Purified CH2 domain proteins in the concentration of 2 mg/ml were coated on 96-well ELISA plates overnight at 4 ° C. Serially concentrations of biotinylated proteins with triplicate samples were added into wells and incubated for 1 h. Bound proteins were detected with HRP conjugated streptavidin (1:1000; Sigma). The o- Phenylenediamine substrate (Sigma) was added and the reaction was read at 450 nm.
O phenylenediamine substrate
Manufactured by Merck Group
Sourced in United States, United Kingdom
O-phenylenediamine substrate is a chemical compound commonly used as a substrate in various analytical and diagnostic applications. It serves as a sensitive and versatile reagent for the detection and quantification of various analytes in laboratory settings. The core function of this product is to facilitate colorimetric or fluorometric reactions, enabling the measurement and analysis of target substances.
Automatically generated - may contain errors
Lab products found in correlation
Peroxidase conjugated anti mouse igg antibody, by Merck Group (4 mentions)
Anti mouse monoclonal icam 1 antibodies, by Merck Group (3 mentions)
Nunc immuno microwell 96 well plates, by Thermo Fisher Scientific (2 mentions)
Ez read 400 microplate reader, by Harvard Bioscience (2 mentions)
Hrp conjugated streptavidin, by Merck Group (1 mentions)
Maxisorp plate, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase hrp conjugated anti human igg, by Jackson ImmunoResearch (1 mentions)
Synergy ht reader, by Agilent Technologies (1 mentions)
Maxisorb, by Thermo Fisher Scientific (1 mentions)
Imark plate reader, by Bio-Rad (1 mentions)
Maxisorb immunoplates, by Thermo Fisher Scientific (1 mentions)
Spectra mr plate reader, by Dynex (1 mentions)
Tnf α, by R&D Systems (1 mentions)
Il 1β, by R&D Systems (1 mentions)
Mcp 1, by R&D Systems (1 mentions)
Peroxidase conjugated anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Nunc immuno microwell, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Mouse anti his tag antibody, by Bio-Rad (1 mentions)
Goat anti mouse horseradish peroxidase hrp antibody, by Jackson ImmunoResearch (1 mentions)
19 protocols using o phenylenediamine substrate
1
Protein Binding Quantification Assay
2
SARS-CoV-2 RBD Protein Characterization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The recombinant SARS-CoV-2 RBD gene was cloned, expressed, and purified, and ELISAs were performed as previously described (29 (link)). Briefly, purified RBD was coated on MaxiSorp plates (Thermo Fisher Scientific, #439454) at a concentration of 1 μg/ml in phosphate-buffered saline (PBS) at 4°C overnight. The plates were washed extensively with PBS containing 0.05% Tween 20 (PBST). Three-fold serially diluted plasma or purified mAb was added to the plates and incubated at room temperature for 1 hour. After incubation, the plates were washed, and the SARS-CoV-2 RBD-specific IgG, IgM, and IgA signals were detected by incubating with horseradish peroxidase (HRP)–conjugated anti-human IgG (Jackson ImmunoResearch Laboratories, #109-036-098), IgM (Jackson ImmunoResearch Laboratories, #109-036-129), or IgA (Jackson ImmunoResearch Laboratories, #109-036-011). Plates were then washed thoroughly and developed with o-phenylenediamine substrate (Sigma-Aldrich, #P8787) in 0.05 M phosphate-citrate buffer (Sigma-Aldrich, #P4809) pH 5.0, containing 0.012% hydrogen peroxide (Thermo Fisher Scientific, #18755). Absorbance was measured at 490 nm.
