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Goat anti human igg fc

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Goat anti-human IgG Fc is a laboratory reagent that binds to the Fc region of human immunoglobulin G (IgG) antibodies. It can be used in various immunoassays and other applications that require the detection or capture of human IgG.

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Lab products found in correlation

Phosphatase substrate, by Merck Group (3 mentions) Pnpp disodium salt hexahydrate, by Merck Group (2 mentions) Streptavidin coated plate, by Thermo Fisher Scientific (1 mentions) 384 well plate, by Greiner (1 mentions) Gentlemacs m tube, by Miltenyi Biotec (1 mentions)

9 protocols using goat anti human igg fc

1

Recombinant Protein Capture ELISA

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For recombinant protein capture (ELISA), 384-well plates were incubated with 2 μg ml−1 of antigen overnight at 4°C. The plates were blocked for one hour with 2% non-fat dry milk supplemented with 2% goat serum. Plates were washed three times with PBS-T and primary mAbs or hybridoma cell culture supernatants were applied to wells for one hour. Plates were washed with PBS-T four times before applying 25 μl secondary alkaline phosphatase-conjugated antibody (goat anti-human IgG Fc, Meridian Life Science) at a dilution of 1:4,000 in blocking solution. After a 1 h incubation, the plates were washed five times with PBS-T and 25 μl of substrate solution (1 mg ml−1 pNPP disodium salt hexahydrate, Sigma) was added to each well. The plates were incubated at room temperature for approximately 30 min before reading the optical density at 405 nm on a Biotek plate reader. Experiments with the RSV F mutants were conducted similarly.
Mousa J.J., Kose N., Matta P., Gilchuk P, & Crowe JE J.r. (2017). A novel pre-fusion conformation-specific neutralizing epitope on the respiratory syncytial virus fusion protein. Nature microbiology, 2, 16271.
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2

Recombinant Protein Capture ELISA Protocol

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For recombinant protein capture ELISA, 384-well plates were treated with 2 μg/mL of antigen for one hour at 37°C or overnight at 4°C. Following this, plates were blocked for one hour with 2% milk supplemented with 2% goat serum. Primary mAbs and culture supernatants were applied to wells for one hour following three washes with PBS-T. Plates were washed with PBS-T four times before applying 25 μL secondary antibody (goat anti-human IgG Fc, Meridian Life Science) at a dilution of 1:4,000 in blocking solution. After a one-hour incubation, the plates were washed five times with PBS-T, and 25 μL of phosphatase substrate solution (1 mg/mL phosphatase substrate in 1 M Tris HCl pH 9.6, Sigma) was added to each well. The plates were incubated at room temperature before reading the optical density at 405 nm on a BioTek plate reader. ELISA experiments using biotintylated peptides were conducted by coating pre-blocked streptavidin-coated plates (Fisher) with 10 μg/mL peptide for two hours. After three washes with PBS-T, plates were coated with primary mAbs for one hour. The remaining steps were conducted as described above.
Mousa J.J., Binshtein E., Human S., Fong R.H., Alvarado G., Doranz B.J., Moore M.L., Ohi M.D, & Crowe JE J.r. (2018). Human antibody recognition of antigenic site IV on Pneumovirus fusion proteins. PLoS Pathogens, 14(2), e1006837.
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3

ELISA Assay for Antigen-Specific Antibodies

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384-well plates were coated with 2 µg/mL of antigen overnight at 4 °C. Plates were blocked for with 2% milk supplemented with 2% goat serum for one hour, followed by three washes with PBS-T. Primary mAbs or B cell culture supernatants were applied to wells for two hours. Plates were washed with PBS-T four times before applying secondary antibody (goat anti-human IgG Fc, Meridian Life Science) at a dilution of 1:4,000 in blocking solution. After a one-hour incubation, the plates were washed six times with PBS-T, and phosphatase substrate solution (1 mg/mL phosphatase substrate in 1 M Tris aminomethane, Sigma) was added to each well. The plates were incubated at room temperature before reading the optical density at 405 nm on a Biotek plate reader.
Wen X., Mousa J.J., Bates J.T., Lamb R.A., Crowe JE J.r, & Jardetzky T.S. (2017). Structural basis for antibody cross-neutralization of respiratory syncytial virus and human metapneumovirus. Nature microbiology, 2, 16272.
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4

