SARS-CoV-2 spike RBD protein and SARS-CoV-2 S protein (200 ng/well) were coated on 96-well ELISA plates, and samples without coating served as controls. Nanobody was used for ELISA detection. Mouse anti-HA polyclonal antibody (Solarbio, Beijing, China) was used as the secondary antibody, and HRP-conjugated Goat Anti-Mouse IgG (Solarbio, Beijing, China) was used as the last antibody. Chromochrome solution was added for 15 min. The termination solution was added to terminate the reaction and the absorbance was measured at 450 nm wavelength.
Hrp conjugated goat anti mouse igg
Manufactured by Solarbio
Sourced in China
HRP-conjugated goat anti-mouse IgG is a secondary antibody that binds to mouse immunoglobulin G (IgG) and is conjugated with horseradish peroxidase (HRP). This reagent can be used for various immunoassay techniques that require detection of mouse IgG.
Automatically generated - may contain errors
Lab products found in correlation
96 well microplate, by Corning (1 mentions)
Elisa reader, by Molecular Devices (1 mentions)
Hrp conjugated goat anti rabbit igg, by Solarbio (1 mentions)
Mouse anti gapdh, by Proteintech (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Sds polyacrylamide gel electrophoresis, by Solarbio (1 mentions)
Enhanced chemiluminescence, by Solarbio (1 mentions)
3 3 5 5 tetramethylbenzidine tmb solution, by Solarbio (1 mentions)
Nunc maxisorb, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ecl reagent, by Thermo Fisher Scientific (1 mentions)
0.45 um pvdf membrane, by Merck Group (1 mentions)
Pvdf membrane, by Solarbio (1 mentions)
Tmb solution, by Tiangen Biotech (1 mentions)
Model 550, by Bio-Rad (1 mentions)
Sulfuric acid, by Sangon (1 mentions)
8 protocols using hrp conjugated goat anti mouse igg
1
SARS-CoV-2 Spike Protein ELISA Detection
2
Tick Antigen ELISA for Antibody Quantification
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Antibody levels in rabbit sera were determined by enzyme-linked immunosorbent assays (ELISA). Between each successive step, the wells were washed 3 times with PBS-Tween-20 (PBST). The 96-well microplates (Costar, New York, USA) were coated with 1 μg of tick antigens in 1 M carbonate buffer and incubated at 4 °C overnight. After blocking with 200 μl/well of blocking buffer (10% Bovine Serum Albumin in PBST) at 37 °C for 1 h, the plates were incubated with 100 μl/well of the rabbit sera which were serially double diluted from 1:200 to 1:204,800 in PBST for 45 min at 37 °C. Then 100 μl/well of HRP-conjugated goat anti-mouse IgG (Solarbio, Beijing, China) diluted 1:10,000 in PBST was added and incubated for 30 min at 37 °C. Finally, immune complexes were revealed by incubating with 100 μl/well of TMB Color liquid (Solarbio, Beijing, China) for 15 min at 37 °C in dark. The reaction was stopped with 50 μl/well of 1 M H2SO4, and the absorbance was measured at 450 nm with an ELISA reader (Molecular Devices, Sunnyvale, CA, USA). All samples were run in triplicate.
3
Western Blot Analysis of Acod1 and Nrf2
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The PMVECs were lysed with cell lysates and then separated by SDS-polyacrylamide gel electrophoresis (Solarbio, Beijing, China) and transferred onto polyvinylidenedifluoride membranes (Millipore, USA). The samples were incubated with anti-rabbit Acod1 antibody (1:1000; Abcam, Shanghai, China) or anti-rabbit Nrf2 antibody (1:1000; ABclonal, Shanghai, China) at 4℃ overnight. Anti-rabbit Histone H3 (1:5000; Gene Tex, USA) or anti-mouse GAPDH (1:10000; Proteintech, Wuhan, China) were used as the controls. Subsequently, these cells were subjected to incubation with HRP-conjugated goat anti-rabbit IgG (1:3000; Solarbio, Beijing, China) or HRP-conjugated goat anti-mouse IgG (1:3000; Solarbio, Beijing, China) at 37℃ for one hour. The proteins were visualized by enhanced chemiluminescence (Solarbio, Beijing, China).
