The protocol was adapted from Langowski et al.103 (link) First, recombinant I53_dn5 nanoparticle or H1 MI15-foldon protein was incubated for 1 h on 96-well Nunc MaxiSorp plates (Thermo Scientific) (2.0 μg/mL, 50 μL per well). Then, 200 μL of Tris-Buffered Saline Tween (TBST: 25 mM Tris pH 8.0, 150 mM NaCl, 0.05% (v/v) Tween 20) with 2% (w/v) BSA was added to each well and incubated for 1 hr. Plates were washed 3× in TBST using a robotic plate washer (BioTek). Then, 50 μL of a 1:2,500 serum dilution in TBST with 2% (w/v) BSA was added to each well and incubated for 1 h. To test for avidity, 50 μL of 2 M sodium thiocyanate (NaSCN) or PBS (control) was added to wells for 15 min. After washing plates 3× with TBST, 50 μL of anti-mouse HRP-conjugated goat secondary antibody (CellSignaling Technology) diluted 1:2,000 in TBST with 2% (w/v) BSA was incubated in each well for 1 hr. Following a final 3× TBST plate wash, 100 μL of TMB (SeraCare) was added to each well and rested for 2 min before quenching with 100 μL of 1 N HCl. Absorbance at 450 nm was immediately collected for each well on a SpectraMax M5 plate reader (Molecular Devices). All steps were performed at ambient temperature. Percentage OD450 in the corresponding NaSCN/PBS wells were used to determine the avidity index.
Hrp conjugated goat anti mouse secondary antibody
Manufactured by Cell Signaling Technology
Sourced in United States
The HRP-conjugated goat anti-mouse secondary antibody is a laboratory reagent designed for the detection of mouse primary antibodies in various immunoassays. It contains a horseradish peroxidase (HRP) enzyme conjugated to a goat-derived antibody that specifically binds to mouse immunoglobulins. This secondary antibody can be used to amplify and visualize the signal from mouse primary antibodies, facilitating the detection and quantification of target proteins or antigens.
Automatically generated - may contain errors
Lab products found in correlation
Nunc immuno plate, by Thermo Fisher Scientific (3 mentions)
O phenylenediamine dihydrochloride substrate, by Merck Group (3 mentions)
Robotic plate washer, by Agilent Technologies (2 mentions)
3 3 5 5 tetramethylbenzidine (tmb), by LGC (2 mentions)
Protein assay, by Bio-Rad (2 mentions)
Nunc maxisorp plate, by Thermo Fisher Scientific (1 mentions)
Spectramax m5, by Molecular Devices (1 mentions)
Ni nta plate, by Qiagen (1 mentions)
Spectramax m5 plate reader, by Molecular Devices (1 mentions)
Mouse anti human β actin, by Merck Group (1 mentions)
Immun blot pdvf membrane, by Bio-Rad (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Phosphatase inhibitor, by Merck Group (1 mentions)
Mouse anti human β actin antibody, by Merck Group (1 mentions)
Horseradish peroxidase hrp conjugated goat anti mouse secondary antibody, by Cell Signaling Technology (1 mentions)
Ripa buffer, by Cell Signaling Technology (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Cytokine specific elisa kits, by BD (1 mentions)
Protease and phosphatase inhibitor cocktail, by MedChemExpress (1 mentions)
Rabbit anti gsk 3β, by Cell Signaling Technology (1 mentions)
11 protocols using hrp conjugated goat anti mouse secondary antibody
1
Nanoparticle-based Antibody Avidity Assay
2
His-tagged Protein Binding Assay
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The protocol was adapted from Brouwer et al.33 (link) First, 50 μL of 6.5 nM His-tagged protein per well was incubated for 1 h in 96-well Ni-NTA plates (Qiagen). Then, 200 μL of Tris-Buffered Saline Tween (TBST: 25 mM Tris pH 8.0, 150 mM NaCl, 0.05% v/v Tween 20) with 2% (w/v) BSA was added to each well and incubated for 1 h. Plates were washed 3× in TBST using a robotic plate washer (BioTek). Then, 50 μL of serum dilutions starting at 1:100 and serially diluting 5-fold seven times using TBST with 2% (w/v) BSA (8 total dilutions) were added to each well and incubated for 1 h. After washing plates 3× with TBST, 50 μL of anti-mouse HRP-conjugated goat secondary antibody (CellSignaling Technology) diluted 1:2,000 in TBST with 2% (w/v) BSA was incubated in each well for 1 h. Following a final 3× TBST plate wash, 100 μL of TMB (SeraCare) was added to each well and rested for 2 min, then 100 μL of 1 N HCl was added to each well to quench the reaction. Absorbance at 450 nm was immediately collected for each well on a SpectraMax M5 plate reader (Molecular Devices). Data were plotted in Prism (GraphPad) to determine AUC values. All steps were performed at ambient temperature.