3
EPCR Expression Modulation in HUVECs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Modified whole-cell ELISA was performed as previously described for determination of expression levels of EPCR on HUVECs (28) (link). Briefly, confluent monolayers of HUVECs were treated with or without OroA for 6 h, followed by treatment with PMA, tumor necrosis factor (TNF)-α or interleukin (IL)-1β for 1 h. Media were then removed and cells were washed with PBS and fixed with 50 μl of 1% paraformaldehyde for 15 minutes at room temperature. After washing, 100 μl of EPCR antibodies (Abnova) was added, and, 1 h (37℃, 5% CO2) later, cells were washed three times, followed by treatment with 100 μl of 1：2,000 peroxidase-conjugated anti-rabbit IgG antibodies (Sigma) for 1 h. Cells were then washed three times and developed using o-phenylenediamine substrate (Sigma). Colorimetric analysis was performed by measurement of absorbance at 490 nm. All measurements were performed in triplicate wells.
4
Quantifying ICAM-1 Expression in Lung Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The expression of intercellular adhesion molecule-1 (ICAM-1) on lung tissues was determined by a direct ELISA. The lysed lung tissues were coated onto Nunc-Immuno™ MicroWell™ 96 well plates and incubated overnight at 4 °C. After washing, anti-mouse monoclonal ICAM-1 antibodies (Millipore Corporation, Billerica, MA, 1:50 each) were added. After 1 h (37 °C, 5% CO2), the cells were washed three times and then 1:2000 peroxidase-conjugated anti-mouse IgG antibody (100 μL; Sigma, Saint Louis, MO) was added for 1 h. The cells were washed again three times and developed using the o-phenylenediamine substrate (Sigma, Saint Louis, MO). Colorimetric analysis was performed by measuring absorbance at 490 nm. All measurements were performed in triplicate wells.
5
Quantifying Cell Surface CA IX Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fifty thousand cells were seeded in six parallels onto 96-well plate and incubated under normoxic or hypoxic conditions for 24 h.
Analysis of surface CA IX protein on live cells: The cells were incubated with M75 antibody [77 (link)] (10 μg/μl in DMEM with 10% FCS, 50 μl/well) at 37°C for 2 h. The cells were quickly washed twice with PBS pH 7.2 and then incubated with secondary antibody (1:5000; 50 μl/well). After the incubation the samples were again washed 3× with PBS pH 7.2. The bound antibody was visualized by 10 mg of o-phenylenediamine substrate (Sigma-Aldrich) with 10 μl H2O2 in citric buffer (pH 5) for 5–15 min in the dark. The reaction was stopped by 10 mM H2SO4 (50 μl/well) and absorbance was measured at 492 nm in Synergy HT reader (Bio Tek instruments, Winooski, VT, USA).
Analysis of total CA IX on fixed cells: The cells were washed twice with PBS pH 7.2 and then fixed with methanol for 5 min at –20°C and washed twice again. Non-specific binding was blocked with 10 % FCS in DMEM medium at 37°C for 30 min (50 μl/well). Following steps were identical with the protocol on live cells.
Analysis of surface CA IX protein on live cells: The cells were incubated with M75 antibody [77 (link)] (10 μg/μl in DMEM with 10% FCS, 50 μl/well) at 37°C for 2 h. The cells were quickly washed twice with PBS pH 7.2 and then incubated with secondary antibody (1:5000; 50 μl/well). After the incubation the samples were again washed 3× with PBS pH 7.2. The bound antibody was visualized by 10 mg of o-phenylenediamine substrate (Sigma-Aldrich) with 10 μl H2O2 in citric buffer (pH 5) for 5–15 min in the dark. The reaction was stopped by 10 mM H2SO4 (50 μl/well) and absorbance was measured at 492 nm in Synergy HT reader (Bio Tek instruments, Winooski, VT, USA).
Analysis of total CA IX on fixed cells: The cells were washed twice with PBS pH 7.2 and then fixed with methanol for 5 min at –20°C and washed twice again. Non-specific binding was blocked with 10 % FCS in DMEM medium at 37°C for 30 min (50 μl/well). Following steps were identical with the protocol on live cells.
6
Binding Affinity of Biotinylated HA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Nunc Maxisorb microtiter plates were coated with purified hLYVE-1 monomer or dimer species, prepared as described above, each at a concentration of 4 μg/ml in PBS at room temperature overnight. Plates were then blocked (with 1% w/v BSA in PBS, 0.05% Tween 20) before the addition of bHA in 2-fold dilutions from 5 μg/ml to 39.06 ng/ml. Streptavidin HRP (Pierce) was added to detect bound bHA before developing the plate with o-phenylenediamine substrate (Sigma) and quenching with 1 m H2SO4. Absorbance at 490 nm was recorded using an i-MarkTM plate reader (Bio-Rad).