Recombinant Protein Capture ELISA

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For recombinant protein capture (ELISA), 384-well plates were incubated with 2 μg ml−1 of antigen overnight at 4°C. The plates were blocked for one hour with 2% non-fat dry milk supplemented with 2% goat serum. Plates were washed three times with PBS-T and primary mAbs or hybridoma cell culture supernatants were applied to wells for one hour. Plates were washed with PBS-T four times before applying 25 μl secondary alkaline phosphatase-conjugated antibody (goat anti-human IgG Fc, Meridian Life Science) at a dilution of 1:4,000 in blocking solution. After a 1 h incubation, the plates were washed five times with PBS-T and 25 μl of substrate solution (1 mg ml−1 pNPP disodium salt hexahydrate, Sigma) was added to each well. The plates were incubated at room temperature for approximately 30 min before reading the optical density at 405 nm on a Biotek plate reader. Experiments with the RSV F mutants were conducted similarly.
Mousa J.J., Kose N., Matta P., Gilchuk P, & Crowe JE J.r. (2017). A novel pre-fusion conformation-specific neutralizing epitope on the respiratory syncytial virus fusion protein. Nature microbiology, 2, 16271.
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5

Recombinant Protein Capture ELISA

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For recombinant protein capture ELISAs, 384-well plates (Greiner Bio-One) were treated with 2 μg/ml of antigen in PBS for 1 hr at 37°C or overnight at 4°C. Following this, plates were washed once with water before blocking for 1 hr with 2% blocking buffer. Primary mAbs or culture supernatants were applied to wells for 1 hr following three washes with water. Plates were washed with water three times before applying 25 μl of secondary antibody (goat anti-human IgG Fc; Meridian Life Science) at a dilution of 1:4,000 in blocking solution. After incubation for 1 hr, the plates were washed five times with PBS-T, and 25 μl of a PNPP (p-nitrophenyl phosphate) solution (1 mg/ml PNPP in 1 M Tris base) was added to each well. The plates were incubated at room temperature for 1 hr before reading the optical density at 405 nm on a BioTek plate reader. Binding assay data were analyzed in GraphPad Prism using a nonlinear regression curve fit and the log(agonist)-versus-response function to calculate the binding EC50 values.
Huang J., Diaz D, & Mousa J.J. (2020). Antibody recognition of the Pneumovirus fusion protein trimer interface. PLoS Pathogens, 16(10), e1008942.
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6

ELISA Assay for Antigen-Specific Antibodies

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384-well plates were coated with 2 µg/mL of antigen overnight at 4 °C. Plates were blocked for with 2% milk supplemented with 2% goat serum for one hour, followed by three washes with PBS-T. Primary mAbs or B cell culture supernatants were applied to wells for two hours. Plates were washed with PBS-T four times before applying secondary antibody (goat anti-human IgG Fc, Meridian Life Science) at a dilution of 1:4,000 in blocking solution. After a one-hour incubation, the plates were washed six times with PBS-T, and phosphatase substrate solution (1 mg/mL phosphatase substrate in 1 M Tris aminomethane, Sigma) was added to each well. The plates were incubated at room temperature before reading the optical density at 405 nm on a Biotek plate reader.
Wen X., Mousa J.J., Bates J.T., Lamb R.A., Crowe JE J.r, & Jardetzky T.S. (2017). Structural basis for antibody cross-neutralization of respiratory syncytial virus and human metapneumovirus. Nature microbiology, 2, 16272.
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7

