Cellular protein fractions were immobilized onto the ELISA microplate for overnight at 4 °C. After blocking with 2% skim milk, culture supernatant or the immunized mouse serum containing primary antibodies; anti-E. histolytica (anti-Eh4), anti-E. moshkovskii (anti-Em6) and anti-pan-human Entamoeba; E. histolytica, E. moshkovskii and E. dispar (anti-Ehmd4) mAb or 1:5000 diluted pre-immunized serum (a control) were applied. The reactions were incubated for 1 h at room temperature. Entamoeba binding partner-mAb complexes were detected with 1:5000 diluted anti-mouse immunoglobulins (Thermo Fisher Scientific), anti-mouse IgG (Sigma-Aldrich, St. Louis, MO, USA) and anti-mouse IgA (Sigma-Aldrich) conjugated with horse radish peroxidase (HRP) enzyme (Thermo Fisher Scientific) for 1 h. The colorimetric signal was developed using 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (Thermo Fisher Scientific) and quenched with 1N hydrochloric acid (HCl). The color intensity was recorded at a wavelength of 450 nm using a Sunrise microplate reader (Tecan, Männedorf, Switzerland). The measurement was performed in triplicate and presented as means of optical density (OD) ± standard error of the mean (SEM).
3 3 5 5 tetramethylbenzidine tmb substrate
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
3,3′,5,5′-tetramethylbenzidine (TMB) substrate is a chromogenic substrate used in enzyme-linked immunosorbent assays (ELISA) and other colorimetric detection methods. It provides a blue-colored product upon oxidation by peroxidase enzymes.
Automatically generated - may contain errors
Lab products found in correlation
Bovine serum albumin (bsa), by Merck Group (5 mentions)
Goat anti human igg conjugated with hrp horseradish peroxidase, by Southern Biotech (5 mentions)
96 well plate, by Thermo Fisher Scientific (4 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (4 mentions)
Multiskan fc, by Thermo Fisher Scientific (4 mentions)
Prism 7, by GraphPad (3 mentions)
Elisa plate reader, by Tecan (3 mentions)
Multiskan mk3, by Thermo Fisher Scientific (2 mentions)
Rabbit anti mouse f ab horseradish peroxidase hrp, by Jackson ImmunoResearch (2 mentions)
Prism software, by GraphPad (2 mentions)
Elisa plate, by Thermo Fisher Scientific (2 mentions)
Fab specific secondary horseradish peroxidase hrp antibodies, by Jackson ImmunoResearch (2 mentions)
Neutravidin, by Thermo Fisher Scientific (2 mentions)
Horseradish peroxidase hrp conjugated streptavidin, by Thermo Fisher Scientific (2 mentions)
Tween 20, by Thermo Fisher Scientific (2 mentions)
Prism v8, by GraphPad (2 mentions)
Tmb stop solution, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Sybr green master mix, by Thermo Fisher Scientific (2 mentions)
Expifectamine 293 transfection kit, by Thermo Fisher Scientific (2 mentions)
69 protocols using 3 3 5 5 tetramethylbenzidine tmb substrate
1
Entamoeba-Specific Antibody Detection Assay
2
ELISA Evaluation of Refolded Antibody
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To reassure the ability of the refolded product to neutralize antigens, ELISA analyses were performed. 96 well ELISA plates were either coated with 100 ng/well PT-gliadin or coated with 1% w/v PEG 6000 as negative control. We described the coating protocol as well as the ELISA in detail in our previous study [11 (link)]. To reduce unspecific interactions, samples containing refolded tscFv or tscFv IBs were diluted with Tris-buffered saline (24.8 mM Tris, 136.9 mM NaCl and 2.7 mM KCl, pH 8.0) containing 0.05% Tween 20 (TBST). 100 μL sample/well were incubated for an hour at 25 °C and 450 rpm. Every well was washed three times with 300 μL TBST. Subsequently, 100 μL of a 1:1000 dilution of Anti-Chicken IgG (H + L), F(ab′)2 fragment-Peroxidase antibody produced in rabbit (Sigma, Vienna, Austria) with TBST were added per well and incubated at 37 °C and 450 rpm for an hour (THERMOstar microplate incubator, BMG Labtech, Ortenberg, Germany). Each well was washed four times with 300 μL TBST. A color reaction was mediated by the addition of 100 μL premixed 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (Thermo Scientific, Vienna, Austria), which reacted with the peroxidase. After 15 min, 50 μL of 0.9 M HCl were added as stop reagent. Absorbance was measured at 450 nm in a Multiskan FC Microplate Photometer (Thermo Scientific, Vienna, Austria).
