ACE-1 protein level was measured in mouse brain tissue homogenates using a commercially available mouse ACE-1 ELISA duoset following manufacturer’s guidelines (R&D Systems, Abington, Oxford, UK). In brief, a NUNC maxisorp 96-well plate (R&D Systems, Abington, Oxford, UK) was coated with a capture antibody (100μl) that was diluted in PBS (1600 ng/ml) and incubated overnight at room temperature. After washing (x5) and blocking for 1 h at room temperature with 1 % BSA:PBS, brain homogenates diluted in 1 % BSA:PBS (ACE-1: 1/80), a serial dilution of ACE-1 (125–8000 pg/ml), and 1 % BSA:PBS blanks were added to respective wells for 2 h at room temperature. After further washing (x5), the detection antibody diluted in 1 % BSA:PBS (400 ng/ml) was incubated for 2 h at room temperature. The plate was washed (x5) and streptavidin horse-radish peroxidase (1/200 in PBS:0.01 % Tween-20) was added for 20 min at room temperature in the dark. After a final wash step, TMB substrate (R&D Systems, Abington, Oxford, UK) was added to each well for 30 min at room temperature and 2 N sulphuric acid was added to stop the reaction. Absorbance was read at 450 nm for each well using a microplate reader (FLUOstar OPTIMA; BMG Labtech, Aylesbury, UK). ACE-1 concentration was interpolated from the serial dilution of recombinant mouse ACE-1. The average value for each sample was obtained from duplicates.
Tmb substrate
Manufactured by R&D Systems
Sourced in United Kingdom, United States
TMB substrate is a chromogenic substrate used in various immunoassays for the detection and quantification of target analytes. It undergoes an enzymatic reaction, resulting in a colored product that can be measured spectrophotometrically. The core function of TMB substrate is to provide a colorimetric readout in immunoassays.
Automatically generated - may contain errors
Lab products found in correlation
Fluostar optima, by BMG Labtech (7 mentions)
Streptavidin hrp, by R&D Systems (6 mentions)
Nunc maxisorp 96 well plate, by R&D Systems (5 mentions)
Streptavidin horseradish peroxidase, by R&D Systems (3 mentions)
Tween 20, by Merck Group (3 mentions)
Nonfat dry milk, by Southern Biotech (2 mentions)
Medisorp plates, by Merck Group (2 mentions)
Infinite m1000, by Tecan (2 mentions)
Non fat dry milk, by Bio-Rad (2 mentions)
Synergy htx plate spectrophotometer, by Agilent Technologies (2 mentions)
Nunc maxisorp 96 well plate, by Thermo Fisher Scientific (2 mentions)
Ang 2, by Abcam (1 mentions)
Ang 2, by Cloud-Clone (1 mentions)
Protease, by Merck Group (1 mentions)
Sodium phosphate dibasic, by Merck Group (1 mentions)
Sodium phosphate monobasic, by Merck Group (1 mentions)
Sodium formate, by Merck Group (1 mentions)
Zinc nitrate, by Merck Group (1 mentions)
Hrp conjugated goat anti mouse igg1 igg2a, by Southern Biotech (1 mentions)
Skim milk, by BD (1 mentions)
20 protocols using tmb substrate
1
Quantification of Mouse Brain ACE-1 Levels
2
Quantification of Brain ACE-2 Levels
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ACE-2 protein level was measured in brain tissue homogenates using a commercially available mouse ACE-2 ELISA duoset (R&D Systems, Abington, Oxford, UK) following manufacturer’s guidelines with minor modifications. Capture antibody was diluted at 4 µg/ml in PBS (2-fold higher than recommended) in a NUNC maxisorp 96-well plate (R&D Systems, Abington, Oxford, UK) and coated overnight at room temperature. After a wash and block step, brain tissue homogenates diluted 1 in 10 in PBS:1% BSA, a serial dilution of recombinant ACE-2, or a blank, was added to the wells in duplicate for 2 h at room temperature. Following a wash step, detection antibody at 200 ng/ml (2-fold higher than recommended) was added to the plate for 2 h at room temperature. Following a further wash step, streptavidin horse-radish peroxidase at 1 in 20 (2-fold higher than recommended) was incubated for 20 min. TMB substrate (R&D Systems, Abington, Oxford, UK) was added to each well for 30 min at room temperature and 2 N sulphuric acid was added to stop the reaction. Absorbance was read at 450 nm for each well using a microplate reader (FLUOstar OPTIMA; BMG Labtech, Aylesbury, UK). The concentration of ACE-2 was interpolated from the standard curve. Each sample was measured in duplicate and the average calculated.