4
Vip3Aa11 Protein Binding Assay
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EPS-HD270 (2.0 mg/mL, 100 μL) was loaded into 96-well ELISA plates (Nunc Maxisorb, Thermo) and immobilized at 4 °C overnight. The ELISA plates were washed three times with Tris-buffered saline (TBS) and blocked with TBST (TBS containing 0.1% Tween-20) containing 2.0% BSA (200 μL) at 37 °C for 2 h. The plates were washed with TBST three times and incubated with different concentrations of Vip3Aa11 (0, 0.5, 1, 2, 4, 8, 16, 32 nmol/L) (100 μL) for 1 h at 37 °C. After washing with TBST buffer 3 times, anti-Vip3A antibody (TBST 1:5000 dilution, 100 μL) was added. TBST (100 μL) containing 1/10,000 HRP-conjugated goat anti-mouse IgG (Solarbio Life Sciences, Beijing, China) was incubated at 37 °C for 1 h. For each treatment, three replicates were performed. After washing, the reaction was tested with 3,3′,5,5′-tetramethylbenzidine (TMB) solution (Solarbio Life Sciences, Beijing, China) (100 μL) for 15 min in the dark at 37 °C. The reaction was terminated with HCl (2.0 mol/L, 100 μL), and the absorbance was immediately read at 450 nm using a microplate reader. The equilibrium dissociation constant (Kd) was analyzed using Sigma-plot Software (Version 12.0).
5
Quantifying C. albicans Enolase1 Antibodies
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Candida albicans enolase1 IgG antibody in rabbit plasma was measured by indirect ELISA, as previously described (He et al., 2015 (link)). Recombinant CaEno1 was then coated onto each well of blank ELISA plates for overnight at 4°C after being diluted to 1 μg/ml in CBS. After washing trice with PBST, the plates were then blocked with PBST plus 3% (w/v) BSA solution, and incubated for 2 h at 37°C. After washing, rabbit plasma samples with a 1:500 dilution by PBST plus 1% (w/v) BSA were added and incubated at 37°C for 1 h. After three washes with PBST, 1:5,000 diluted HRP-conjugated goat anti-mouse IgG (Solarbio, Beijing, China) was added and incubated for 1 h at 37°C. After another round of washing, 100 μl of TMB substrate solution was applied to each well and incubated for 10 min at 37°C. Thereafter, the reaction was terminated and the OD450 values were determined.
6
Specificity Evaluation of Anti-ASFV p30 McAb
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Western blotting was used to determine the specificity of anti-ASFV p30 McAb for the purified p30 recombinant protein as well as for the ASFV HLJ/18 strain (Inactivated ASFV Pig/HLJ/2018 strain-infected porcine alveolar macrophages (PAMs) were provided by Harbin Veterinary Research Institute and stored at -80 °C.). The purified p30 protein or inactivated samples was mixed with 5 × loading buffer (ABclonal) separately, denatured by heating at 98 °C for 5 min, separated by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) (on a 15 % gel (Epizyme, Shanghai, China), and then blotted to a 0.4 µm polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA). PVDF membrane was blocked with 5 % skimmed milk in PBST (0.05 % Tween 20 in PBS) for 1 hour at room temperature (RT). Membranes were washed three times with PBST for 10 min, then incubated with p30 McAb (1:1000) as primary antibodies for 1 h at 37 °C, and then washed three times. The membranes were incubated with HRP-conjugated goat anti-mouse IgG (Solarbio, Beijing, China) (1:5000) as a secondary antibody and were incubated together for 1 h at 37 °C. Then PBST was used three times, followed by enhanced chemiluminescence (NCM Biotech, Suzhou, China) color development.
7
Immunoblotting of His-tagged Proteins
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Harvested cells and supernatants were separated by 12% SDS-PAGE gels and transferred onto the 0.45 um PVDF membranes (Millipore, United States). After the PVDF membranes had been blocked with TBST containing 5% non-fat dry milk at RT for 2 h and incubated with the His-tag antibody (Solarbio, China; dilution, 1: 2,000) at 4°C overnight. The next day, the HRP-conjugated goat anti-mouse IgG (Solarbio, China; dilution, 1: 5,000) was added to incubate for 2 h after washing with TBST. At last, Signals were detected using a chemiluminescent ECL reagent (Thermo Fisher Scientific, United States).
8
ELISA for Recombinant VP27 Protein
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Recombinant VP27 protein was diluted in carbonate-bicarbonate buffer (pH 9.5) to a final concentration of 2 µg/mL and coated on 96-well plates (100 μL per well). Following adsorption of the antigen after incubation at 4°C overnight, the plates were washed with PBST 3 times and then blocked with 5% non-fat milk at 37°C for 1.5 h. Next, 100 μL of serum from immunized mice was incubated in the plate at a 1:1,000 dilution at 37°C for 1 h, and subsequently washed 3 times with PBST. Then, 100 μL of HRP-conjugated goat anti-mouse IgG (1:10,000; Solarbio, Beijing, China) was added into the plates and incubated at 37°C for 1 h. After washing with PBST 3 times, 100-μL TMB solution (TIANGEN, Beijing, China) was added into the plates at 37°C for 15 min. The reaction was stopped with 50-μL sulfuric acid (2 mol/L; Sangon Biotech Co., Ltd, Shanghai, China), and absorbance was measured at 450 nm using a microplate reader (Model550; Bio-Rad, Hercules, CA).
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