3
ELISA Assay for Phosphorylated Peptides
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96-well ELISA plates (Corning Life Sciences, Corning, NY) were coated with 100 ng in 100 μL PBS per well of each peptide (see Table 1 ). All wells were washed with PBS four times and blocked with PBS with 5% FBS. Primary antibodies were added to blocking solution and incubated for 1 h. After PBS washes, plates were incubated with horseradish peroxidase-conjugated goat anti-mouse antibody (Vector Labs Inc., Burlingame, CA) in blocking solution for an hour. Plates were washed with PBS and 3,3′,5,5′-tetramethylbenzidine (TMB substrate, Thermo Fisher Scientific, Waltham, MA) was added to each well. The reactions were stopped with 0.2 M HCl and the optical density was measured at 450 nm with a plate reader. All ELISA experiments were performed in quadruplicates.
Table of synthetic peptides used for ELISAs
| Peptide Name | Peptide Sequence |
|---|---|
| pSer199, pSer202, pSer205 | 193DRSGYS- |
| pSer199 | 193DRSGYS- |
| pSer202 | 193DRSGYSSPG- |
| pThr205 | 193DRSGYSSPGSPG- |
| pSer208 | Cys-202SPGTPG- |
| pThr205/pSer208 | Cys-202SPG- |
| Ser208 | Cys-202SPGTPGSRSRTP213 |
4
Anti-RBD Antibody ELISA for Viral Detection
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Anti-RBD antibody (Fakhra Co., Iran) diluted at 1:1000 (0.005 g/L in PBS) was added into each well (96 well plate, Thermo Fisher Scientific Inc, USA), followed by an incubation of 24 h/4 °C and blocking with 3% skim milk in PBS containing 20% Tween (0.2%) (PBST) for 1 h. After washing with PBST, 100 μl of the inactivated virus cocktail was added to each well at 37 °C/1 h, followed by washing with PBST. Then, 100 μl of biotinylated single-stranded aptamer 70 pM was added to each well with 2 h/RT/slight shaking. In the next step, each well-received streptavidin conjugated horse radish peroxidase (HRP) diluted at 1:1000 1 h/RT/slight shaking (Thermo Fisher Scientific, Germany). The 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate (Thermo Fisher Scientific, Germany) was added to each well, and the reaction was stopped by sulfuric acid (4 N). The optical density of the wells was measured at 450 nm.
5
SARS-CoV-2 S Protein Quantification
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Whole ectodomain of SARS-CoV-2 S protein with His Tag (Sino Biological Inc., Beijing, China; catalogue number: 40589-B08V1) was diluted with coating buffer (0.1 M NaHCO3, 34 mM Na2CO3) and a total of 20 ng of protein was loaded into individual wells of a 96 well plate (Nunc, Roskilde, Denmark) and allowed to coat overnight at 4 °C. Plates were then washed four times with 0.05% Tween 20 in PBS (PBST) and blocked with 5% bovine serum albumin (BSA)/PBST for 30 min before murine antibodies serially diluted with blocking buffer were added to desired wells for 1 hour. Plate were washed four times with PBST before incubation for 1 hour with HRP-conjugated goat anti-mouse IgG (Thermo Fisher Scientific) secondary antibodies diluted in blocking buffer, and washed four times with PBST. Visualisation of bound secondary antibodies was done by the addition of 3,3',5,5'-tetramethylbenzidine (TMB) substrate (Thermo Fisher Scientific) for 5 min in the absence of light and the reaction was stopped with 2 M sulphuric acid. Optical density at 450 nm (OD450nm) was determined by a Tecan (Männedorf, Switzerland) Infinite M1000 reader and normalised OD450nm was obtained by subtracting background absorbances determined in BSA coated wells.
6
Quantifying Salivary IgA Titers
7
SARS-CoV-2 S1 Domain IgG ELISA
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ELISA plates were coated with 50 μL (0.1 μg per well) of SARS-CoV-2 spike protein S1 domain (Sino Biological, Wayne, PA, USA) in PBS and placed on a microplate overnight at 4 °C, followed by washing with PBS with Tween 20 (PBS-T) and blocking in 5% non-fat dry milk (LabScientific, Danvers, MA, USA). The ELISA plates were washed again with PBS-T, and diluted serum samples were serially diluted 2-fold (1:100–1:12,800). After washing, S1 domain-specific IgG antibodies were detected using horseradish peroxidase (HRP) conjugated anti-hamster antibodies (Jackson ImmunoResearch, Laboratories, Inc, West Grove, Pennsylvania, USA) and 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate (Thermo Fisher Scientific) (Malherbe et al., 2021 (link)). Reciprocal endpoint titers were determined in Prism software, version 9 (GraphPad, La Jolla, CA, USA), using a sigmoidal 4-parameter logistic fit curve. Endpoint titers were calculated at the point where the curve crossed the ELISA cutoff value. Data are presented as the mean and standard deviation of two independent ELISA runs.
