Cell lysates obtained as described above were separated using 12% SDS-PAGE and transferred onto a PVDF membrane (Millipore, USA). Then, the membrane was incubated with PBST containing 5% skim milk for 2 h at room temperature, followed by incubation with a primary antibody (anti-HA tag rabbit polyclonal antibody, 1:2000, Proteintech, USA; anti-S1 rabbit monoclonal antibody, 1:500, Future Biotech, China; or anti-RBD mouse monoclonal antibody 13E10D5, 1:1000, Genscript, China) at 4 °C overnight and four washes with TBST. Thereafter, the membrane was reacted with an HRP-conjugated goat anti-rabbit IgG (H + L) (1:10,000, Zsbio, China) or HRP-conjugated goat anti-mouse IgG (H + L) secondary antibody (1:5000, Bioss, China) at room temperature for 1 h and washed with PBST four times. Subsequently, the bands were visualized with ECL reagent (Thermo Fisher Scientific, USA).
Hrp conjugated goat anti rabbit igg h l
Manufactured by ZSGB-BIO
Sourced in China
HRP-conjugated goat anti-rabbit IgG (H + L) is a laboratory reagent used for the detection and quantification of rabbit immunoglobulin G (IgG) in various immunoassays. The product consists of goat-derived antibodies that have been conjugated to horseradish peroxidase (HRP), a commonly used enzyme label. The H + L designation indicates that the antibodies recognize both the heavy and light chains of rabbit IgG.
Automatically generated - may contain errors
Lab products found in correlation
Hrp conjugated goat anti mouse igg h l, by ZSGB-BIO (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Anti ha tag rabbit polyclonal antibody, by Proteintech (1 mentions)
Ecl reagent, by Thermo Fisher Scientific (1 mentions)
Pierce bca protein quantitation assay kit, by Thermo Fisher Scientific (1 mentions)
Ecl kit, by Merck Group (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Anti tlr4, by Affinity Biosciences (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Ripa lysis buffer, by Applygen (1 mentions)
3 protocols using hrp conjugated goat anti rabbit igg h l
1
Western Blotting Analysis of Viral Proteins
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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