ELISA plates were coated overnight at 4 °C with 100 μL of inactivated PRV HeN1 strain (1 μg/mL) diluted in bicarbonate coating buffer (1.59 g/L Na2CO3 and 2.93 g/L NaHCO3, pH = 9.6). The wells of the plate were washed four times with PBST (PBS containing 0.05% Tween-20) and then blocked with 5.0% skimmed milk in PBST for 2 h at 37 °C. After gradient dilution of mouse serum, 100 μL was added per well, and the plates were incubated for 30 min at 37 °C. The wells were then washed four times with PBST, and 100 μL of horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (H + L) (Zsbio, Beijing, China) was added at a dilution of 1:5000 for 30 min at 37 °C. Next, plates were washed four times with PBST, and ELISAs were developed using 100 μL of TMB single solution (Life Technologies) for 15 min at 37 °C. The reactions were then terminated with 50 μL/well 2 M H2SO4, and the absorbance was measured at 450 nm.
Hrp conjugated goat anti mouse igg h l
Manufactured by ZSGB-BIO
Sourced in China
The HRP-conjugated goat anti-mouse IgG (H + L) is a secondary antibody used in various immunoassays and immunodetection techniques. It is produced by conjugating horseradish peroxidase (HRP) to goat-derived antibodies that recognize both the heavy (H) and light (L) chains of mouse immunoglobulin G (IgG).
Automatically generated - may contain errors
Lab products found in correlation
Hrp conjugated goat anti rabbit igg h l, by ZSGB-BIO (2 mentions)
Tmb single solution, by Thermo Fisher Scientific (1 mentions)
Ecl kit, by Merck Group (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Pierce bca protein quantitation assay kit, by Thermo Fisher Scientific (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Anti tlr4, by Affinity Biosciences (1 mentions)
Ripa lysis buffer, by Applygen (1 mentions)
3 protocols using hrp conjugated goat anti mouse igg h l
1
Quantitative Anti-PRV IgG ELISA Protocol
2
Western Blot Analysis of Synaptic Proteins
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The PFC protein were solubilized in RIPA lysis buffer with protease inhibitors. The protein concentrations were analyzed through Pierce BCA protein quantitation assay kit (Thermo Fisher Scientific, Massachusetts, United States). The proteins were separated with 12% SDS-PAGE gel and then transferred by electroblotting onto a 0.22 μm polyvinylidene fluoride (PVDF) membrane (Bio-Rad, United States). After blocking in 5% skim milk for 2 h at room temperature, the membrane was incubated with primary antibody (anti-syntaxin1A; anti-synaptophysin, anti-Tubulin, proteintech, United States) at 4°C overnight. Furthermore, the blot was incubated with HRP-conjugated goat Anti-Rabbit IgG (H + L) or HRP-conjugated goat Anti-Mouse IgG (H + L) (ZSGB-BIO, China) for 2 h at room temperature. Blots were developed using an ECL Kit (Millipore, United States) following the manufacturer’s protocol.
3
Western Blot Analysis of Nampt and TLR4 Expression
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Tissue samples were homogenized using liquid nitrogen and lysed in RIPA lysis buffer (Applygen Technologies Inc.) for 30 min. Lysates were then centrifuged at 4˚C for 10 min at 12,000 x g, and the supernatants were collected. Total proteins were separated by SDS-polyacrylamide gel electrophoresis, and transferred onto PVDF membranes (Millipore). The membranes were incubated with the appropriate primary antibodies, including rabbit polyclonal anti-Nampt (cat. no. DF6059; Affinity Biosciences; dilution, 1:500), rabbit polyclonal anti-TLR4 (cat. no. bs-20594R; Beijing Biosynthesis Biotechnology Co., Ltd.; dilution, 1:1,000) and mouse monoclonal anti-GAPDH (cat. no. TA-08; ZSBIO; dilution, 1:2,000) at 4˚C overnight, followed by incubation with HRP-conjugated goat anti-rabbit IgG (H+L) (cat. no. ZB-2301; ZSBIO; dilution, 1:2,000) or HRP-conjugated goat anti-mouse IgG (H+L) (cat. no. ZB-2305; ZSBIO; dilution, 1:2,000) at 4˚C for 2 h. Signals were visualized with the SuperSignal® West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Inc.), and the band density was quantified using Quantity One software (version 4.6.9; Bio-Rad Laboratories, Inc.).
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