To detect autoantibodies against autoantigens, 96-well plates coated with Sm, or RNP (The Binding Site) were incubated with serially diluted sera at 37°C for 2h. The plates were then washed and incubated with HRP-conjugated goat anti-mouse IgG (Southern Biotechnology, Birmingham, AL) at 37°C for 1h, followed by development with TMB peroxidase EIA substrate (Bio-Rad, Hercules, CA). The reaction was stopped with 1N H2SO4 and the optical density at 450 nm was measured using an ELISA reader. A mixture of sera from MRL-lpr mice was used to establish standard curves in each plate and antibody levels were shown as relative titers.
Tmb peroxidase eia substrate
Manufactured by Bio-Rad
Sourced in United States
TMB Peroxidase EIA Substrate is a chromogenic substrate used in enzyme immunoassays (EIAs) and enzyme-linked immunosorbent assays (ELISAs) to detect the presence of an enzyme-conjugated analyte. The substrate undergoes an enzymatic reaction that results in a colored product, which can be measured using a spectrophotometer.
Automatically generated - may contain errors
Lab products found in correlation
Duoset elisa kit, by R&D Systems (3 mentions)
Hrp conjugated goat anti mouse igg, by Southern Biotech (1 mentions)
Goat anti mouse ige, by Southern Biotech (1 mentions)
Epoch microplate spectrophotometer, by Agilent Technologies (1 mentions)
Goat anti mouse igg, by Santa Cruz Biotechnology (1 mentions)
Horseradish peroxidase conjugated streptavidin, by BD (1 mentions)
Ez link sulfo nhs lc biotinylation kit, by Thermo Fisher Scientific (1 mentions)
Tween 20, by Merck Group (1 mentions)
H2so4, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Imark microplate reader, by Bio-Rad (1 mentions)
Goat anti mouse secondary antibody, by Bio-Rad (1 mentions)
Rabbit polyclonal antibody against hg csf, by Abcam (1 mentions)
Recombinant hg csf, by Abcam (1 mentions)
Rabbit anti β glucuronidase, by Merck Group (1 mentions)
9 protocols using tmb peroxidase eia substrate
1
Autoantibody Detection ELISA Protocol
2
DNCB-induced Ig Levels Quantification
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DNCB-induced serum IgE and IgG levels were assessed using an enzyme-linked immunosorbent assay (ELISA) kit. Briefly, blood samples of mice were obtained from facial veins on the last day of the experiment (day 17) (n = 3 per group). Collected samples were incubated at room temperature for 30 min and centrifuged at 3500 rpm for 25 min, and supernatants were stored at − 80 °C until use. The 96-well plates were pre-coated with 0.17 mg/ml DNP-ovalbumin (Santa Cruz, Dallas, TX, USA), and then blocked with 2% BSA for 1 h at 37 °C, followed by washing with PBS-T. Diluted serum samples and standards were added to each well and incubated for 2 h at room temperature. After washing with PBS-T, goat anti-mouse IgG (Santa Cruz, Dallas, TX, USA) and goat anti-mouse IgE (Southern Biotech, Birmingham, AL, USA) in 1% PBS were added at ratios of 1:4000 and 1:2000, respectively. Following several wash steps, TMB Peroxidase EIA Substrate (Bio-Rad, Hercules, CA, USA) was added to each well for colorimetric development, and the reaction was subsequently terminated with 1 N H2SO4. Absorbance was detected at 450 nm using a microplate reader (Epoch Microplate Spectrophotometer, BioTek, Winooski, VT, USA).
3
Quantification of Inflammatory Cytokines
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The release of IL-1β, IL-8, and TNF-α into the supernatant of THP-1 monocultures (t=24 h) or the basolateral supernatant of triple cultures (t=48 h) was analyzed using R&D systems DuoSet ELISA kits for these proinflammatory cytokines. Antibodies were diluted according to the manufacturer’s protocol and high-protein-binding 96-well plates were incubated with capture antibody in coating buffer (0.1 M NaHCO3, pH 8.2) overnight. After blocking with 3% BSA/PBS, 100 µl of supernatants, diluted if necessary (IL-1β: 1:5 and IL-8: 1:10), were incubated for 2 h. Detection antibody, horseradish peroxidase (1:40 in 1% BSA/PBS), and BioRad TMB Peroxidase EIA Substrate was consecutively incubated for 2 h, 0.5 h, and 5-20 min, respectively, before stopping the reaction with 50 µl 1 M H2SO4. Absorbance was measured at 450 nm and 540 nm. The standard curve was plotted as a four-parameter log fit.
