The isolated protein was diluted in 0.1M sodium carbonate-bicarbonate (pH 9.4) coating buffer. 100μl of 5 μg/ml protein was coated on flat bottom 96 well microtest plate and incubated for 3 hr at 37°C. After incubation, excess antigen was removed by washing with neutral phosphate buffer saline (PBS). Then wells were blocked over night at 4°C by 3% bovine serum albumin (BSA), incubated for 2 hrs at room temperature with 1:100 dilution of HSP70 primary antibody (sc-1060, Santa Cruz Biotechnology, USA). Any excess antibody was removed by washing with PBS and incubated with horseradish peroxidase—conjugated secondary antibody of 1:1000 dilution (sc-2357, Santa Cruz Biotechnology, USA) for 2 hr. The wells were incubated with 3, 3’, 5, 5’- tetramethyl benzidine (TMB) (eBiosciences lnc. San Dieago, CA, USA) for 30 min at 37°C after washing. The reaction was stopped by 0.2 M sulphuric acid and optical density at 450 nanometre (nm) wavelength was measured by ELISA reader TECAN (Infinite® 200 PRO) and numeric data expressed as relative light unit (RLU), data was expressed as the mean RLU ± SD.
Tetramethylbenzidine tmb
Manufactured by Thermo Fisher Scientific
Sourced in United States
Tetramethylbenzidine (TMB) is a colorimetric substrate used for the detection and quantification of peroxidase enzyme activity in various immunoassay applications, such as enzyme-linked immunosorbent assays (ELISA). TMB produces a blue-colored product upon oxidation by peroxidase, which can be measured spectrophotometrically.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Sc 1060, by Santa Cruz Biotechnology (1 mentions)
Sc 2357, by Santa Cruz Biotechnology (1 mentions)
Infinite 200 pro, by Tecan (1 mentions)
Streptavidin hrp, by Agilent Technologies (1 mentions)
P nitrophenylphosphate p npp solution, by Merck Group (1 mentions)
Igg1 and igg2a, by BD (1 mentions)
Streptavidin hrp, by Thermo Fisher Scientific (1 mentions)
Microplate reader, by Bio-Rad (1 mentions)
Sst microtainer tubes, by BD (1 mentions)
Anti human iga hrp, by Southern Biotech (1 mentions)
Anti human igg hrp, by Jackson ImmunoResearch (1 mentions)
96 well plate, by Corning (1 mentions)
Maxisorp, by Thermo Fisher Scientific (1 mentions)
Microplate reader, by Tecan (1 mentions)
Immuno plate, by Thermo Fisher Scientific (1 mentions)
Bradford assay, by Merck Group (1 mentions)
Versamax microplate reader, by Molecular Devices (1 mentions)
Opteia kit, by BD (1 mentions)
22 protocols using tetramethylbenzidine tmb
1
Quantification of HSP70 Protein Levels
2
ELISA for Autoantibody Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Example 17
ELISA plates (Nunc Immunoplate MaxiSorp; Fisher Scientific) were coated with 3 μg/ml MP4, 10 μg/ml MOG:35-55 or 3 μg/ml ovalbumin (OVA) in bicarbonate buffer overnight at 4° C. The plates were blocked for 2 hours with phosphate-buffered saline (PBS), containing 0.05 Tween 20 (PBST) and 1% BSA at room temperature. Subsequently, serum samples diluted in PBST/BSA, were added to the plate for overnight incubation at 4° C. Secondary antibodies used were biotinylated rat anti-mouse IgG (eBioscience, San Diego, Calif.), IgG1 and IgG2a (BD-Pharmingen, San Jose, Calif.). Streptavidin-AP or Streptavidin-HRP served as tertiary reagents (DakoCytomation, Glostrup, Denmark), both diluted at 1:1000 in PBST/BSA. Plates were developed with 100 μl/well of freshly prepared p-nitrophenylphosphate (p-NPP) solution (Sigma-Aldrich) or tetramethylbenzidine (TMB) (eBioscience).
3
Plasma Biomarkers in Longitudinal Study
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
At baseline and at every half-yearly follow-up visit (up to 10-year follow-up), peripheral venous blood samples were collected and processed within 2 h by Ficoll separation and divided into plasma and peripheral blood mononuclear cells (PBMCs) fractions. Plasma samples were subsequently stored at -80 degrees.
The concentrations of inflammatory markers (VEGF-A, TGFβ, CXCL-9, CXCL-13, IL-1β, IL-6, IL-8, IL-10) in plasma were determined in duplicate by ELISA (R&D systems Europe, Abingdon, UK) (Additional file1 : Table 1). Streptavidin-HRP (eBioscience) and tetramethylbenzidine (TMB) substrates (eBioscience) were used to develop the ELISA. Optical densities were measured at 450 nm using a Microplate Reader (Bio-Rad, Hercules, CA, USA).