3
Quantifying Brain Angiotensin-II by ELISA
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Ang-II concentration was measured by direct ELISA using an in-house assay as previously described (14 (link),24 (link)). Brain homogenates were diluted in PBS (1:20) and incubated alongside a serial dilution of Ang-II (Abcam, Cambridge, UK; 78–5 000 ng/mL) in a NUNC maxisorp 96-well plate (R&D Systems) for 2 hours with shaking at room temperature. The plate was washed 5 times in 0.5% Tween-20:PBS, blocked with 1% BSA:PBS for 1 hour at room temperature, washed again as above and a biotinylated antibody specific for Ang-II (1 µg/mL; Cloud-Clone, Wuhan, China) was incubated for 2 hours. After a further wash step, streptavidin horse-radish peroxidase (1:200, R&D Systems) was added for 20 minutes at room temperature, the plate was further washed, and TMB substrate (R&D Systems) was added for 30 minutes at room temperature. 2N sulphuric acid was added to stop the color change. Absorbance was read using a microplate reader (FLUOstar OPTIMA; BMG Labtech) at 450 nm for each well. Ang-II concentration was interpolated from the standard curve, and each sample was measured in duplicate and the average calculated.
4
SARS-CoV-2 Serological Assay Development
5
Ang-II Measurement by Direct ELISA
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Angiotensin II (Ang-II) concentration was measured by direct ELISA using an in-house assay as previously described [36] (link), [37] (link). In brief, brain homogenates diluted in PBS (1:20), a serial dilution of recombinant angiotensin II (Abcam, Cambridge, UK) (78–5000 ng/ml), and PBS alone as a blank, were all incubated in a NUNC maxisorp 96-well plate (R&D Systems, Abington, Oxford, UK) for 2 h with shaking at room temperature. The plate was washed with 0.5 % Tween-20:PBS (x5) and blocked with 1 % BSA:PBS for 1 h at room temperature. The plate was then incubated with an Ang-II biotinylated detection antibody (1 µg/ml) (Cloud-Clone, Wuhan, China) for 2 h. After a further wash step, streptavidin horse-radish peroxidase (1:200, R&D Systems, Abington, Oxford, UK) was added for 20 min at room temperature, the plate was further washed, and TMB substrate (R&D Systems, Abington, Oxford, UK) was added for 30 min at room temperature in the dark. 2 N sulphuric acid was added to stop the colour change and absorbance was read using a microplate reader (FLUOstar OPTIMA; BMG Labtech, Aylesbury, UK) at 450 nm for each well. Concentrations of Ang-II were interpolated from the standard curve. Each sample was measured in duplicate and the average calculated.
6
ELISA for Mouse Antibody Responses
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Flat bottom 384‐well MediSorp plates (Sigma‐Aldrich) were coated with 0.4 μg/mL (20 µL/well) of HEK293T‐produced E1E2 antigen in PBS and incubated overnight at 4 °C. After extensive washing with PBS containing 0.05% Tween 20 (Sigma‐Aldrich), plates were blocked for 1 h, at RT with PBS containing 5% nonfat dry milk (BioRad) and then washed as before. Six binary dilutions, starting from 1:200, of serum in PBS containing 5% nonfat dry milk were added to the antigen‐coated plates (40 μL/well) and incubated for 2 h, at RT. The plates were washed, incubated with HRP‐conjugated goat anti‐mouse IgM/IgG (Southern Biotech, AL), diluted 6000‐fold or with HRP‐conjugated goat anti‐mouse IgG1/IgG2a (Southern Biotech) diluted 1000‐fold in PBS containing 5% nonfat dry milk, for 2 h, at RT. Enzymatic reactions were developed following the addition of the TMB substrate (R&D Systems), for 20 min at RT then stopped with 20 μL/well of 1 m H2SO4. Absorbance was measured at 450 nm using a Tecan Infinite M1000 microplate reader. Normalization across ELISA plates was performed by pooling all the immune sera from day 49 which served as an internal standard. Serum pool dilution starting from 1:100 was used as an internal standard on each plate and a 4‐parameter logistic regression model was fitted to the measured values.