7
Anti-IFN-γ Antibody ELISA Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Anti-IFN-γ antibody in sera was determined by indirect enzyme-linked immunosorbent assay (ELISA), as previously detailed [9] (link). Briefly, Maxisorb immunoplates (Nalge Nunc International, Penfield, NY, USA) were coated with 1 µg/mL of recombinant human IFN-γ in bicarbonate buffer (pH 9.6) and kept overnight at 4°C. Plates were blocked with phosphate-buffered saline (PBS) containing 1% non-fat milk powder at 37°C for 1 hour. Sera diluted 1∶1,000 were incubated for 1 hour at 37°C in duplicate. Plates were subsequently developed with anti-human IgG horseradish peroxidase conjugate (Caltag Laboratories, Invitrogen Life Technologies, Thermo Fisher Scientific, Paisley, UK) followed by o-phenylenediamine substrate (Sigma-Aldrich, St. Louis, MO, USA). The enzyme reaction was terminated with sulfuric acid (2N) and read for absorbance at 492 nm on a Spectra MR plate reader (Dynex Technologies, Chantilly, VA, USA).
8
HUVEC Surface Receptor Expression Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Expression of VCAM, ICAM, and E-selectin on HUVECs was determined by whole-cell ELISA, as described previously [28 (link), 29 (link)]. Binding monolayers of HUVECs were treated with TGFBIp (5 μg/ml) for 6 h, followed by CNS treatment for 6 h. After removing the medium and washing the cells with PBS, they were fixed with 50 μl of 1% paraformaldehyde at room temperature for 15 min. After washing, 100 μl of murine anti-human monoclonal antibodies (VCAM, ICAM and E-selectin); Temecula, CA; 1:50 each) was applied. After 1 h (37 °C, 5% CO2), cells were washed 3 times and then 100 μl of 1:2000 peroxidase-conjugated anti-mouse IgG antibody (Sigma) was administered for 1 h. The cells were washed again 3 times and developed using O-phenylenediamine substrate (Sigma). Chromaticity analysis was performed by measuring absorbance at 490 nm. All measurements were performed in 3 wells. The same experimental procedure was used to monitor the cell surface expression of αv β3 and αv β5 using specific antibodies obtained from EMD Millipore (MA).
9
Cytokine Quantification via ELISA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell culture supernatants were collected and the levels of TNF-α, IL-1β, and MCP-1 (R&D Systems, Minneapolis, MN, USA) were measured using ELISA kits according to the manufacturer's instructions. All samples were run in triplicate. After the reaction, plates were washed and 100 μL of o-phenylenediamine substrate (Sigma-Aldrich) was added to each well. Plates were incubated for 30 min at room temperature, after which 50 μL of 4 N sulfuric acid was added to each well. The plates were read at 490 nm on a microplate reader (Spectra MAX 340PC), and the data were analyzed.
10
Quantification of Lung ICAM-1 Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The expression of intercellular adhesion molecule-1 (ICAM-1) on lung tissues were determined by a direct ELISA. The lysed lung tissues were coated onto Nunc-Immuno™ MicroWell™ 96 well plates and incubated overnight at 4 °C. After washing, anti-mouse monoclonal ICAM-1 antibodies (Millipore Corporation, Billerica, MA, USA, 1:50 each) were added. After 1 h (37 °C, 5% CO2), the cells were washed three times and then 1:2000 peroxidase-conjugated anti-mouse IgG antibody (100 μL; Sigma, Saint Louis, MO, USA) was added for 1 h. The cells were washed again three times and developed using the o-phenylenediamine substrate (Sigma, Saint Louis, MO, USA). Colorimetric analysis was performed by measuring absorbance at 490 nm. All measurements were performed in triplicate wells.
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!