gE (2 μg/mL) was used to coat 96-well plates (Corning) at 4 °C overnight. After blocking with 5% (w/v) skim milk at 37 °C for 1 h, the plates were incubated with serial dilutions of mouse sera at 37 °C for 1 h. Bound antibodies were detected with goat anti-mouse IgG-horseradish peroxidase (HRP) conjugate (1:5000, Bio-Rad, Hercules, CA, USA) as a secondary antibody. Ten minutes after the addition of the substrate 3,3′,5,5′-tetramethylbenzidine (BD), 1 mol/L phosphoric acid was added to terminate the reaction. The absorbance at 450 nm was detected with a spectrophotometer (BioTek Instruments, Inc., Winooski, VT, USA). IgG titers were defined as end-point dilutions showing cutoff signals above OD450 = 0.1, and IgG titers lower than 100 were defined as 100 for calculations.
Goat anti mouse igg horseradish peroxidase hrp conjugate
Manufactured by Bio-Rad
Sourced in United States
Goat anti-mouse IgG-horseradish peroxidase (HRP) conjugate is a secondary antibody that binds to mouse immunoglobulin G (IgG) and is conjugated to the enzyme horseradish peroxidase. It can be used in various immunoassay techniques to detect and quantify mouse IgG in samples.
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4 protocols using goat anti mouse igg horseradish peroxidase hrp conjugate
1
ELISA for Mouse Antibody Titers
2
Determination of IgG Subtypes in Mice
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At day 8 pi, blood was withdrawn from retro‐orbital cavity of mock‐infected or infected mice and IgG subtypes were determined using an ELISA technique. Polystyrene microtiter 96‐well plates (Nunc‐Immuno™ MicroWell™ plates, Sigma Aldrich, United States) were coated with a solution of 2.5 µg/mL goat anti‐mouse IgG (IgG1 or IgG2a) (Bio‐Rad, Richmond, CA) in 0.135 mol/L NaCl pH 7.2 and incubated at 37°C overnight. The plates were washed four times with 0.1% Tween 20 in 10 mmol/L NaH2PO4, 154 mmol/L NaCl, pH 7.0 (TPBS) and blocked with 5% bovine serum albumin (BSA) in PBS for 1 hour at 37°C. After four washings in TPBS, serial dilutions of murine plasma in 50 mmol/L sodium borate, pH 8.5, were added and incubated for 1 hour at 37°C. After four washings in TPBS, 100 µL of goat anti‐mouse IgG‐horseradish peroxidase (HRP) conjugate (Bio‐Rad), diluted 1:1000 in PBS, were added. After incubation for 1 hour at 37°C, serum IgG subtypes were determined using a color development solution containing 2.2 mmol/L o‐phenylenediamine. Absorbance was measured at 492 nm on a Model 2550 enzyme immunoassay (EIA) reader.
3
ELISA Protocol for Antibody Quantification
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After whole blood clotting at 4 °C overnight, immunized sera were collected after centrifugation at 3000 rpm for 20 min. gE (2 µg/mL, supplied by AtaGenix Laboratory Co., Ltd., Wuhan, China) was precoated on 96-well plates (Corning, Corning, NY, USA) at 4 °C overnight. After blocking with 5% (w/v) skim milk at 37 °C for 1 h, plates were incubated with 2 serial dilutions of mouse sera at 37 °C for 1 h. Bound antibodies were detected with goat anti-mouse IgG-horseradish peroxidase (HRP) conjugate (1:10,000, Bio-Rad, Hercules, CA, USA) as a secondary antibody. Ten min after the addition of the substrate 3,3′,5,5′–tetramethylbenzidine (TMB, BD, USA), 2 mol/L sulfuric acid was added to terminate the reaction. The absorbance at 450 nm was detected with a spectrophotometer (BioTek Instruments, Inc., Winooski, VT, USA). IgG titers were defined as end-point dilutions showing cutoff signals above OD450 = 0.15, and IgG titers lower than 200 were defined as 200 for calculations.
4
Western Blot Immunodetection Protocol
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The nitrocellulose membranes were blocked with 3% BSA in Tris-buffered saline with 0.1% Tween-20 (TBS-T) for 1 h and then incubated for 1 h with primary antibody. The following primary antibodies were used: polyclonal anti-SopB antibody (1:1,000; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), polyclonal anti-p65 antibody (1:500; Santa Cruz Biotechnology Inc.), polyclonal anti-phospho-Akt antibody (1:500; Abcam Inc., Cambridge, MA, USA), polyclonal anti-Akt antibody (1:500; Abcam Inc.), monoclonal anti-phospho-p38 MAPK (Thr180/Tyr182) (1:1000; Cell Signaling Technology, Beverly, MA, USA), polyclonal anti-p38 MAPK antibody (1:1000; Cell Signaling Technology), polyclonal anti-lamin-B antibody (1:1,000; Santa Cruz Biotechnology Inc.), polyclonal anti-GAPDH antibody (1:3,000; Abcam Inc.), and polyclonal anti-beta actin antibody (1:5000; Abcam Inc.). After washing the membranes three times with TBST, the secondary antibody of goat anti-mouse IgG horseradish peroxidase (HRP) conjugate, goat anti-rabbit IgG HRP conjugate, or rabbit anti-goat IgG HRP conjugate (1:3,000; Bio-Rad) was incubated with the membranes for 1 h. The blots were developed using a BM chemiluminescence blotting substrate (POD; Roche, Mannheim, Germany).
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