Microtiter wells were coated overnight at 4°C with DC-SIGN or DC-SIGNR (5, 2.5, 1.25, and 0.625 µg/well) in carbonate–bicarbonate buffer, pH 9.6, and left overnight at 4°C. Wells were blocked with 100 µL of 2% w/v BSA in PBS for 2 h at 37°C. Following three washes with PBS + 0.05% Tween 20, ghA, ghB, ghC, or its substitution mutants (2.5 µg/100 μl) was added to each well in the buffer containing 50 mM NaCl, 100 mM Tris–HCl, pH 7.5, and 5 mM CaCl2. MBP (Sigma) was used as a negative control. The plate was incubated at 37°C for 1.5 h and then at 4°C for 1.5 h. The wells were washed and the bound protein was detected with anti-MBP monoclonal antibodies in PBS (1:5,000, Sigma) followed by rabbit anti-mouse IgG-HRP conjugate (1:5,000; Sigma) for 1 h. The color was developed using o-phenylenediamine dihydrochloride (OPD, Sigma) and read at 415 nm.
Rabbit anti mouse igg hrp conjugate
Manufactured by Merck Group
Sourced in United States
Rabbit anti-mouse IgG HRP conjugate is a secondary antibody reagent used in immunoassay and immunodetection applications. It consists of a rabbit-derived antibody that binds to mouse immunoglobulin G (IgG) and is conjugated to horseradish peroxidase (HRP), an enzyme that can be used to generate a detectable signal.
Automatically generated - may contain errors
Lab products found in correlation
Anti mbp, by Merck Group (1 mentions)
O phenylenediamine dihydrochloride opd, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Microtiter plate, by Greiner (1 mentions)
Dab substrate, by Roche (1 mentions)
Goat anti mouse igg hrp conjugate, by Bio-Rad (1 mentions)
Microplate reader, by Bio-Rad (1 mentions)
6 protocols using rabbit anti mouse igg hrp conjugate
1
Quantifying DC-SIGN/R Binding Interactions
2
Influenza Virus Protein Binding Assay
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Purified H1N1/H3N2 (1.36 × 106 pfu/ml) virus particles were run on a 12% (w/v) SDS-PAGE, and transferred onto a PVDF membrane for 2 h at 320 mA in transfer buffer (25 mM Tris–HCl, pH 7.5, 20% v/v methanol, and 190 mM glycine). Membrane was blocked with PBS+5% w/v BSA (Sigma-Aldrich) at room temperature, followed by PBST washes. The membrane was then incubated with 10 μg/ml of factor H or VCP overnight at 4°C, and probed appropriately with either monoclonal mouse anti-human factor H (MRCOX23) (MRC Immunochemistry Unit, Oxford) (1 mg/ml) or rabbit anti-VCP polyclonal antibody (0.5 mg/ml) (King Faisal Specialist Hospital and Research Center, Saudi Arabia) at room temperature for 1 h. Following PBST washes (three times, 10 min each), the membrane was incubated with secondary antibody, rabbit anti-mouse IgG HRP conjugate (1:1,000) (Sigma-Aldrich) or Protein A-HRP-conjugate for 1 h at room temperature. The secondary antibody was removed, followed by PBST washes; then the membrane was developed using 3,3′-diaminobenzidine (DAB).
3
Quantifying Antibody Responses to Influenza Virus
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Measurement of HA-specific IgM and IgG in sera of A(H1N1)pdm09 virus infected mice was performed as below. The microtiter plates (Greiner) were coated with pandemic influenza virus HA protein [200 ng/well; rHA of the H1N1 (A/California/7/2009); ThermoFisher Scientific, Waltham, MA] by keeping the plates overnight at 4°C; control wells were coated with similar amount of BSA. The wells were then blocked by adding 5% milk, washed once with PBS-T and incubated with 1:50 diluted heat inactivated mouse sera for 1 hr at room temperature. Thereafter, the wells were washed three times with PBS-T and incubated with goat anti-mouse IgM HRP conjugate (1:1000 dilution; Sigma Aldrich) or rabbit anti-mouse IgG HRP conjugate (1:1000 dilution; Sigma Aldrich) for 30 min at room temperature. The unbound conjugate was then washed, ABTS was added, and optical density was measured at 415 nm. Mouse sera and conjugates were diluted in PBS-T containing 0.5% milk and 0.5% BSA.
