Analysis DNA samples were denatured at 95°C for 5 min and spotted onto Hybond-N+ nitrocellulose membranes (GE Healthcare). After vacuum-baked at 80°C for 2 h and ultraviolet cross-linking for 15 min, membranes were blocked with 5% non-fat milk in TBST for 1 h and incubated with antibodies against 5mC (Epigentek, 1:1000) and against 5hmC (Active motif, 1:10,000) overnight at 4°C. The antibodies were diluted in blocking buffer. Membranes were washed three times with TBST, further incubated with HRP-conjugated goat anti-mouse secondary antibody (Beyotime, 1:1000) and HRP-conjugated goat anti-rabbit secondary antibody (Beyotime, 1:1000) at 37°C for 1 h respectively. Finally, the membranes were washed with TBST again, and detected using an ECL detection system (Thermo Scientific).
Hrp conjugated goat anti mouse secondary antibody
Manufactured by Beyotime
Sourced in China
The HRP-conjugated goat anti-mouse secondary antibody is a laboratory reagent used for the detection and quantification of mouse primary antibodies in various immunoassays. It consists of a goat-derived antibody that binds to mouse immunoglobulins, conjugated with the enzyme Horseradish Peroxidase (HRP). This allows for the amplification and visualization of the target mouse antibody signal in applications such as Western blotting, ELISA, and immunohistochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Hybond n nitrocellulose membrane, by GE Healthcare (1 mentions)
Ecl detection system, by Thermo Fisher Scientific (1 mentions)
Hrp conjugated goat anti rabbit secondary antibody, by Beyotime (1 mentions)
Mouse anti glyceraldehyde 3 phosphate dehydrogenase gapdh, by Santa Cruz Biotechnology (1 mentions)
Imagequant las 4000 mini system, by GE Healthcare (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (1 mentions)
Mouse anti human hif 1α monoclonal antibody, by BD (1 mentions)
Mouse anti human β actin antibody, by Beyotime (1 mentions)
Hrp conjugated goat anti rabbit secondary antibody, by Cell Signaling Technology (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Ly294002, by Beyotime (1 mentions)
Ab189403, by Abcam (1 mentions)
Ab44976, by Abcam (1 mentions)
Ab71973, by Abcam (1 mentions)
Ab182651, by Abcam (1 mentions)
Enhanced chemiluminescence western blotting detection system, by Merck Group (1 mentions)
Sc 493, by Santa Cruz Biotechnology (1 mentions)
5 protocols using hrp conjugated goat anti mouse secondary antibody
1
Quantifying DNA Methylation and Hydroxymethylation
2
Antibody and Cytokine Assay for Pseudorabies Virus Immunization
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Antibody titers against PRV were detected with indirect ELISA in the sera of immunized mice. Briefly, PRV-AH virions (purified by ultracentrifugation) were diluted appropriately with carbonate buffer solution to 1:800, which was confirmed by the preliminary experiment, and added to 96-well microplates. The microplates were placed at 37 °C for 1 h, then overnight at 4 °C. The coating solution was removed, and the microplates were washed three times with PBST. Then, 5% skim milk powder was added to the microplates and they were incubated at 37 °C for 2 h. After washing, the sera with a dilution of 1:200 were added to the microplates and incubated at 37 °C for 30 min, followed by incubation with HRP-conjugated goat anti-mouse secondary antibody (Beyotime, Shanghai, China, 1:250) for 30 min at 37 °C. Tetramethylbenzidine (TMB) substrate was added for a color reaction, followed by stopping with 1 mol/L H2SO4. The optical density (OD) was measured at 450 nm, and a statistical analysis was carried out.
The cytokines IFN-γ and IL-4 in the sera of immunized mice were detected using a commercial mouse cytokine immunoassay ELISA kit (Meimian, Yancheng, China) according to the manufacturer’s instructions.
The cytokines IFN-γ and IL-4 in the sera of immunized mice were detected using a commercial mouse cytokine immunoassay ELISA kit (Meimian, Yancheng, China) according to the manufacturer’s instructions.