3
Western Blotting Protocol for LC3 Detection
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Western blotting was carried out as previously described [25 (link)]with some modifications. Briefly, cells were lysed in lysis buffer (50mM Tris.HCl, pH 6.8, 10% glycerol, 2% sodium dodecyl sulfate, 0.001% bromophenol blue, 100mM DTT, 10X protease inhibitor cocktail (Roche) and phosphatase inhibitors (Sigma)). Samples were heated to 95°C for 10 minutes and stored at -80°C. Lysates were subjected to SDS/PAGE on 15% Tris-Tricine Criterion (Bio-Rad, Hercules, CA) minigels followed by transfer to 0.2-μm-pore polyvinylidene difluoride membrane (Immun-Blot PDVF membrane; Bio-Rad). Membranes were probed with antibodies to mouse anti-human LC3 (2G6, nanoTools Antikörpertechnik GmbH, Teningen Germany) and mouse anti-human β–actin (Sigma). Following exposure to HRP-conjugated goat anti-mouse secondary antibody (Cell Signaling Technology, Danvers, MA, USA), proteins were visualized by enhanced chemiluminescence using Supersignal West Pico chemiluminescent substrate (ThermoScientific, Rockford, IL, USA).
4
Quantifying Protein Expression in Transfected Cells
5
Detecting Parasite-Specific Antibodies in Serum
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To detect parasite-specific antibodies, protein extracts from blood stages obtained by saponin lysis (0.1%) of parasite pellets were sonicated in lysis buffer (10 mM Tris, pH 7.4, 150 mM NaCl, 0.02% NaN3, 20 mM MgCl2, 1 % Triton X-100, and complex protease inhibitors) and centrifuged (10 ,000 g for 30 min at 4°C). The total amount of proteins in the supernatant was measured using a Bio-Rad Laboratories protein assay. 96-well plates (Nunc-immuno plate; Thermo Fisher Scientific) were coated with 2 µg/ml PbNK65 WT protein extracts in carbonate buffer, pH 9.6, for 2 h at 37°C and then saturated with 1% (wt/vol) BSA (Sigma-Aldrich). Serum samples were assayed using serial dilutions and incubated for 2 h at 37°C. Specific binding was detected using HRP-conjugated goat anti–mouse secondary antibody (diluted 1:2,000; Cell Signaling Technology) followed by the addition of o-phenylenediamine dihydrochloride substrate (Sigma-Aldrich). HCl 1N was used to block the reaction. The optical density was read at 490–630 nm. Each sample was tested against nonimmune serum and PBS as background controls. Amounts of IL-6 in the serum were analyzed following the instructions provided by the ELISA kit supplier (BD).
6
Parasite Antibody Detection ELISA Protocol
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To detect parasite-specific antibodies, 96-well plates (Nunc-immuno plate; Thermo Scientific, Rockford, IL) were coated with parasite protein extract from asexual blood stages in carbonate buffer, pH 9.6, for 2 h at 37 °C. After the plates were saturated with 1% (w/v) pork gelatine, each serum was assayed at serial dilutions and incubated overnight for 2 h at 37 °C. Specific binding was detected using HRP-conjugated goat anti-mouse secondary antibody (Cell Signalling technology®, Danvers, MA) followed by the addition of o-phenylenediamine dihydrochloride (OPD) substrate (Sigma-Aldrich; St.Louis, MO). Hydrogen chloride (HCl) 1 N was used to block the reaction. The optical density (OD) was read at 490–655 nm. Each sample was tested against non-immune serum and PBS as background controls. Amounts of IL-12p70, IFN-γ and IL-6 in the serum were analysed by cytokine-specific ELISA kits (BD Biosciences, Mountain View, CA).
