Serum ELISAs were performed by coating Corning 96-well clear flat bottom high bind microplates with 100 μL of protein at 5 μg/mL in PBS. Plates were incubated overnight at 4°C. Coating solution was removed, and plates were blocked using 1% BSA in PBS with 1% Tween for 60 minutes at room temperature. Blocking solution was removed. Sera were diluted 1:40 in PBS, and 5-fold serial dilution was performed. CR3022 IgG at a starting dilution of 5 μg/mL with 5-fold serial dilution was used as a positive control. 40 μL of primary antibody solution was applied to each well. Primary incubation occurred for 90 minutes at room temperature. Plates were then washed three times with PBS-Tween. HRP-conjugated rabbit anti-mouse IgG antibody (Abcam) at a concentration of 1:20,000 in PBS and a volume of 150 μL was used as a secondary antibody. Secondary incubation occurred for 60 minutes at room temperature. Plates were then washed three times with PBS-Tween. 1xABTS development solution (ThermoFisher) was applied as outlined in the manufacturer’s recommendations. Development was stopped after 30 minutes with a 1% SDS solution. Plates were read at 405 nm using a SectraMax iD3 plate reader (Molecular Devices).
Hrp conjugated rabbit anti mouse igg antibody
Manufactured by Abcam
Sourced in United States, United Kingdom
HRP-conjugated rabbit anti-mouse IgG antibody is a secondary antibody used for detection in immunoassays. It is produced by immunizing rabbits with mouse IgG and conjugating the resulting antibodies to horseradish peroxidase (HRP).
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6 protocols using hrp conjugated rabbit anti mouse igg antibody
1
Serum ELISA for IgG Antibody Detection
2
Ni-NTA EDIII Purification and Western Blot
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Ni-NTA Purified EDIII was run on 10% SDS-PAGE, along with pre-stained protein marker on adjacent lane and transferred onto nitrocellulose membrane using a semidry transfer apparatus. The membrane was incubated in blocking buffer of 5% skimmed milk at 4°C, overnight. Then, the membrane was incubated in the blocking buffer containing primary antibody (anti-HisTag mAb (Abcam)/anti-dengue mAb (Abnova, Taiwan) at a 1:500 dilution) with gentle shaking for 2 hr at 37°C. The membrane was washed by PBST (PBS containing 0.1% Tween 20) three times and then incubated in secondary antibody (a 1:5000 dilution of HRP-conjugated rabbit anti mouse IgG antibody (Abcam) in blocking buffer), with gentle shaking for 1 hr at room temperature (22°C). After washing with PBST for 15 min, detection was performed using DAB (diaminobenzidine) as a substrate.
3
Serum ELISA for Antibody Detection
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Serum ELISAs were performed using 96-well, clear, flat-bottom, high bind microplates (Corning). These plates were coated with 100 mL of protein, which were adjusted to a concentration of 5 mg/mL (in PBS). Plates were incubated overnight at 4°C. After incubation, plates had their coating solution removed and were blocked using 1% BSA in PBS with 1% Tween. This was done for 60 min nutating at room temperature. This blocking solution was removed, and sera was diluted 40-fold in PBS. A 4-fold serial dilution was then performed. CH67 IgG, similarly serially diluted (4-fold) from a 5 mg/mL starting concentration, was used as a positive control. 40 μL of primary antibody solution was used per well. Following this, samples were incubated for 90 min at room temperature. Plates were rinsed three times using PBS-Tween. 150 μL of HRP-conjugated rabbit anti-mouse IgG antibody, sourced commercially from Abcam (1:20,000 dilution in PBS), was used for the secondary incubation, performed for one hour, similarly nutating at room temperature. Plates were then rinsed three times using PBS-Tween. 150 μL of 1xABTS development solution (ThermoFisher) was used according to the manufacturer’s protocol, and stopped after 30 min using 100 μL of a 1% SDS solution, and plates were read using a SectraMaxiD3 plate reader (Molecular Devices) at 405 nm.
4
Western Blot Analysis of CHAP-amidase
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Western blot analysis was conducted as previously reported (Fahimi et al., 2014 (link); Ferdosian et al., 2015 (link); Hosseini et al., 2016 (link)). Briefly, the lysate of CHAP-amidase-expressing cells was run on 10% SDS-PAGE (Bioneer Co., South Korea) and transferred onto nitrocellulose membrane using a semi-dry transfer system. The membrane was blocked overnight in 5% skimmed milk at 4°C and then incubated with primary antibody (anti-His tag monoclonal antibody (mAb) (Abcam Inc., MA, USA), while shaking for 2 h at room temperature (22°C). The membrane was washed three times with PBST (PBS containing 0.05% Tween 20) and incubated with secondary antibody (1:4,000 dilution of HRP-conjugated rabbit anti-mouse IgG antibody) (Abcam Inc., MA, USA) with gentle shaking for 1 h at room temperature. The membrane was washed three times with PBST and the signal was detected by adding DAB (3,3′-diaminobenzidine) as a chromogenic substrate.
5
Serum ELISA for Antibody Detection
6
Western Blot and Dot Blot Analysis of Recombinant Proteins
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For Western blot analysis the purified proteins were applied on a 10 % SDS-PAGE and transferred onto nitrocellulose membrane by using a semidry (Biorad, California, USA) system (Tris 12.5 mM, Glycine 96 mM, SDS 0.03 %, and methanol 10 % with pH 8.3). The membrane was incubated in the blocking buffer of 5 % skimmed milk at 4 ºC overnight. It was then incubated in a 1:1000 diluted primary antibody, anti-HisTag (Abcam, UK) or pan-dengue specific monoclonal antibody (cat no. MAB4043, Abnova), with gentle shaking for 2 hours at room temperature (RT). The membrane was washed three times with PBST (PBS containing 0.1 % Tween 20) and then incubated in secondary antibody, a 1:2000 dilution of HRP-conjugated rabbit anti mouse IgG antibody (Abcam, UK), with gentle shaking for 1 h at RT. After a 15 min washing with PBST, the detection was performed using diaminobenzidine (DAB) as a chromogenic substrate. Dot blotting analysis was performed using a very similar method like Western blotting. Briefly, recombinant proteins were dotted on nitrocellulose membrane strips and were reacted with diluted serum samples from the immunized animals. Blocking, antibody incubation, and detection methods were identical with Western blotting, except the serum dilutions were 1:10 and the chromogenic substrate was 4-Chloro-1-naphthol (Sigma-Aldrich, St. Louis, MO, USA).
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