For SIV Gag Western blot analysis, TeloRF cell lysates were separated by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) transfer membranes (Amersham) using standard laboratory procedures. The membranes were blocked for nonspecific protein binding overnight at room temperature in 5% skim milk and 0.1% Tween20 in PBS and then probed using SIV Gag p27 monoclonal antibody [42 (link)]. An anti-mouse IgG peroxidase conjugated antibody (Sigma clone: A5906) and 3,3’-diaminobenzidine (DAB; Vector Laboratories) were used for immunodetection. Western blot analysis for β-Actin (C4) (Santa Cruz Biotechnology; sc-47778 HRP) was also performed on the TeloRF cell lysates according to the manufacturer’s instructions.
Peroxidase conjugated anti mouse igg antibody
Manufactured by Merck Group
Sourced in United States
The Peroxidase-conjugated anti-mouse IgG antibody is a laboratory reagent used in various immunoassay techniques. It consists of an anti-mouse IgG antibody that has been conjugated to the enzyme peroxidase. This antibody can be used to detect the presence of mouse immunoglobulin G (IgG) in samples, as the peroxidase enzyme can catalyze a color-producing reaction when exposed to the appropriate substrate.
Automatically generated - may contain errors
Lab products found in correlation
O phenylenediamine substrate, by Merck Group (4 mentions)
Anti mouse monoclonal icam 1 antibodies, by Merck Group (3 mentions)
Benchmark plus, by Bio-Rad (3 mentions)
Nunc immuno microwell 96 well plates, by Thermo Fisher Scientific (2 mentions)
3 3 diaminobenzidine (dab), by Vector Laboratories (1 mentions)
Pvdf transfer membranes, by Cytiva (1 mentions)
β actin c4, by Santa Cruz Biotechnology (1 mentions)
Pip strip, by Echelon Biosciences (1 mentions)
Anti gst monoclonal antibody, by Santa Cruz Biotechnology (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Ecl prime reagent, by Cytiva (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Dc protein assay kit, by Bio-Rad (1 mentions)
Nupage, by Thermo Fisher Scientific (1 mentions)
Skimmed milk, by Bio-Rad (1 mentions)
Bhk 21 cells, by American Type Culture Collection (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Skim milk, by Bio-Rad (1 mentions)
Nitrocellulose membrane, by GE Healthcare (1 mentions)
Anti 6xhis monoclonal antibody, by Merck Group (1 mentions)
17 protocols using peroxidase conjugated anti mouse igg antibody
1
SIV Gag Protein Detection Protocol
2
Exploring GST-CT1 Lipid Interactions
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The interaction of GST–CT1 with phospholipids was investigated using the PIPStrip™ (Echelon Biosciences) according to manufacturer's protocol with slight modifications. The PIPstrips were blocked for 1 h in 1% skim dry milk in Tris-buffered saline with 0.1% v/v Tween-20 (TBS-T) at room temperature before incubation with purified GST–CT1 in 5% bovine serum albumin (BSA) in TBS-T at 4°C overnight. The strips were washed three times for 10 min with TBS-T and then probed with anti-GST monoclonal antibody (Santa Cruz) at a dilution of 1:1000, followed by anti-mouse-IgG peroxidase-conjugated antibody at a dilution of 1:5000 (Sigma-Aldrich). Both antibodies were prepared with 5% BSA in TBS-T. Enhanced chemiluminescence detection was performed with SuperSignal West Pico Chemiluminescent Substrate (Pierce-Thermo Scientific).
3
Western Blot Analysis of ZEB2 in SN4741 Cells
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For western blot analysis, SN4741 cells were washed twice with PBS (pH 7.2) and lysed in RIPA buffer (150 mM NaCl, 50 mM Tris·Cl pH 7.4, 1 mM EDTA, 0.5% NP-40, 1 × protease inhibitor cocktail [Roche]). Protein concentrations were determined using DC Protein Assay Kit (Bio-Rad, Hercules, CA). Equal amounts of total protein were subjected to 10% SDS-polyacrylamide gel electrophoresis, and electro-transferred onto polyvinyl difluoride membrane. After blocking with 5% bovine serum albumin, the membranes were incubated with goat anti-ZEB2 antibody (LifeSpan BioSciences, Inc., Seattle, WA) followed by a secondary antibody (anti-goat IgG, peroxidase conjugated, from Sigma-Aldrich, Saint Louis, MO). Bands were visualized by luminescence using the ECL prime reagent (Amersham, Aylesbury, UK) according to manufacturer’s instructions and the Chemi-Doc XRS system (Bio-Rad). After detection of ZEB2, membranes were stripped in 62.5 mM Tris/HCl (pH 6.8), 2% SDS, and 100 mM β-mercaptoethanol, washed, re-blotted with a mouse anti-GAPDH antibody, followed by an anti-mouse IgG peroxidase-conjugated antibody (Sigma-Aldrich), and detected by luminescence.