3
Measuring Wnt-3 Protein Levels in Np65-KO Mice
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Adult Np65-KO and WT mice (4 months old, n = 3/genotype) were used for western blotting. Briefly, animals were decapitated after deep anesthesia with 1% pentobarbital sodium (30 mg/kg i.p.). Forebrains were collected and frozen in nitrogen and then stored at −80°C until use. The total proteins were extracted using RIPA lysis buffer (P0013B, Beyotime) with 1 mmol/L PMSF (ST506, Beyotime). Protein concentrations were measured using the BCA Protein Assay Kit (P0010, Beyotime, Jiangsu, China), then 10 ng of total protein was separated by SDS-PAGE and transferred to the PVDF membrane. After blocking with 5% bovine serum albumin (BSA), the membranes were incubated overnight at 4°C with primary antibody against Wnt-3 (1:1,000, Santa Cruz Biotechnology, Dallas, TX) and mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:1,000, Santa Cruz Biotechnology, Dallas, TX). Subsequently, the membranes were incubated with HRP-conjugated goat anti-mouse secondary antibody (1:1,000, Beyotime) for 2 h at room temperature. The labeled proteins were detected by using the ImageQuant LAS 4000mini system (GE Healthcare Life Sciences, Chicago, IL). The protein levels were normalized to that of GAPDH from three independent experiments.
4
Transcription Factor Expression Analysis
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Transfection reagent (LipofectamineTM 2000) was obtained from Invitrogen Corporation (Carlsbad, CA). Complete protease inhibitor cocktail was from Roche (Mannheim, Germany). Mouse anti-human HIF-1α monoclonal antibody was from BD Transduction Laboratories (San Diego, CA, USA). Rabbit anti-human ZEB1, Snail1, Slug, and Twist1 primary antibodies and goat anti-rabbit HRP-conjugated secondary antibody were purchased from Cell Signaling Technology (Beverly, MA, USA). Mouse anti-human β-actin antibody, goat anti-mouse HRP-conjugated secondary antibody, LY294002, and lysis buffer were purchased from Beyotime Biotechnology Corporation, Shanghai (Shanghai, China). PrimeScriptTM RT reagent Kit with gDNA Eraser (Perfect Real Time) and SYBR®Premix Ex TaqTM II (Tli RNaseH Plus) qPCR reagent Kit were purchased from TaKaRa Biotechnology (Dalian) Co., LTD (Dalian, China).
5
Protein Expression Analysis in HUVECs
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HUVECs were homogenized in RIPA buffer (Beyotime). Proteins were extracted according to the manufacturer׳s instruction (Beyotime Biotech). After protein quantification, proteins (50 µg) were separated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis gels (Mini-PROTEAN® 3 Dodeca™ Cell, Bio-Rad Laboratories) and subsequently transferred onto polyvinylidene fluoride membranes [21] . After blocking, the membranes were incubated with primary antibody against PI3K (1:1000; Ab189403, Abcam, Cambridge, MA, USA), p-PI3K (1:800; Ab182651, Abcam), Akt (1:1000; #9272, Cell Signaling Technology [CST], Danvers, MA, USA), p-Akt (1:1000; #9271, CST), Caspase-3 (1:500; Ab44976, Abcam), Bax (1:300; Sc-493, Santa Cruz Biotechnology, Dallas, TX, USA), Bcl2 (1:300; Sc-492, Santa Cruz Biotechnology), PHLPP2 (1:5000; Ab71973, Abcam), or GAPDH (1:2000; #5174, CST) at 4°C overnight, followed by incubation with goat anti-rabbit, donkey anti-goat, or goat anti-mouse HRP-conjugated secondary antibody (1:1000; Beyotime Biotech) for 2 h at room temperature. Immunoreactive proteins were visualized using the enhanced chemiluminescence Western blotting detection system (Millipore, Billerica, MA, USA) as described previously [22, 23] . Staining intensity of the bands was measured using a gel image analysis system (Tanon-5200, Shanghai, China).
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