Serum from vaccinated mice was diluted and added to parental or human HER2-expressing NMUMG cells for 1 hour at room temperature. Plates were washed and cells were fixed with 1% formalin. A secondary antimouse IgG-HRP conjugated antibody (Cell Signaling Technology) was used prior to developing with 3,3',5,5'-Tetramethylbenzidine (TMB) substrate (Biolegend) and absorbance determined using a Bio-Rad Model 680 microplate reader (Bio-Rad).
3 3 5 5 tetramethylbenzidine tmb substrate
Manufactured by BioLegend
Sourced in United States
3,3′,5,5′-tetramethylbenzidine (TMB) substrate is a chromogenic substrate used in enzyme-linked immunosorbent assay (ELISA) and other immunoassay applications. It undergoes an enzymatic reaction to produce a colored product that can be measured spectrophotometrically.
Automatically generated - may contain errors
Lab products found in correlation
Spectramax plate reader, by Molecular Devices (2 mentions)
Secondary antimouse igg hrp conjugated antibody, by Cell Signaling Technology (1 mentions)
Model 680 microplate reader, by Bio-Rad (1 mentions)
H2so4 stop solution, by BioLegend (1 mentions)
Envision system hrp, by Agilent Technologies (1 mentions)
Mrx 2, by Dynex (1 mentions)
Hek293, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase conjugated goat anti rabbit antibody, by Cell Signaling Technology (1 mentions)
Rabbit anti flag m2 antibody, by Cell Signaling Technology (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Mouse igg and igm elisa kits, by Fortis Life Sciences (1 mentions)
Capiject tubes, by Terumo (1 mentions)
96 well elisa plates, by BD (1 mentions)
Softmax pro 7, by Molecular Devices (1 mentions)
Spectramax id5 spectrophotometer, by Molecular Devices (1 mentions)
Pierce lal chromogenic endotoxin quantitation kit, by Thermo Fisher Scientific (1 mentions)
Calf thymus dna, by Merck Group (1 mentions)
Mouse igg elisa kit, by Fortis Life Sciences (1 mentions)
Anti mouse il 10, by R&D Systems (1 mentions)
Avidin hrp, by BioLegend (1 mentions)
10 protocols using 3 3 5 5 tetramethylbenzidine tmb substrate
1
HER2-expressing Cell Binding Assay
2
Peptide-HLA-E Binding Assay
3
ELISA-Based Serum IgG Titer Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The total serum IgG titers against the recombinant DIII proteins or virions were determined by ELISA. The ELISA plate was coated with 0.2 μg of the recombinant DIII proteins or 105 FFU formalin-inactivated virus (100 μl/well) in 0.05M carbonate buffer at 4oC overnight. After being washed with PBST three times, each well of the plate was blocked with 150 μl/well of 1% bovine serum albumin (BSA) in PBS for 1 h at room temperature (RT) and then washed with 200 μl PBST three times. Next, the mouse sera were 4-fold serially diluted from a dilution factor of 1:100 with dilution buffer (PBS containing 1% BSA and 0.05% Tween 20) and added to the ELISA plate for 1 h of incubation at RT. After washing with PBST three times, each well of the ELISA plate was treated with 100 μl HRP-conjugated goat anti-mouse IgG antibody at a dilution factor of 1:10000 at RT for 1 h and washed with PBST three times. Finally, 100 μl/well of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (BioLegend) was added to the ELISA plate for coloration and allowed to react in the dark for 15 min. The reaction was stopped by 100 μl/well of 2 N sulfuric acid. The absorbance for each well was measured at 450 nm using an ELISA reader (DYNEX MRX II). The end-point titers of total IgG were defined as the maximal serum dilutions that produced an OD value of over 0.2.
4
Quantifying Cell Surface Receptor Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To quantify cell surface receptor expression, HEK293 (ThermoFisher Scientific, Nr. R70507) cells transfected with G-CASE and pcDNA or N-terminally FLAG-tagged H1/2R constructs were grown for 48 h in transparent 96-well plates (Brand, Wertheim, Germany) and washed once with 0.5% BSA (Merck KGaA, Darmstadt, Germany) in PBS. Next, cells were incubated with a rabbit anti-FLAG M2 antibody (142 ng/mL) (Cell Signaling Technology, Danvers, MA, USA) in 1% BSA–PBS for 1 h at 4 °C. Following incubation, the cells were washed three times with 0.5% BSA–PBS and incubated with a horseradish peroxidase-conjugated goat anti-rabbit antibody (30 ng/ml) (Cell Signaling Technology, Danvers, MA, USA) in 1% BSA–PBS for 1 h at 4 °C. The cells were washed three times with 0.5% BSA/PBS, and 50 µl of the 3, 3′, 5, 5′ tetramethyl benzidine (TMB) substrate (BioLegend, San Diego, CA, USA) was added. Subsequently, the cells were incubated for 30 min and 50 µl of 2 M HCl was added. The absorbance was read at 450 nm using a BMG ClarioStar Plus plate reader.