4
T. cruzi Antibody Detection Protocol
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T. cruzi -specific antibodies were used as reported by Caldas et al. (32 (link)). Briefly, enzyme-linked immunosorbent assay plates were coated with T. cruzi antigen alkaline extracted from strain Y during the exponential growth phase in liver infusion tryptose (LIT) medium. Anti-mouse IgG peroxidase-conjugated antibody (Sigma Chemical Co.) was used as the secondary antibody. The mean absorbance for 10 negative-control samples was used to determine the reactivity index value, which was obtained by dividing the absorption value (optical density [OD] value) of each serum sample by the mean value of the differential control sample.
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5
Serodetection of T. cruzi Infection
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Blood samples from treated and infected mice were collected from the orbital venous sinus (500 μL) at 15 months after infection. T. cruzi-specific antibodies were detected using the technique described by Voller [33 (link)]. Enzyme-linked immunosorbent assay (ELISA) plates were coated with T. cruzi antigen prepared from the alkaline extraction of the Y strain at exponential growth in LIT medium. Anti-mouse IgG peroxidase-conjugated antibody (Sigma Chemical Co.) was used. The mean absorbance for 10 negative control samples plus two standard deviations was used as the cut-off to discriminate positive and negative results.
6
Quantifying ICAM-1 Expression in Lung Tissues
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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.
7
Measuring ZIKV E Antibody Binding Affinity
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To measure equilibrium binding affinity, anti-ZIKV E IgG antibodies were titrated through ELISA. High binding 96-well polystyrene plates (Costar, USA) were coated with 2 µg/mL of recombinant ZIKV E protein (Native Antigen, UK) for 16h at 4°C, and blocked with assay buffer (5% skimmed milk in PBS-T buffer) for 30 min at RT. Samples were serially diluted (1:50 – 1:25.600) and added to the plates. Plates were washed five times with PBS-T followed by peroxidase-conjugated anti-mouse IgG antibody (Sigma Aldrich, USA) for 1h at RT. After a final wash step, the reaction was revealed with TMB (KPL SureBlue ReserveTM) substrate for 30min at RT, stopped with 1N HCl and read at 450nm (OD 450nm) using a microplate spectrophotometer (Benchmark Plus, Bio-rad, USA). All samples were tested in duplicate, and the results were considered valid when intra-assay variability was below 20%. Antibody titers are expressed as EC50 and were calculated using a three-parameter nonlinear model. The data was plotted using GraphPad Prism version 7.0.
8
Western Blot Detection of Prion Protein
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PK-treated (50 μg/mL for 1 hour at 37°C) and untreated brain homogenates corresponding to 0.7 mg or 0.3 mg brain tissue, respectively, were separated on 12% Bis/Tris Acrylamide gels (NuPAGE, Invitrogen) and transferred to nitrocellulose membranes (Protran, Schleicher & Schüll, Germany). Detection of macaque PrPSc was performed using the monoclonal anti-PrP antibody 11C6 and a Peroxidase conjugated anti-mouse IgG-antibody (Sigma-Aldrich, Germany). Signal was visualized using a chemiluminescent substrate (Super Signal West Pico, Pierce) and high sensitivity films (Amersham). Densitometric analysis of PrPSc was performed using the Image J program 1.37v.
9
ZIKV Envelope Protein Detection Assay
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Monolayers of BHK-21 cells (ATCC, USA) were transfected with the constructs using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer’s instructions. After 48h, cells were fixed using 80% acetone for 1h at room temperature (RT) and blocked with assay buffer containing 5% skimmed milk (Bio-Rad, USA) in PBS-T buffer (1X PBS with 0.05% Tween-20) for 1h at RT. For ZIKV E detection, the 4G2 monoclonal antibody (34 (link)) (1μg/mL) was added in assay buffer for 2h at 37°C. Plates were washed five times with PBS-T, followed by peroxidase-conjugated anti-mouse IgG antibody (Sigma Aldrich, USA) for 1h at RT. After a final wash step, the reaction was revealed with tetramethylbenzidine (TMB, KPL SureBlue Reserve, USA) substrate for 45 min at RT, stopped with 1N HCl and read at 450nm (OD 450nm) using a microplate spectrophotometer (Benchmark Plus, Bio-rad, USA). A plasmid coding for YFV proteins (pL/YFV) was used as a positive control (18 (link)). All samples were tested in duplicate, and the results were considered valid when intra-assay variability was below 20%.
10
HUVEC Surface Receptor Expression Assay
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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).
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