The concentrations of inflammatory markers (VEGF-A, TGFβ, CXCL-9, CXCL-13, IL-1β, IL-6, IL-8, IL-10) in plasma were determined in duplicate by ELISA (R&D systems Europe, Abingdon, UK) (Additional file
4
Serum-based Virus-specific ELISA and HI Assay
5
SARS-CoV-2 RBD Protein Binding Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
96-well plates (Corning) were coated with 2 ug/mL of recombinant SARS-CoV-2 RBD protein diluted in PBS and incubated at 4°C overnight. Plates were washed with PBS-T (PBS containing 0.05% Tween-20) and incubated with blocking buffer (PBS-T and 3% milk) for 1h at room temperature (RT). Plasma, culture supernatants or monoclonal antibodies were serially diluted in dilution buffer (PBS-T and 1% milk) in triplicate, added to plates, and incubated at RT for 2 h. Secondary antibodies were diluted in dilution buffer as follows: anti-human IgG-HRP (Jackson ImmunoResearch) at 1:3000 or anti-human IgA-HRP (Southern Biotech) at 1:1500. Plates were incubated with secondary antibodies for 1h at RT, then detected with 1X 3,30,5,50-Tetramethylbenzidine (TMB) (Invitrogen) and quenched with 1M HCl. Sample optical density (OD) was measured by a spectrophotometer at 450 nm and 570 nm.
6
Quantifying Omicron RBD-Specific Antibodies
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Plasma samples were analyzed by ELISA to detect Omicron RBD-specific antibodies of various isotypes, including IgM, IgG1, IgG2, IgG3, and IgG4; 96-well Maxisorp plates (Thermo Fisher Scientific, Waltham, MA, USA) were coated with recombinant Omicron RBD protein (Acro Biosystem, SPD-C522e) at 2μg/mL overnight. Plates were blocked with 2% Bovine serum albumin in PBS at RT for 1 h before the addition of serially diluted plasma samples and incubated for 2 h at RT. RBD-specific antibodies of various isotypes were detected with Horseradish peroxidase (HRP)–conjugated mouse anti-human IgM (SouthernBiotech #2023-05), IgG1 (#9054-05), IgG2 (#9060-05), IgG3 (#9210-05), or IgG4 (#9200-05) antibodies. Plates were developed with stabilized chromogen tetramethylbenzidine (TMB) (Invitrogen) and stopped with 3N hydrochloric acid before reading at 450 nm on a microplate reader (Tecan). Quantification of each antibody isotype was performed using a 6-point standard curve generated from a reference control plasma sample.
7
Quantification of IL-6 and TNF-α by ELISA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
IL6 and TNFα were quantified by sandwich ELISA using commercial kits (ELISA MAX 430502, Biolegend, and BD OptEIA 555212, respectively), following the manufacturer's instructions. Briefly, 96-well, flat-bottom ELISA plates (Nunc ImmunoPlate) were coated with the respective capture antibody and incubated overnight at 4 °C in carbonate buffer (pH 9.6). Non-specific binding sites were blocked by incubating for 30 min at RT with PBS-1% albumin. Culture medium was added and incubated for 2 h at RT. The plates were then incubated with the corresponding horseradish peroxidase (HRP)-conjugated anti-cytokine detection antibody for 30 min at RT. Bound complexes were detected by reaction with tetramethylbenzidine (TMB) (Invitrogen, Carlsbad, CA, USA) after 30 min incubation in the dark. The reaction was stopped with 2 N H2SO4, and absorbance was measured at 450 nm at 37 °C on a VERSAmax ELISA plate reader. Standard curves were built to calculate the concentration of each cytokine. Concentrations were expressed as pg/mg protein employing the Bradford assay (Sigma, B6916).
8
ELISA Quantification of IL-6 in Cell Culture
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell culture medium was collected after a 12-h culture, and IL-6 concentration was measured by ELISA (OptEIA kit BD-Biosciences, Franklin Lakes, NJ, USA) following the manufacturer’s directions. Briefly, 96-well plates were sensitized with the capture antibody overnight at 4 °C. The plates were blocked with PBS-FBS 10% for 1 h at room temperature. Then, either medium or the IL-6 standard was added and incubated at room temperature for 2 h, followed by incubation for 1 h at room temperature with the horseradish peroxidase-coupled detection antibody. The chromogenic substrate tetramethylbenzidine (TMB) (Invitrogen) was added, and the reaction was stopped 30 min later with H2SO4 2N. Absorbance was determined at 450 nm at 37 °C using a VERSAmax microplate reader (Molecular Devices, San Jose, CA, USA). IL-6 concentration was calculated according to a standard curve.
The results obtained were used to plot a concentration–response curve and to determine the maximal effect (Emax) and effective 50% concentration (EC50) values for each subfraction.
The results obtained were used to plot a concentration–response curve and to determine the maximal effect (Emax) and effective 50% concentration (EC50) values for each subfraction.
9
Influenza-Specific Antibody Titers by ELISA
10
IgG Subclass Detection in Anti-α3(IV)NC1
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The detection of IgG subclasses against α3(IV)NC1 was performed as previously reported [7] . In brief, the recombinant human α3(IV)NC1 proteins were diluted at 1μg/mL and coated onto half of the wells of a polystyrene microtiter plate. The other half of the wells were coated with BSA as antigen-free wells. Diluted serum samples at 1:100 were then added to the antigen and BSA coated wells respectively, and incubated at 37°C for 60 min. After washing, horseradish peroxidase labeled mouse monoclonal antibodies against human IgG1, IgG2, IgG3, and IgG4 (Fc speci c, Southern Biotech, USA, 1:2000) were added at 37°C for 60 min. After washing, color was developed by adding 50 μL of substrate (Tetramethyl benzidine, TMB, Invitrogen, USA) into the wells for 15 min. Reaction was terminated by adding stop solution and color measured at 450 nm.
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!