100 ng of hyperglycosylated or wild-type recombinant hemagglutinin proteins were adhered to high-capacity binding, clear bottom, 96-well plates (Corning) in PBS overnight at 4°C. Plates were blocked with 1% BSA in PBS containing 0.1% Tween-20 (PBS-T) for 1 hour at room temperature (RT), shaking. Blocking solution was removed, and plates were incubated with 10-fold dilutions of structurally characterized IgGs in PBS for 1 hour at RT, shaking. After 1 hour, plates were washed three times with PBS-T, then incubated with 1:20,000 diluted HRP-conjugated anti-human IgG (Abcam) for 1 hour at RT. Plates were washed three times with PBS-T. 1-Step ABTS substrate (ThermoFisher) was added to plates for 20-30 mins at RT, then stopped with 100 µL of 1% SDS. Absorbance measurements from each well at 410 nm were recorded using a plate reader. Data were plotted using Prism 9 (Graphpad) and relative binding was determined.
Hrp conjugated anti human igg
Manufactured by Abcam
HRP-conjugated anti-human IgG is a secondary antibody that is conjugated with horseradish peroxidase (HRP). It is designed to detect and bind to human immunoglobulin G (IgG) in various immunoassays and immunochemical techniques.
Automatically generated - may contain errors
Lab products found in correlation
Abts substrate, by Thermo Fisher Scientific (1 mentions)
Prism 9, by GraphPad (1 mentions)
Spectramax id3 plate reader, by Molecular Devices (1 mentions)
Hrp conjugated anti mouse igg, by Abcam (1 mentions)
Ab6759, by Abcam (1 mentions)
Hrp conjugated goat anti mouse igg, by Abcam (1 mentions)
Semi dry transfer unit, by Bio-Rad (1 mentions)
4 protocols using hrp conjugated anti human igg
1
Recombinant Hemagglutinin Protein Binding
2
Serum ELISA Protocol for Antibody Detection
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Serum
ELISAs were performed as previously
described.14 (link) Briefly, plates were coated
with 100 μL of protein at 5 μg/mL overnight at 4 °C,
then blocked with 150 μL of 1% BSA in PBS-Tween for 1 h at room
temperature. Sera was added at a starting dilution of 1:40 with a
serial 5-fold dilution and incubated for 90 min at room temperature.
Plates were washed, and 150 μL of HRP-conjugated antimouse IgG
(Abcam) or HRP-conjugated antihuman IgG (Abcam) was added at 1:20,000.
After a 1 h secondary incubation at room temperature, the plates were
washed and 150 μL of 1× ABTS development solution (Thermo
Fisher) was added. Plates were developed for 30 min at room temperature
before stopping with 100 μL of 1% SDS to read on a SpectraMaxiD3
plate reader (Molecular Devices) for absorbance at 405 nm.
ELISAs were performed as previously
described.14 (link) Briefly, plates were coated
with 100 μL of protein at 5 μg/mL overnight at 4 °C,
then blocked with 150 μL of 1% BSA in PBS-Tween for 1 h at room
temperature. Sera was added at a starting dilution of 1:40 with a
serial 5-fold dilution and incubated for 90 min at room temperature.
Plates were washed, and 150 μL of HRP-conjugated antimouse IgG
(Abcam) or HRP-conjugated antihuman IgG (Abcam) was added at 1:20,000.
After a 1 h secondary incubation at room temperature, the plates were
washed and 150 μL of 1× ABTS development solution (Thermo
Fisher) was added. Plates were developed for 30 min at room temperature
before stopping with 100 μL of 1% SDS to read on a SpectraMaxiD3
plate reader (Molecular Devices) for absorbance at 405 nm.
3
Immunoblotting of Flag-GlyRS in HEK293
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Human HEK293 cells overexpressing Flag-tagged GlyRS were lysed in RIPA buffer and separated by 10% SDS-PAGE and transferred to PVDF membrane. We tested the membrane in immunoblotting with an anti-EJ positive serum as reference serum diluted in 1:1000. The secondary antibody was HRP-conjugated anti-human IgG (1:5000, ab6759, Abcam).
4
Western Blot Analysis of Recombinant Proteins
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The expressed recombinant protein was boiled for 10 min at 100 °C in SDS-loading buffer. The sample was subjected to SDS-PAGE electrophoresis on 12% acrylamide gel. Protein samples were transferred onto PVDF membranes using semidry transfer unit (Bio-Rad, Corning Inc., NY, USA) and the membranes were then blocked at 37 °C water-jacketed incubator for 2 h with 5% (w/v) skim milk diluted in TBST. After being washed three times with TBST, PVDF membranes were incubated for 2 h at 37 °C with mouse anti-His monoclonal antibody (1:500) (CWBIO, CW0082) or mixed TB-positive serum (1:200) diluted in TBST. After being washed, PVDF membranes were incubated at 37 °C for 1 h with HRPconjugated goat anti-mouse IgG (1:10000) (Abcam, ab6728) or HRP-conjugated anti-human IgG (1:5000) (Abcam, ab6728), respectively. In the end, PVDF membranes was developed with electrochemiluminescence (ECL) using a chemiluminescence imaging system.
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