Supernatant containing the αvβ3-Fc fusion protein (500 µl) was incubated with an excess of protein A-sepharose beads (50 µl) during 1 h at 4°C. The solution was then centrifuged at 2000 rpm × 5 min. This process was repeated four times, to ensure complete depletion of the fusion protein. Each supernatant and precipitate obtained was evaluated by Western blotting using anti-human IgG (Fc specific)-HRP antibody (1:2000; Sigma). After the fourth incubation/centrifugation cycle, the level of the αvβ3-Fc protein was undetectable in the precipitate (precipitate 4); thus the fourth supernatant was used in the miniTweezers experiments.
Anti human igg fc specific hrp antibody
Manufactured by Merck Group
Sourced in United States
The Anti-human IgG (Fc specific)-HRP antibody is a biotechnology tool used in various immunoassay techniques. The antibody is specific to the Fc region of human immunoglobulin G (IgG) and is conjugated with horseradish peroxidase (HRP), an enzyme that can be used for colorimetric or chemiluminescent detection.
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Lab products found in correlation
3 protocols using anti human igg fc specific hrp antibody
1
Depletion of αvβ3-Fc Fusion Protein
2
SARS-CoV-2 Antibody Quantification
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HM specific IgA, IgM, and IgG antibody levels targeted to the receptor‐binding domain (RBD) of the SARS‐CoV‐2 spike protein were included from the previous COVID‐19 study.[3 ] Briefly, the analysis was carried out as follows: 96‐well immunoplates (Corning, Kennebunk, ME, USA) were coated with 2 µg mL−1 of RBD overnight at 4 °C, blocked in 3 % (w/v) milk powder in PBS containing 0.1% Tween 20. Samples were incubated at a 1:4 dilution, incubated for 2 h and after three washing steps incubated with antihuman IgA (α‐chain‐specific) horseradish peroxidase (HRP) antibody (Thermo‐Fisher Scientific, Bremen, Germany; # A18781; 1:6000), antihuman IgM (µ‐chain‐specific) HRP antibody (Sigma‐Aldrich, St. Louis, MO, USA; # A0420; 1:4000), and antihuman IgG (Fc‐specific) HRP antibody (Sigma‐Aldrich, St. Louis, MO, USA; # A0170; 1:4000) for 1 h. Plates were developed with 3,3′,5,5′‐Tetramethylbenzidine and reactions were stopped with 50 µL of 2 M sulfuric acid. Absorbance at 450 nm was read in a ClarioStar (BMG Labtech, Ortenberg, Germany) microplate reader.
3
VNAR Protein Binding Detection ELISA
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A sandwich ELISA format was applied to test the ability of lead VNAR F3 to detect SEED homodimer present in solution, mimicking intended application as a bioprocessing reagent.
ELISA plate was coated overnight at 4°C with the VNAR protein as a capture ligand at 2.5 µg/mL in 1x PBS, 100 µL/well. SEED protein was added at top concentration of 10 µg/mL and serially diluted in 1x PBS, incubated for 1 h at room temperature. Binding was detected indirectly with 1/1,000 dilution of goat anti-human IgG (Fc specific) antibody (Sigma-Aldrich, 2136) and rabbit anti-goat IgG-HRP (Sigma-Aldrich, A8919).
The VNAR proteins were further evaluated for their applicability as detection reagents capturing target from a heterogeneous mix of SEED proteins in solution. VNARs were immobilised on an ELISA plate at a sub-saturating concentration in 1x PBS, 100 µL/well, incubated overnight at 4°C. SEED proteins were pre-mixed at different proportions of homodimer and heterodimer in a final combined concentration of 5 µg/mL prior to their addition to a blocked ELISA plate at 100 µL/well in duplicate, then incubated for 1 h at room temperature and washed as standard. Binding signal was generated following standard protocols and via detection of bound SEED protein with a 1/200 dilution of anti-human IgG (Fc specific)-HRP antibody (Sigma-Aldrich, A0170).
ELISA plate was coated overnight at 4°C with the VNAR protein as a capture ligand at 2.5 µg/mL in 1x PBS, 100 µL/well. SEED protein was added at top concentration of 10 µg/mL and serially diluted in 1x PBS, incubated for 1 h at room temperature. Binding was detected indirectly with 1/1,000 dilution of goat anti-human IgG (Fc specific) antibody (Sigma-Aldrich, 2136) and rabbit anti-goat IgG-HRP (Sigma-Aldrich, A8919).
The VNAR proteins were further evaluated for their applicability as detection reagents capturing target from a heterogeneous mix of SEED proteins in solution. VNARs were immobilised on an ELISA plate at a sub-saturating concentration in 1x PBS, 100 µL/well, incubated overnight at 4°C. SEED proteins were pre-mixed at different proportions of homodimer and heterodimer in a final combined concentration of 5 µg/mL prior to their addition to a blocked ELISA plate at 100 µL/well in duplicate, then incubated for 1 h at room temperature and washed as standard. Binding signal was generated following standard protocols and via detection of bound SEED protein with a 1/200 dilution of anti-human IgG (Fc specific)-HRP antibody (Sigma-Aldrich, A0170).
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