), was measured by using 96-well Nunc MaxiSorp™ Flat-Bottom plate (Thermo Fisher Scientific). First, 100 μl TNFα solution was coated with a concentration of 100 ng/mL in sodium carbonate buffer (pH 9.6) overnight at 4 °C. The wells were washed three times with PBS-Tween solution (PBS buffer with 0.05% Tween 20) and blocked with blocking buffer (PBS-Tween containing 5% skim milk) at room temperature for 1 h. After washing three times with the PBS-Tween solution, 100 μl of adalimumab sample in serial dilution was added to each well and incubated at room temperature for 1 h. Then, the wells were washed again three times with PBS-Tween solution and incubated with 1:8000-diluted Anti-IgG (H + L chain) (Human) pAb-HRP antibody at room temperature for 1 h. The plate was washed four times with PBS-Tween solution and incubated with 100 μl ELISA POD Substrate TMB Solution (Nacalai) at room temperature. The reaction was stopped by adding an equal volume of 1 M H2SO4, and the absorbance was measured at 450 nm using Multiskan FC microplate reader (Thermo Fisher Scientific).
3
Comparative E2 Protein Binding Assay
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The anti-E2 antibodies were assayed for their comparative affinity for the J6CF E2-Fc and TH E2-Fc proteins. All steps were performed at room temperature. A sample of each of the various monoclonal antibodies was subjected to either a 3- or 10-fold serial dilution, and the dilutions were distributed to an EIA plate (Thermo Fisher Scientific) in which the J6CF E2-Fc or TH E2-Fc protein had been immobilized (0.5 μg/well). An antibody, HR1-007 raised against Protobothrops flavoviridis venom hemorrhagic factor was employed as a control human IgG. After incubation for 1 hour, the plates were washed with PBS containing 0.05% Tween 20 (PBS-T), and horseradish peroxidase (HRP) -conjugated goat anti-human IgG F(ab’)2 (1:5000; Thermo Fisher Scientific) was added to each well. After incubation for 1 hour, the plates were washed with PBS-T, and color was developed with ELISA POD substrate TMB solution (Nacalai Tesque, Kyoto, Japan); reactions were quenched with 1 mol/L sulfuric acid, and absorbance (at 450 nm) was measured using a microplate reader.
4
Silk-Based ELISA Binding Assay
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The gene-transfected DO-11.10 cells were lysed using RIPA buffer (50 mM Tris–HCL pH 7.6, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, and protease inhibitor cocktail; Nacalai Tesque) on ice for one hour. Cell lysates were centrifuged at 10,000 × g for ten minutes at 4 °C and the supernatants were used for ELISA. The stock silk solutions (10 mg/mL in 2 M LiBr) were diluted with 1 mM Tris–HCl (pH 8.0) to a concentration of 0.2 mg/mL. One hundred microliters of the cell lysate, culture supernatants from hybridoma cells, and diluted silk solutions were applied to 96 well plates and incubated overnight at 4 °C. After washing thrice with PBS, each well was blocked using ELISA Diluent (BioLegend, San Diego, CA, USA) at room temperature for one hour. After five washes with PBS and Tween 20, formalin-inactivated As (1.8 × 108 CFU/mL) was diluted, applied to the wells, and incubated at room temperature for 90 min. Binding was detected using sequential incubation of plates with anti-As-4A MAb and HRP-conjugated anti-mouse IgG Fc (abcam, ab97265), followed by incubation with ELISA POD Substrate TMB solution (Nacalai Tesque). After color development, the reaction was stopped with 2N H2SO4, and the absorbance was read at 450 nm using a microplate reader (iMark™ Microplate Reader; Bio-Rad).
5
ACE2 Binding Affinity Assay for BC-PIVs
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Recombinant BC-PIVs (1×106 TCID50) in PBS on the 96-well plates were immobilized at 4°C overnight. After immobilization, the plates were incubated with 4% Block Ace (DS Pharma Biomedical)/PBS for 2 h at 4°C. After blocking, serially diluted recombinant human ACE2 protein with Fc Tag (ACROBiosystems) was added to each well, and the plate was incubated for 2 h at 4°C, followed by washing 6 times with PBST and subsequent 1-h incubation with HRP-conjugated protein A (1:1,500 dilution; BioLegend) in PBS in the dark at RT. The plates were then washed further 6 times with PBST, and 100 μL of TMB Start solution of ELISA POD substrate TMB solution (Nacalai Tesque) was added to each well. The plate development was halted by the addition of 50 μL of 1N H2SO4 per well. The absorbance at 450 nm was recorded using a microplate reader.
6
ELISA for Evaluating β2-m Binding
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ELISA was used for evaluating the β2-m-binding activity of ApoE NTD-MHC α3 and ApoE NTD-scFv, as described previously, 2 with minor modifications. Briefly, ApoE NTD-MHC α3/scFv samples in PBS at different concentrations (0, 50, 100, 150, 200, 300, 400, and 500 nM) were coated onto a 96-well assay polystyrene plate (AGC Techno Glass, Japan), which was then washed five times with PBS containing 0.05% (v/v) Tween 20 (PBS-T). After blocking with a blocking buffer (ELISA assay diluent; BioLegend, CA), β2-m (0 or 1 µg mL -1 ) in the blocking buffer or in fetal bovine serum (FBS) (Equitech-Bio, TX) was added to the protein-coated plate. After incubation and washing, rabbit anti-human β2-m PAb (13511-1-AP; Proteintech, IL), followed by HRP-conjugated swine anti-rabbit Ig PAb (P0399; Dako, Denmark), was added to the plate. The plate was then washed with PBS-T, and β2-m bound to the protein-coated plate was detected using ELISA POD substrate TMB solution (Nacalai Tesque, Japan). Absorbance at 450 nm was measured using a plate reader (Varioskan™; Thermo Fisher Scientific, MA). Background (0 µg mL -1 β2-m) absorbance was subtracted from the sample (1 µg mL -1 β2-m) absorbance. Three different wells were used for each condition (n = 3).
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