5
Quantifying Anti-dsDNA IgG in Plasma
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Blood was collected into anticoagulant-coated Capiject tubes (Terumo Medical, Somerset, NJ) and centrifuged at 15,000×g for 30 sec. Plasma was collected and stored at -80°C. For detection of anti-double-stranded DNA (dsDNA) IgG, the plate was coated with 0.1 mg/ml of calf DNA (Sigma) in 1 saline-sodium citrate (SSC) buffer at 4°C overnight, followed by washing with PBS containing 0.05% Tween-20 (PBS-T). Wells were then blocked with PBS containing 1% bovine serum albumin (BSA) for 1 h at room temperature and washed. Samples were added and incubated for 1 h at room temperature. After additional washing, HRP-conjugated goat anti-mouse IgG-Fc secondary antibody (Bethyl Laboratories, Montgomery, TX) was added and incubated for 1 h at room temperature, following by more washes with PBS-T. 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate (Biolegend) was then added. After the reaction was stopped, the plate was read at 450 nm with SpectraMax plate reader (Molecular Devices, Sunnyvale, CA). Total IgG and IgM concentrations were determined with mouse IgG and IgM ELISA kits according to the manufacturer’s instructions (Bethyl Laboratories).
6
Quantifying Antibody Levels in Respiratory Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human IgG1 antibody levels in serum and BAL fluid were determined by IgG1 Ready-SET-Go! ELISA (Affymetrix, eBioscience) according to the manufacturer’s instructions. After heat inactivation (56°C for 30 min) samples were diluted 1:40 (serum) and 1:2 (BAL fluid). Influenza-specific human Ab titers in serum and BAL fluid were determined by ELISA as previously described (20 (link)) with the following modifications. The IgG ELISA was performed in 96-well ELISA plates (BD Biosciences) coated with 1 × 106 PFU/ml of A/swine/England/1353/09 over night at 4°C. Twofold dilutions of BAL fluid samples or serum (heat inactivated for 30 min at 56°C) were added, starting from 1:2 or 1:10 dilution, respectively. Binding of influenza-specific Abs was detected using a monoclonal anti-human IgG (hIgG) (Fc) (Biorad) and 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (BioLegend). Optical density (OD) readings were taken at 450 and 570 nm (wavelength correction). Ab values were expressed as endpoint titers defined as the highest dilution at which the OD was higher than twice the background OD.
7
Peanut Allergy IgE Disruption by mAbs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
An indirect competitive ELISA was used to evaluate the ability of mAbs to disrupt peanut allergy serum IgE binding to Ara h 2. Microtiter plates were coated with 5 μg/mL natural Ara h 2 (Indoor Biotechnologies) overnight at 4°C and then blocked (PBST, consisting of 0.05% Tween-20 and 1% BSA) for 2 hours. Purified Ara h 2–specific mAbs (0.625–5 μg/mL) were added for 2 hours, followed by a 2-hour incubation with a 1:50 dilution of IgG-depleted peanut allergy pooled plasma from placebo-treated OIT participants (n = 6). For IgE detection, anti-IgE conjugated to HRP (1:10,000, Bethyl Laboratories) was added for 1 hour. Plates were washed with PBST between each step of the protocol, and all incubations were performed at room temperature, unless otherwise specified. For the colorimetric assay, 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (BioLegend) was incubated for 45 minutes, and the reaction was stopped with 1 M phosphoric acid (Thermo Fisher Scientific). Absorbance was measured at 450 nm on a SpectraMax iD5 spectrophotometer (Molecular Devices) with SoftMax Pro 7.1 (Molecular Devices). Before analysis, the background was subtracted from OD values. The percentage of inhibition was calculated after normalization to the control (without a mAb).
8
Anti-dsDNA IgG ELISA Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Serum samples were obtained following whole blood collection and coagulation, and stored at −20°C. Anti-double stranded (ds)DNA IgG levels in diluted serum samples was detected following a previously reported ELISA assay (12 (link)). Briefly, the plate was coated with 0.1 mg/ml of calf thymus DNA (Sigma) in 1× saline-sodium citrate buffer and incubated at 4°C overnight, followed by washing with PBS containing 0.05% Tween-20 (PBS-T). Wells were then blocked with PBS containing 1% BSA for 1 h at room temperature. Samples were added and incubated for 1 h at room temperature. After additional washes in PBS-T, horseradish peroxidase-conjugated goat anti-mouse IgG-Fc secondary antibody (Bethyl Laboratories, Montgomery, TX) was added and incubated for 1 h at room temperature, followed by further washing. 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate (Biolegend) was then added, and the reaction was stopped by adding 2N H2SO4 stop solution. The plate was read at 450 nm using SpectraMax plate reader (Molecular Devices, Sunnyvale, CA). Total IgG levels were determined using a mouse IgG ELISA kit (Bethyl Laboratories) according to the manufacturer's instructions. Serum endotoxin (lipopolysaccharide, or LPS) level was measured using Pierce LAL Chromogenic Endotoxin Quantitation Kit (Thermo Scientific) following the manufacturer's procedures.
9
Quantitative Mouse IL-10 ELISA Protocol
10
Quantification of Mitofusin-1 Antibodies
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!