7
SARS-CoV-2 RBD Binding Assay
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ELISA was performed as described previously to examine the binding of mAb CR3022 with RBD26 (link) with some modifications. Briefly, 50 μl (2 μg/ml) of the plant-purified SARS-CoV-2 RBD or commercial recombinant CHO-derived SARS-CoV-2 spike RBD-His protein (10534-CV, R & D Systems, USA) was coated on 96-well microplates (Greiner Bio-One GmbH, Germany) and incubated at 4 °C overnight. After washing, the plates were blocked with 5% skim milk (BD, Franklin Lakes, NJ) in 1X PBS for 2 h at 37 °C. Then, the plant-produced mAb CR3022 was added in triplicate twofold serial dilutions to the plate. After 2 h incubation at 37 °C, sheep anti-human kappa light chain conjugated with HRP (The Binding Site, UK) at a dilution of 1:1000 in 1X PBS was added and samples were incubated for 1 h at 37 °C. The plate was then washed three times with PBST, and the signal developed with TMB substrate (R&D system, USA) and the absorbance read at 450 nm. Positive convalescent serum collected from a COVID-19 patient was used as positive control. The commercially available human IgG1 (ab206198; Abcam, UK), a plant produced anti-PD1 antibody28 (link) and negative serum were used as negative controls.
8
Quantifying Complement Activation by PolySia-NP
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Wells were blocked (1% bovine serum albumen (BSA)), followed by 3 h incubation with zymogen (potent activator of the alternative complement pathway) in defibrinated plasma (serum), plus PolySia-NP or vehicle (10% sucrose) control. Complement deposition was visualized after 1 h incubation with human C3b antibody (Complement Tech-A114), then 20 min streptavidin-HRP (R&D Systems) incubation, and 20 min TMB substrate (R&D Systems) reaction. Results were read using a BioTek Synergy HTX plate spectrophotometer (Agilent) at 490 nm absorbance.
9
Quantification of Angiotensin (1-7) in Human Brain
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Ang (1–7) levels were measured in human brain tissue homogenates in 1% SDS lysis buffer (see above) using an in-house direct ELISA kit. Recombinant human Ang (1–7) (Enzo Life Sciences) or human brain tissue homogenates (diluted 1:40 In PBS) were incubated for 2 h in a clear, high binding capacity Nunc MaxiSorp plate (Thermo Fisher Scientific, Waltham, MA, USA) at 26 °C with shaking. The wells were washed five times in PBS with 0.05% Tween-20 and blocked for 1 h in PBS:1% bovine serum albumin (Sigma-Aldrich). After another five washes, the wells were incubated with biotinylated anti-human Ang 1–7 (2 μg/ml in PBS) (Cloud-Clone, Wuhan, China) for 2 h at 26 °C with shaking, followed by a further wash step. Streptavidin/HRP (1:200) in PBS/0.01% Tween-20 was added to each well, which was incubated at room temperature for 20 minutes in the dark. TMB substrate (R&D Systems) was added after a further wash and left to develop in the dark for 20 minutes. Absorbance at 450 nm was read following the addition of 2 N sulphuric acid (‘stop’ solution) using a FLUOstar OPTIMA plate reader. Ang (1–7) concentration was interpolated from a standard curve generated by serially diluting recombinant human Ang (1–7) (5000–78.125 pg/ml). The assay showed minimal cross-reactivity with a number of closely related peptides, including Ang I, Ang II and Ang III.
10
ELISA Assay for p53-R175H Antibodies
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ELISA plates were coated with p53-R175H antigen, which was dissolved in coated buffer (R&D Systems, Minneapolis, MN, USA) overnight at 4 °C. The plates were washed three times with PBST (pH 7.4) containing 0.05% (v/v) Tween-20 and then blocked with 3% BSA in PBS for 1 h. Serum was then added and incubated at room temperature for 2 h. The binding was detected using a horseradish peroxidase-conjugated second antibody (Cell Signaling Technology). The reaction was developed using a TMB substrate (R&D Systems) and then stopped with 2 N H2SO4. The absorbance at 450–650 nm was measured using a plate reader (CLARIOstar, BMG Labtech, USA).
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