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Tmb substrate

Manufactured by Solarbio
Sourced in China, United States

TMB substrate is a chromogenic substrate used in enzyme-linked immunosorbent assays (ELISA) and other immunoassays. It undergoes a color change reaction when catalyzed by the enzyme horseradish peroxidase (HRP), which is commonly used as a reporter enzyme in these assays.

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15 protocols using tmb substrate

1

Quantifying Osteocalcin and Alkaline Phosphatase

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The anti-osteocalcin (OCN) and anti-alkaline phosphatase (ALP) antibodies for ELISA were obtained from Santa Cruz Biotechnology. The primary antibodies (1:1000) were diluted with ELISA coating solution (SolarBio, Beijing, China), 0.1 mL was added into each well and the cells were incubated at 4°C. Next day, each well was washed three times with PBS before being lysed for assay.
The cell lysates were diluted with PBS and 0.1 mL diluted cell lysate was added into appropriate wells. After the plates were incubated at 37°C for 2 h, the lysate was removed and washed four times with 200 μL PBS/ well. Then, 100 μL of the appropriate primary antibodies (1:3000) were added to each well and the plate was incubated at 37°C for 1 h. After washing four times with PBS, 100 μL HRP-linked secondary antibody was added to each well and the plate was incubated at 37°C for 30 min. After washing four times with PBS, 100 μL of TMB Substrate (SolarBio) was added to each well and the plate was incubated at 37°C for 10 min, and at 25°C for 30 min. Then 100 μL of ELISA STOP Solution (SolarBio) was added to each well and the plate was shaken gently for a few seconds. Finally, the absorbance at 450 nm was read using a BioRad microplate reader.
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2

ELISA-based Quantification of HB27 Protein

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Evaluation of HB27 expression in vitro and in vivo was performed by ELISA as described previously.15 (link) Briefly, 96-well microtiter plates were coated with the recombinant RBD protein (Sino Biological) overnight at 4 °C. The coated plates were washed once with PBS and blocked with 5% BSA at 37 °C for 1 h. Plates were then washed twice with PBS and incubated with calibrators and serial dilutions of cell culture media or mouse sera at 37 °C for 1 h, prior to three further washes and subsequent 1 h incubation with HRP-conjugated goat anti-human IgG-Fc antibody (Biodragon) as secondary antibody, followed by incubation with TMB substrate (Solarbio). The absorbance at 450/620 nm was measured and accurate quantification were conducted using SpectraMax iD3 (Molecular Devices). Each quantitative test produces a standard curve for back-calculation of accurate concentration of HB27.
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3

SARS-CoV-2 Antibody ELISA Quantification

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Specific IgG antibody endpoint GMT were measured by ELISA. Briefly, sera serially diluted in ELISA buffer (1% BSA with 0,05% Tween-20) were added (100 μl/well) to 96-well plates (Costar) coated with recombinant SARS-CoV-2 S protein antigen (Novozan Biotechnology Co., Ltd.) and blocked with ELISA buffer for 120 min at 37 °C. After three washes with wash buffer (PBS with 0.05% Tween-20), the plates were incubated at 37 °C for 30 min with horseradish peroxidase-conjugated goat anti-mouse IgG (Proteintech, SA00001-1) or goat anti-hamster IgG (Shanghai Universal Biotech Co., Ltd., 127-035-160) diluted in ELISA buffer at final concentrations of 0.08 μg/μl. The plates were then washed 3 times with wash buffer. Signals were developed using TMB substrate (Solarbio). The colorimetric reaction was stopped by the addition of 2 M H2SO4. Finally, the absorbance (450 nm) was measured with a Varioskan LUX Multimode Microplate Reader (Thermo). The IgG endpoint GMT were defined as the dilution fold with an optical density exceeding the average background plus 3 times the standard deviation (sera from the PBS control groups). The Limit of Detection (LoD) was defined as the reciprocal of the highest concentration of sera tested.
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4

SARS-CoV-2 Spike Protein IgG Assay

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Recombinant SARS-CoV-2 Spike protein (50 ng, Sino Biological) diluted in carbonate buffer (0.1 M, pH 9.6) were coated into 96-well EIA/RIA plates (Coning) overnight at 4 °C. The plates were then washed with PBS-T (0.05% Tween-20) and were blocked with 10% goat serum in PBS for 2 h at 37 °C. Then, serum samples serially diluted in PBST containing 2% goat serum were added and incubated for 2 h at 37 °C. After washing, total IgG was evaluated using HRP-conjugated goat anti-mouse IgG Ab (1:10,000) or HRP-conjugated goat anti-NHP IgG Ab (1:50,000) for 1 h. In mice experiments, IgG subclasses were evaluated using biotinylated anti-mouse IgG2a mAb (clone: MG2a, Mabtech), biotinylated anti-mouse IgG1 mAb (clone: MG1, Mabtech), biotinylated anti-mouse IgG2c mAb (clone: MTG2c, Mabtech) for 1 h and further analyzed with Streptavidin-HRP (1:1000, Mabtech). TMB substrate (Solarbio) was used for development and the absorbance was read at 450 nm using SPECTROsta Nano (BMG) microplate reader. Endpoint titers were calculated as the dilution that exceed 2.1-folded value of the background.
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5