7
Serological Assay for Parasite-Specific Antibodies
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To detect parasite-specific antibodies, 96-well plates (Nunc-immuno plate; Thermo Scientific, Rockford, IL) were coated with parasite protein extract from isolated SPZ from A. stephensi salivary glands in carbonate buffer, pH 9.6, for 2 h at 37 °C. After the plates were saturated with 1% (w/v) pork gelatine, each serum sample was assayed at serial dilutions and incubated overnight for 2 h at 37°C. Specific binding was detected using horseradish peroxydase (HRP)-conjugated goat anti-mouse secondary antibody (Cell Signaling technology®, Danvers, MA) followed by the addition of o-phenylenediamine dihydrochloride (OPD) substrate (Sigma-Aldrich; St. Louis, MO). Hydrogen chloride (HCl) 1 N was used to block the reaction. The optical density (OD) was read at 490–655 nm. Each sample was tested against non-immune serum and PBS as background controls.
8
Western Blot Analysis of Cell Signaling
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HEK 293T, HRECs or mouse lung tissue were sonicated three times in lysis buffer with the addition of protease and phosphatase inhibitor cocktail (Med Chem Express, USA). The lysates were mixed with 4 × sodium dodecyl sulfate (SDS) sample buffer (Solarbio, CN) and then loaded on 12% polyacrylamide gel. Western blotting was conducted, and signals were detected by iBright™ CL1500 Imaging System (Thermo Fisher Scientific, USA). The following antibodies were used: rabbit anti-CyclinD1 (MA5-14512, Thermo Fisher Scientific, USA), rabbit anti-c-Myc (T55150, Abmart, CN), rabbit anti-FZD4 (A8161, ABclonal Technology, CN), rabbit anti-p-GSK3β-Ser-9 (5558, Cell Signaling Technologies, USA), rabbit anti-GSK3β (12456, Cell Signaling Technologies, USA), rabbit anti-β-actin (3700, Cell Signaling Technologies, USA), mouse anti-FLAG (F3165, Merck, GER), rabbit anti-LRP5 (5731, Cell Signaling Technologies, USA), rabbit anti-FZD6 (A10503, ABclonal Technology, CN), HRP-conjugated goat anti-mouse secondary antibody (7076, Cell Signaling Technologies, USA), and HRP-conjugated goat anti-rabbit secondary antibody (7074, Cell Signaling Technologies, USA).
9
Western Blot Antibody Reagents
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Anti-caspase 9 and anti-caspase 3 antibodies were purchased from Cell Signaling and anti-α-tubulin antibody was procured from Sigma, aoat anti-rabbit HRP conjugated secondary antibody was purchased from Bio-Rad, and goat anti-mouse HRP conjugated secondary antibody was purchased from Cell Signaling.
10
Quantifying AZ304-Induced p-ERK Activation
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A375 cells were seeded into 96-well micro plates (Costar, Corning, and Lowell, MA) at 2 × 105 cells/well in phenol red free DMEM (Invitrogen, Carlsbad, CA) supplemented with 10% foetal bovine serum. After 48 h, the cells were treated with DMSO or multiple concentrations of AZ304 and then returned to the incubator for 75 min. Medium were then aspirated and cells were fixed with a 6% formaldehyde solution for 20 min at room temperature. Cells were washed once with PBS containing 0.05% Triton X-100 (PBST) and 0.6% hydrogen peroxide added for 20 min at room temperature. After washing again in PBST, cells were blocked with 10% FBS/PBST solution for 1 h at room temperature. After washing, p-ERK monoclonal antibody was added and the plates were placed at 4 °C for overnight. Plates were then washed in PBST, incubated with goat anti-mouse HRP-conjugated secondary antibody (Cell Signalling Technology, Danvers, MA) for 2 h at room temperature, washed in PBST, ABTS solution (Sigma, St. Louis, MO) added and plates incubated for 2 h at 30 °C. Quantification of signal was determined at OD405 using a SpectraMax plate reader (Molecular Devices, Sunnyvale, CA). All assays were done in duplicate across different plates. EC50 was calculated using XLift.
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