293T cells were transiently transfected with plasmids expressing soluble Envs using polyethylenimine following a standard protocol. Forty-eight to seventy-two hours post-transfection, cellular supernatants and lysates were collected, clarified and analyzed by reducing SDS-PAGE. To test for protein oligomerization, supernatants were analyzed by Blue Native PAGE following ThermoFisher’s protocol. Working dilutions for Western blotting were 1:2,000 goat anti-gp120 polyclonal antibody (ThermoFisher), 1:2,000 4E10 anti-gp41 antibody (Polymun Scientific, NIH AIDS Reagent Program), 1:10,000 mouse anti-β- actin (Abcam), 1:3,000 HRP-conjugated goat anti-human IgG (SantaCruz), 1:3,000 HRP-conjugated rabbit anti-goat IgG (ThermoFisher), and 1:10,000 HRP-conjugated goat anti-mouse IgG (ThermoScientific).
Hrp conjugated goat anti human igg
Manufactured by Santa Cruz Biotechnology
Sourced in United States
HRP-conjugated goat anti-human IgG is a secondary antibody used for the detection of human immunoglobulin G (IgG) in various immunoassays. The antibody is conjugated with Horseradish Peroxidase (HRP), an enzyme that can catalyze a colorimetric reaction, allowing for the visualization and quantification of target IgG.
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Lab products found in correlation
Ecl kit, by Thermo Fisher Scientific (2 mentions)
Hrp conjugated goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Blue nativepage, by Thermo Fisher Scientific (1 mentions)
Goat anti gp120 polyclonal antibody, by Thermo Fisher Scientific (1 mentions)
Hrp conjugated goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Mouse anti β actin, by Abcam (1 mentions)
Pcm 2000 confocal microscope, by Nikon (1 mentions)
7 protocols using hrp conjugated goat anti human igg
1
Transient Transfection of Soluble Envs
2
Autoantibody Detection by Western Blot
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Serum samples that were determined to contain autoantibodies by ELISA were further tested by immunoblotting to confirm the immunoreactivity of the sera. The purified recombinant proteins were transferred to a nitrocellulose membrane and then incubated with patient sera diluted 1 : 200, and finally incubated with HRP-conjugated goat anti-human IgG diluted 1 : 10000 (Santa Cruz Biotechnology, Inc. Santa Cruz, CA, USA). Positive signals were captured by autoradiography using chemiluminescence (Piece Biotechnology, Rockford, IL), according to the manufacturer's instructions. To confirm specificity of Western blotting, sera at the above dilutions were absorbed with recombinant protein (final concentration: 0.03 μg/mL) overnight at 4°C, the mixture was centrifuged and the supernatant was examined in Western blotting.
3
Immunoblotting Confirmation of Autoantibodies
4
Western Blot Analysis of Recombinant MDM2
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Denatured recombinant MDM2 protein was electrophoresed on 10% SDS-PAGE and transferred to nitrocellulose membranes. After blocking in PBS with 5% nonfat milk and 0.05% Tween-20 for 1 h at room temperature, the nitrocellulose membrane was incubated overnight with 1 : 200 dilution of human sera at 4°C. HRP-conjugated goat anti-human IgG (Santa Cruz, USA) was then applied as secondary antibody at a 1 : 10,000 dilution. The immunoreactive bands were detected by ECL kit according to the manufacturer's instructions (Thermo Scientific, Waltham, USA).
5
Quantification of H5N1 HA-stalk Binding
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Sf9 cells (8 × 105) plated in a 2.5-cm culture dish in a synthetic SFX medium were infected with the H5N1 HA-stalk baculovirus at an MOI of 5. At 48 h post-infection, cells were washed with PBS and fixed with 70% cold methanol for 20 min. Next, fixed Sf9 cells were washed twice with PBS buffer and incubated for 1 h at room temperature with 500 µL/well of anti-H1N1 Mab antibodies diluted 1:1000 or with FI6 hMab antibodies diluted 1:1000 with 1% Tween 80 in PBS. Next, 500 µL/well of secondary antibodies—HRP-conjugated goat anti-mouse IgG (cat. No sc-2005, Santa Cruz Biotechnology, Dallas, TX USA) and HRP conjugated goat anti-human IgG (cat. No sc-2907, Santa Cruz Biotechnology, Dallas, TX USA), diluted 1:2000 in PBS and 1% Tween 80 was used. The plate was incubated for 1 h and washed 3 times with 1 mL of washing buffer (Tris-buffered saline pH 7.2/0.1%BSA/0.05%Tween80). The reaction was developed with the NovaRED substrate kit (cat. No SK-4800, Vector Laboratories, Burlingame, CA, USA). The cells were visualized using a Nikon PCM2000 confocal microscope.
6
GRP78 Autoantibody Detection by ELISA
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The recombinant GRP78 protein was purchased from the Cloud-Clone Company. Anti-GRP78 autoantibodies in sera were detected by ELISA as previously described [14 (link)]. Briefly, proteins were diluted in coating buffer to a final concentration of 0.25 mg/ml and then were coated on 96-well microtiter plates (Fisher Scientific, Pittsburgh, PA) at 4°C overnight. The sera at 1 : 100 dilutions were added to the antigen-coated wells and incubated for 2 hours followed by five washes with PBST. HRP-conjugated goat anti-human IgG (Santa Cruz Biotechnology Inc., Dallas, TX) at 1 : 10,000 dilution and the substrate TMB (Sigma, Ronkonkoma, NY) were used as detecting reagents. The optical density (OD) at 450 nm was used for data analysis.
7
Quantifying Autoantibody Levels via ELISA
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The antigenic proteins α-enolase and VDAC1 were diluted to nal concentrations of 1.0 µg/ml. α-enolase and VDAC1 were incubated in 96-well microplates in coating buffer at 4°C overnight,then free unbound sites were blocked with 1% BSA for 2 h at 37°C. The plates were incubated with human serum samples diluted at 1:100 with PBS for 1 h at 37°C, then they were washed and incubated with 100 µl of horseradish peroxidase(HRP)-conjugated goat anti-human IgG diluted at 1:5000 with PBS(Santa Cruz Biotechnology) for 30 min at 37°C. TMB substrate was added and incubated for 5 min at room temperature,and this reaction was terminated by adding the stop solution. The absorbance was measured by spectrophotometry at a wavelength of 450 nm.
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