8
TiO2NPs Impact on Angiogenesis Proteome
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The angiogenesis proteome profiler (R & D Systems) contained 4 membranes, each spotted in duplicate with 55 different angiogenesis antibodies. The assay was performed per the manufacturer’s instructions. This was a qualitative assay that indicates the relative expression of the angiogenesis markers. The concentrations selected were 0 and 100 µg/mL TiO2NPs. The concentration selected represented the control and the no observed adverse effect level (NOAEL) (100 µg/mL TiO2NPs). The membranes were subjected to an ultra-sensitive chromogenic 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate (Thermo Fisher, Waltham, MA, USA) to show sample–antibody complexes labelled with streptavidin-horseradish peroxidase (HRP). Pictures were taken of the membranes after substrate exposure.
9
Recombinant EGFR Ligand Binding Assay
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E. coli were transformed with the respective plasmid encoding EGF, TGF-α and EPRG, respectively, were cultivated in LB medium for two hours in the presence of IPTG (1 mM). Bacteria pellets were resuspended in 1 ml PBS and treated with HRV 3C protease (10 U) overnight at 4 °C in order to release the POI from the FimH part of the fusion protein. Released POIs were concentrated by using of an ultra-0.5 centrifugal Amicon device (Merck KGaA, Darmstadt, Germany). The detection of the proteins of interest was performed by using ELISA kits specific for human EGF, TGF-α and EPRG, respectively (CUSABIO BIOTECH, Houston, TX, USA).
E. coli produced human EGF, TGF-α and EPRG, were tested for binding to EGFR. Therefore, microtiter wells were coated with released EGFR ligands (1.0 µg/ml) in PBS and incubated with human EGFR-hIgG and as control with mouse EGFR-hIgG (each 1.0 µg/ml, R&D Systems, Minneapolis, MN, USA), respectively. A human IgG antibody of irrelevant specificity served as control. Bound EGFR-IgG fusion protein was detected by an IgG-specific biotin-labeled antibody (BioLegend) and streptavidin-HRP (Sigma-Aldrich). Specific binding was visualized by using of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (Thermo Fisher Scientific) and analyzed by using of the Multiscan GO ELISA reader (Thermo Scientific); all measurements were performed in triplicates.
E. coli produced human EGF, TGF-α and EPRG, were tested for binding to EGFR. Therefore, microtiter wells were coated with released EGFR ligands (1.0 µg/ml) in PBS and incubated with human EGFR-hIgG and as control with mouse EGFR-hIgG (each 1.0 µg/ml, R&D Systems, Minneapolis, MN, USA), respectively. A human IgG antibody of irrelevant specificity served as control. Bound EGFR-IgG fusion protein was detected by an IgG-specific biotin-labeled antibody (BioLegend) and streptavidin-HRP (Sigma-Aldrich). Specific binding was visualized by using of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (Thermo Fisher Scientific) and analyzed by using of the Multiscan GO ELISA reader (Thermo Scientific); all measurements were performed in triplicates.
10
Indirect ELISA for Plasmodium falciparum
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Indirect ELISA with parasites was performed with P. falciparum 3D7 in vitro cultures at 106 parasites/ml, using 96-well microtiter plates (SungwooLS, Uijeongbu-si, Gyeonggi-do, Korea), followed by overnight incubation at 4°C. The plate was washed by addition of 200 μl PBST per well with gentle shaking for 2 min (3 times) and then blocked with blocking buffer (4% BSA dissolved in PBST). 2CF5 and 1G10 mAb (5 μg per 100 μl) were added to each well as the primary antibody. After incubation for 1.5 hr at 37°C, the plate was washed 3 times and incubated with anti-mouse HRP (Abcam, Bristol, UK) (1: 5,000) for 1 hr. Color was developed using 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate (Thermo Fisher Scientific Inc.) and stopped with 0.18 M H2SO4. The absorbance was then read at 450 nm using an Infinite F200 microplate reader (TECAN, Männedorf, Switzerland).
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