4
Western Blot Analysis of GFLV Movement Protein
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Infected and non-infected leaf tissues were ground in 1 × PBS, mixed with an equal volume of 2 × sample buffer and boiled for 5 min. Next, the denatured crude leaf extracts, purified GFLV MP and the protein from non-induced bacteria were separated by 12% SDS-PAGE, blotted onto nitrocellulose membranes using electro-blotter at 80 V for 120 min at room temperature as mentioned previously. After blocking with 5% powdered skim milk in phosphate buffered saline Tween 20 (PBST), the membrane was incubated with the anti-GFLV MP IgG (1:500) overnight at 4°C with gentle shaking. After 4 × washing with PBST, the membrane was incubated with the secondary antibody, mouse anti-rabbit IgG HRP-conjugate (1:1,000; Sigma-Aldrich, St. Louis, MO, USA) at room temperature for about 2 h. The bands of interest were visualized by reaction with freshly prepared DAB substrate (Roche Applied Science, Indianapolis, IN, USA).
This study was approved by the animal ethics committee of Pasteur Institute of Iran (IPI) (approval No. 66465132).
This study was approved by the animal ethics committee of Pasteur Institute of Iran (IPI) (approval No. 66465132).
5
Polyclonal Antibodies for Tick AChE1
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Polyclonal rabbit antibodies specific for rAaChE1 or rBmAChE1 (recombinant acetylcholinesterase 1 of the cattle tick, R. [B.] microplus) [14 (link)] were designed and prepared by GeneScript Biotech Corporation (Piscataway, NJ, USA). Rabbits were immunized with peptide-KLH conjugate (rAaChE peptide, CAKTGNPNRPENGTS; rBmAChE1 peptide, TGKRRFDRAESIEEC). SDS-PAGE gels (Bold 4–12% Bis-Tris Plus (NW04122BOX), Thermo-Fisher Scientific) and Western blot onto PVDF membranes were conducted as previously described [40 (link)] with 12 µL of sample loaded into each well. Blots were blocked by 30 min of incubation in 10% dried goat milk (Hoosier Hill Farm, Fort Wayne, IN, USA), incubated overnight (18 h) with the first antibody (rabbit anti-BmAChE1 or rabbit anti-AaChE1) diluted 1:1000 in BLOTTO2 (2% goat milk, 1% Tween-80), washed 5 times (5 min each) in wash buffer (0.01 M NaPhosphate, pH 7.4, 0.3% Tween-20), incubated 2 h with 2nd antibody (mouse anti-rabbit IgG-HRP conjugate, Sigma Chemical, St. Louis, MO, USA) diluted 1:5000 in BLOTTO2, washed 3 times as before, incubated 30 min with 3rd antibody (goat anti-mouse IgG-HRP conjugate, Bio-Rad Laboratories, Inc., Hercules, CA, USA) diluted 1:5000 in BLOTTO2, washed as previously described, and then color developed for 3 min in DAB/H2O2 urea (Sigma Chemical, ST. Louis, MO, USA).
6
Quantification of MCP-Specific Antibodies
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MCP-specific antibodies present in serum samples from animals of three groups above were quantified by indirect ELISA. Precoated ELISA plates with the purified MCP with a final concentration of 2 µg/mL diluted in 50 mM NaHCO3 (pH 9.6) were incubated with serial dilutions of serum samples. Specific IgY antibodies were detected by using rabbit antiserum against Chinese giant salamander IgY prepared by the laboratory [23 ] and mouse-anti-rabbit-IgG-HRP conjugate (Sigma-Aldrich) as primary and secondary antibodies, respectively. The plates were developed with a peroxidase substrate-ate solution (3,3′,5,5′-tetramethylbenzidine). The optical density at 450 nm of each well was determined using a microplate reader (Bio-Rad). The mean absorbance value in duplicate wells was recorded, and the endpoint titers were expressed as the reciprocal of the highest sample dilution for which the optical density was equal to or greater than the mean optical density of preimmune sera.
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