Quantifying mAb Lung Deposition

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To determine if our mAb was reaching the lungs of the animals we administered PhtD3 mAb as described above. Lungs were collected 24 hrs after mAb administration and rinsed in PBS to remove excess blood and then homogenized using gentleMACS M tube (Miltenyi Biotec). Homogenates were spun down at 4000xg for 10 minutes. Supernatants and serum were collected and serially diluted. 384-well plates were treated with 2 μg/ml of antigen in PBS for 1 h at 37 °C or overnight at 4 °C. Following this, plates were washed once with distilled water before blocking for 1 hr with 2% nonfat milk–2% goat serum in 0.05% PBS-Tween (PBS-T) (blocking buffer). Plates were washed with water three times before serially diluted samples in PBS were applied for 1 hr. Following this, plates were washed with water three times before application of 25 μL of secondary antibody (goat anti-human IgG Fc; Meridian Life Science) at a dilution of 1:4,000 in blocking solution. After incubation for 1 h, the plates were washed five times with PBS-T, and 25 μL of a PNPP (p-nitrophenyl phosphate) solution (1 mg/ml PNPP in 1 M Tris base) was added to each well. The plates were incubated at room temperature for 1 hr before reading the optical density at 405 nm on a BioTek plate reader.
Gingerich A.D., Royer F., McCormick A.L., Scasny A., Vidal J.E, & Mousa J.J. (2022). Synergistic protection against secondary pneumococcal infection by human monoclonal antibodies targeting distinct epitopes. Journal of immunology (Baltimore, Md. : 1950), 210(1), 50-60.
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8

Recombinant-Protein Capture ELISA Protocol

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For recombinant-protein capture ELISAs, 384-well plates were treated with 2 μg/ml of antigen in PBS for 1 h at 37°C or overnight at 4°C. Following this, plates were washed once with distilled water before blocking for 1 h with 2% nonfat milk–2% goat serum in 0.05% PBS-Tween (PBS-T) (blocking buffer). Plates were washed with water three times before serially diluted primary MAbs in PBS were applied for 1 h. Following this, plates were washed with water three times before application of 25 μl of secondary antibody (goat anti-human IgG Fc; Meridian Life Science) at a dilution of 1:4,000 in blocking solution. After incubation for 1 h, the plates were washed five times with PBS-T, and 25 μl of a PNPP (p-nitrophenyl phosphate) solution (1 mg/ml PNPP in 1 M Tris base) was added to each well. The plates were incubated at room temperature for 1 h before reading the optical density at 405 nm on a BioTek plate reader. Binding assay data were analyzed in GraphPad Prism using a nonlinear regression curve fit and the log(agonist)-versus-response function to calculate the binding EC50s.
Huang J., Gingerich A.D., Royer F., Paschall A.V., Pena-Briseno A., Avci F.Y, & Mousa J.J. (2021). Broadly Reactive Human Monoclonal Antibodies Targeting the Pneumococcal Histidine Triad Protein Protect against Fatal Pneumococcal Infection. Infection and Immunity, 89(5), e00747-20.
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9

ELISA Assay for Pneumococcus Antibody Binding

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For ELISAs, 384-well plates were treated with 15 μl (∼107 CFU) of whole-cell pneumococcus in PBS in each well. Cell density was checked by microscope to ensure that a confluent layer of pneumococcus was present. The bacteria were then fixed with 15 μl of 4% paraformaldehyde in each well and placed on a plate shaker for 10 min to mix. The 384-well plates were incubated at 4°C for 24 to 48 h to allow the bacteria to fix to the bottom of the plates. Following this, the plates were washed once with 75 μl of PBS-T in each well. The plates were then blocked with 2% blocking buffer for 1 h at room temperature and washed three times with PBS-T. Next, 25 μl of serially diluted primary antibodies was applied to the wells for 1 h at room temperature; then, plates were washed with PBS-T three times. Following this, 25 μl of secondary antibody (goat anti-human IgG Fc; Meridian Life Science) at a 1:4,000 dilution in blocking buffer was applied to each well for 1 h at room temperature. After the plates were washed with PBS-T five times, 25 μl of PNPP (p-nitrophenyl phosphate) solution (1 mg/ml PNPP in 1 M Tris base) was added to each well for 1 h at room temperature. After 1 h the optical density was read at 405 nm on a BioTek plate reader. Binding assay data were analyzed in GraphPad Prism.
Huang J., Gingerich A.D., Royer F., Paschall A.V., Pena-Briseno A., Avci F.Y, & Mousa J.J. (2021). Broadly Reactive Human Monoclonal Antibodies Targeting the Pneumococcal Histidine Triad Protein Protect against Fatal Pneumococcal Infection. Infection and Immunity, 89(5), e00747-20.
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