4
Sandwich ELISA for Detecting IL-2 Interactions
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Flat Nunc-Immuno 96-well plates (Sigma-Aldrich) were coated overnight at 4 °C with capture antibodies in PBS: purified anti-hIL-2 mAb (clone 5344.111, BD Biosciences) or purified hCD25 (223-2 A/CF, R&D Systems). The plates were then washed with 0.1% Tween 20 (Sigma-Aldrich) and blocked for 1 hour at room temperature (RT) with 1% BSA (Sigma-Aldrich) in 0.1% Tween 20 at 450 rpm. After washing, titrated doses of hIL-2, hIL-2/NARA1 (hIL-2 and NARA1 were pre-mixed and incubated for 20 min) or NARA1leukin were added to plates in blocking solution and incubated for 1 to 2 h at RT and 450 rpm, followed by washing. For detection, purified biotinylated anti-hIL-2 mAb (clones MAB202, R&D System) in blocking solution was incubated for 1 h at RT and 450 rpm. Biotinylation was done using the EZ-link Sulfo-NHS.LC biotinylation kit (Thermo Scientific). Plates were then washed and incubated with streptavidin-conjugated horseradish peroxidase (BD Biosciences) for 45 minutes at RT in the dark. Following a final wash, plates were developed using TMB Peroxidase EIA substrate for 5–10 minutes (BioRad) until the reaction was stopped by the addition of 1.8 M H2SO4 (Sigma-Aldrich). The plates were read at 450 nm absorbance using an iMark microplate reader (BioRad).
5
Quantitative ELISA for Viral NS1 Protein
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MDCK or A549 monolayers (overnight confluent, 48-well plate format, triplicates) were infected with assembled viruses at moi = 10. After virus adsorption at 37 °C, the inoculum was removed and replaced with serum free medium containing antibiotic-antimycotic and TPCK-trypsin. Infected cells were incubated at 37 °C for the indicated time periods and then fixed with 80% acetone in DPBS (ice cold at application) for 15 min at room temperature. Fixed cells were washed three times with phosphate buffered saline with 0.1% tween-20 (PBST) and blocked with 5% milk in PBST for 6 h at 4 °C. After 2 washes, plates were incubated at 4 °C overnight with mouse polyclonal serum raised against recombinant NS1(1–124) protein. Plates were then washed three times with PBST, and goat anti-mouse secondary antibody (Bio-Rad) was added (1 μg/ml final), followed by incubation at 37 °C for 1 h. Immunoreactivities were analyzed by adding TMB Peroxidase EIA Substrate (Bio-Rad), and reactions were stopped with 2 N H2SO4. Optical density was measured at 450 nm with removal of noise (measured at 655 nm) using a CLARIOstar multi-function reader (BMG Labtech).
6
Cytokine Quantification via ELISA
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The concentrations of IL-1β, IL-6, IL-8, TNFα, and IL-18 in apical and basolateral supernatants were analyzed using R&D systems DuoSet ELISA kits as described previously [103 (link)]. Briefly, high-protein-binding 96-well plates were coated with primary antibody, blocked with BSA, and incubated with 100 µL sample. Consecutively, detection antibody, horseradish peroxidase, and BioRad TMB Peroxidase EIA Substrate were incubated. The color reaction was stopped with H2SO4 and absorbance was measured at 450 and 540 nm. The standard curve was plotted using a four-parameter log fit. In case the measured concentrations were below the limit of detection (LOD), half the LODs, i.e. 1.95 pg/mL, 4.69 pg/mL, 15.63 pg/mL, 7.81 pg/mL, and 5.86 pg/mL for IL-1β, IL-6, IL-8, TNFα, and IL-18, respectively, were used for calculations and statistics. Relatively large standard deviations can be explained by variability of the constitutive cytokine releases between the independent experiments. This did not affect the statistical analysis since the experimental run was included as a random factor in the mixed-effects models (see Statistics).
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The concentrations of IL-1β, IL-6, IL-8, TNFα, and IL-18 in apical and basolateral supernatants were analyzed using R&D systems DuoSet ELISA kits as described previously [103 (link)]. Briefly, high-protein-binding 96-well plates were coated with primary antibody, blocked with BSA, and incubated with 100 µL sample. Consecutively, detection antibody, horseradish peroxidase, and BioRad TMB Peroxidase EIA Substrate were incubated. The color reaction was stopped with H2SO4 and absorbance was measured at 450 and 540 nm. The standard curve was plotted using a four-parameter log fit. In case the measured concentrations were below the limit of detection (LOD), half the LODs, i.e. 1.95 pg/mL, 4.69 pg/mL, 15.63 pg/mL, 7.81 pg/mL, and 5.86 pg/mL for IL-1β, IL-6, IL-8, TNFα, and IL-18, respectively, were used for calculations and statistics. Relatively large standard deviations can be explained by variability of the constitutive cytokine releases between the independent experiments. This did not affect the statistical analysis since the experimental run was included as a random factor in the mixed-effects models (see Statistics).