Optimized ELISA for Antibody Quantification

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High‐binding 96‐Well ELISA plates were coated with OVA protein (20 µg mL−1) in a Na2CO3/NaHCO3 buffer (pH = 9.6) at 4 °C overnight. After being washed with 0.05% tween‐PBS solution, the plates were blocked with 3% BSA solution. The antiserum was then diluted to different concentrations using 1% BSA in PBS and added to the plates (100 µL well−1) and incubated for 2 h at 37 °C. The plates were washed for three times, and incubated with rabbit anti‐mouse IgG‐Peroxidase antibodies (SBA, 1:2000 dilution) for 1 h at 37 °C. Then the plates were further washed for three times, followed by the addition of TMB substrate (Solarbio), and reacted at room temperature for 20 min in dark. The corresponding OD450 was measured by Multiskan GO (Thermo Scientific) microplate reader.
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6

SARS-CoV-2 Spike Protein ELISA Assay

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An ELISA assay assessing binding antibody levels against the wild-type SARS-CoV-2 was conducted using the wild-type Spike protein. ELISA plates (Corning Costar; Corning, NY, USA) were coated with S protein (Sanyou Biopharmaceuticals Co., Shanghai, China) at a concentration of 5 μg/well and incubated overnight at 4 °C. During the experiment, ELISA plates were blocked with 5% BSA-phosphate-buffered saline (PBS), incubated with serially diluted serum samples, and visualized by reaction with an HRP-conjugated antibody (Abcam, Waltham, MA, USA) and TMB substrate (Solarbio, Beijing, China), using previously described methods [16 (link)]. The absorbance of each well at 450 nm was measured using an ELISA plate reader (Gene Company, Beijing, China). Antibody serum samples yielding OD values at least 2.1-fold higher than the negative control at a test sample dilution of 1:400 were considered positive. Endpoint titers (ETs) were defined as the highest serum dilutions yielding positive OD values. GMTs were calculated as the geometric mean of the ETs of positive serum samples in each group. ELISA assays assessing binding antibody levels against variant strains of SARS-CoV-2 (Alpha, Beta, Gamma, Delta, and Omicron) were conducted using commercial ELISA kits (ACRO Biosystems Co., Beijing, China) according to the manufacturer’s instructions.
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7

Binding Affinity of GLP-1 Fusion Proteins

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An ELISA was used to determine the binding affinities of the fusion proteins to HSA, following the methodology described by Tan et al. [11 (link)]. An immobilized 96-well plate was prepared by incubating 10 μg of HSA in 100 mM carbonate buffer (pH 9.6) overnight at 4 °C. After three washes with PBST (PBS + 0.05% Tween 20), the plate was blocked with 5% BSA (Solarbio, Beijing, China). A series of GLP-ABD, GLP-ABD-XTEN144 or GLP-ABD-XTEN288 fusion proteins at different concentrations, from 0.032 to 500 nM, were subsequently introduced into the wells and left to incubate for 2 h in a room temperature setting. The GLP-1 present in these combined proteins was identified by employing a mouse anti-GLP-1 antibody (ab23472, Abcam, UK) as well as a HRP-conjugated goat anti-mouse IgG (CW0202S, CWBIO, China). After completing a series of three rinses, a TMB substrate (Solarbio, China) was employed to pinpoint the HRP that had successfully bonded. The reaction was concluded by adding 1 M HCl, followed by a measurement of the absorbance at 450 nm using a SpectraMax M2 microplate reader (Molecular Devices, USA).
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8

ELISA for Serological Antibody Titers

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Flat bottom clear plates (Corning, Catalog CLS3590) were coated overnight at 4°C with 2 µg HA protein (split H1N1, H3N2, B/Victoria viruses, and B/Yamagata) in 100 µL 50 mM Tris-HCl (pH 9.5) per well. After blocking (1% milk powder in PBS) for 1 h at RT, sera were added at 1:100, 1:1 000, 1:10 000, and 1:100 000 dilutions and incubated for 2 h at RT. Thereafter, HRP-conjugated anti-mouse IgG (Proteintech, 1:1000) was added and incubated for 1 h at 4°C before being developed with 100 µl/well of TMB substrate (Solarbio). The reactions were stopped after 10 min with 100 µl 1 M HCl and A450-A630 determined in an ELISA reader (ThermoFisher). Wells were washed (3×) with PBS containing 0.05% Tween-20 between each step. A positive control diluted in ten-fold dilutions was set on every ELISA plate to normalize all the detected values on different plates. Utilizing individual serum with serial dilutions, the area under the curve (AUC) was determined to calculate the IgG antibody titre.
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9

Quantifying Angiogenic Factors by ELISA

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The anti-VEGFA and anti-FGF2 primary antibodies for ELISA were purchased from Abcam (Cambridge, UK). The ELISA coating solution, TMB Substrate, and ELISA STOP solution were purchased from SolarBio (Beijing, China). The detailed procedures were as described previously 34 (link).
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10

SARS-CoV-2 Spike RBD Quantification

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Evaluation of RBD expression in vitro and in vivo was performed by ELISA. Briefly, 96-well microtiter plates were coated with 5 μg/ml of human ACE2 (Kactus Biosystems) overnight at 4°C. The coated plates were washed once with PBS and blocked with 5% BSA at 4°C for 12 hours. Plates were then washed twice with PBS and incubated with serial dilutions of cell culture media or mouse sera at room temperature for 1 hour, prior to three further washes and subsequent 1 hour incubation with SARS-CoV-2 S rabbit mAb (Sino Biological) as primary antibody at room temperature. After three washes with PBS, plates were incubated with HRP-conjugated goat anti-rabbit IgG-Fc antibody as secondary antibody (Sino Biological), followed by incubation with TMB substrate (Solarbio). The absorbance at 450/620 nm was measured and accurate quantification were conducted using SpectraMax iD3 (Molecular Devices).
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