7
ELISA Quantification of Cytokine Secretion
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The release of IL-1β and IL-8 into the supernatant following treatment was analyzed using R&D systems DuoSet ELISA kits. Antibodies were diluted according to the manufacturer’s protocol and high-protein-binding 96-well plates were incubated with capture antibody in coating buffer (0.1 M NaHCO3, pH 8.2) overnight. After blocking with 3% BSA/PBS, 100 µl of supernatants, diluted if necessary (IL-1β: 5 to 20-fold and IL-8: 10 to 20-fold), were incubated for 2 h. Detection antibody, horseradish peroxidase (1:40 in 1% BSA/PBS), and BioRad TMB Peroxidase EIA Substrate was consecutively incubated for 2 h, 0.5 h, and 5–20 min, respectively, before stopping the reaction with 50 µL 1 M H2SO4. Absorbance was measured at 450 and 540 nm. The standard curve was plotted as a four-parameter log fit. After 24 h incubation, cytokine concentrations were quantified via ELISA and compared to the particle-free control. Interference of all materials with the ELISA was assessed by spiking cell-free particle samples (50 µg/cm2) with 500 pg/mL of each cytokine. Interference of materials with the assay was not included in the calculation of the results but was considered when interpreting the data.
8
Quantification of Recombinant hG-CSF Protein
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The total protein was extracted as described above. The protein samples were serially diluted in PBS and loaded into 96-well ELISA plate (0.25, 0.5, 1.0, and 2.0 μg of TSP per well), using recombinant hG-CSF (Abcam, UK) as the reference standard.
The plates were incubated for 2 h at RT, then washed three times with PBST (PBS with 0.1% Tween 20) for 5 min each and blocked in PBST containing 2% bovine serum albumin (1 h at RT). Hybridization with the primary antibody was performed overnight at 4°C. After washing, the secondary antibody was added and plates were incubated for 1 h at RT followed by the washing. Rabbit polyclonal antibody against hG-CSF (Abcam, UK) was diluted at 1:1,000, anti-rabbit IgG conjugated with alkaline phosphatase (Pierce, USA)—at 1:2,000. The plates were developed for 30 min at RT using TMB Peroxidase EIA substrate (BioRad). The plates were read at 405 nm and the amount of plant—expressed hG-CSF was estimated based on reference standards.
The plates were incubated for 2 h at RT, then washed three times with PBST (PBS with 0.1% Tween 20) for 5 min each and blocked in PBST containing 2% bovine serum albumin (1 h at RT). Hybridization with the primary antibody was performed overnight at 4°C. After washing, the secondary antibody was added and plates were incubated for 1 h at RT followed by the washing. Rabbit polyclonal antibody against hG-CSF (Abcam, UK) was diluted at 1:1,000, anti-rabbit IgG conjugated with alkaline phosphatase (Pierce, USA)—at 1:2,000. The plates were developed for 30 min at RT using TMB Peroxidase EIA substrate (BioRad). The plates were read at 405 nm and the amount of plant—expressed hG-CSF was estimated based on reference standards.
9
Quantification of Recombinant Enzyme Expression
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Total soluble protein (TSP) was extracted as described above in extraction buffer without SDS. The protein samples were serially diluted in phosphate buffer saline (PBS) (0.25, 0.5, 1.0, and 2.0 lg TSP per well) and placed into a 96-well microtiter plate, using b-glucuronidase from E. coli (Sigma) as the reference standard. The plates were incubated for 2 h at room temperature, washed three times with washing buffer for 5 min each [PBS plus 0.05 % (v/v) Tween-20] and blocked with blocking buffer (PBS containing 2 % w/v bovine serum albumin and 0.05 % Tween-20) for 1 h at 37 °C. The plates were then incubated with rabbit anti-bglucuronidase (diluted 1:1000, Sigma) polyclonal antibody overnight at 4 °C. After washing, the plates were incubated with anti-rabbit IgG conjugated to alkaline phosphatase (1:3000, Pierce) for 1 h at 37 °C. The plates were washed and developed by the addition of 100 ll per well of TMB Peroxidase EIA substrate (BioRad) for 30 min at room temperature. The plate was read at 405 nm and the amount of plant-expressed M130-b-glucuronidase was estimated based on reference b-glucuronidase standards.
To determine the expression level of M130-b-glucuronidase in the transgenic lines, three samples of duckweed per line were analyzed, and their average expression level calculated. All measurements were performed in duplicate.
To determine the expression level of M130-b-glucuronidase in the transgenic lines, three samples of duckweed per line were analyzed, and their average expression level calculated. All measurements were performed in duplicate.
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