The expression of the HA protein was detected using Western blot analysis. Briefly, CEF cells infected with different passages of recombinant NDVs were prepared in a 2×SDS lysis buffer and subjected to SDS–PAGE, then transferred to a polyvinylidene difluoride (PVDF) membrane (Pall Corporation, NY, USA) under 200 mA for 1.5 h. The membranes were blocked with 3% bovine serum albumin (BSA) in phosphate-buffered saline with 0.5% Tween-20 (PBST) for 4 h at 4 °C and then incubated with the primary antibody overnight at 4 °C; after washing with PBST 3 times, the membranes were incubated with a secondary antibody for 6 h at 4 °C. The specific bands were captured using an enhanced ECL reagent (Thermo Fisher Scientific, Waltham, USA).
Enhanced ecl reagent
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Enhanced ECL reagent is a chemiluminescent detection system used for the sensitive and quantitative analysis of proteins in Western blot applications. The reagent provides a stable, high-intensity signal that can be detected using standard laboratory equipment.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Pall Corporation (1 mentions)
Goat anti bat igg heavy and light chain antibody a140 118b, by Fortis Life Sciences (1 mentions)
Donkey anti goat igg hrp sc 2056, by Santa Cruz Biotechnology (1 mentions)
Cl xposure film, by Thermo Fisher Scientific (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Avantor (1 mentions)
Total gsk3β, by Cell Signaling Technology (1 mentions)
Hdac3, by Cell Signaling Technology (1 mentions)
Hrp conjugated secondary antibody, by Abcam (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Gapdh, by Abcam (1 mentions)
Ripa buffer, by Vazyme (1 mentions)
3 protocols using enhanced ecl reagent
1
Western Blot Analysis of HA Protein
2
Bat-specific IgG Dot Blot Analysis
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Bat-specific IgG was analysed via dot blot. Bat-mice were cheek bled post NP-KLH immunizations, sera samples were dotted onto nitrocellulose membrane (Pore size 0.45 µm; BIO-RAD, USA) and left to dry for 30 minutes. Blocking buffer (Tris-buffered saline with polysorbate 20 (TBS-T) and 5% low-fat milk powder) was used to block non-specific sites for 30 minutes at RT with agitation. After washing, membrane was incubated for 1 hour at RT with goat anti-bat IgG heavy and light chain antibody (A140-118B; Bethyl Laboratories, USA) in PBS-T containing 2.5% milk and for 1 hour with agitation, followed by 3 times 5 minutes wash with TBS-T. Secondary antibody incubation was done with donkey anti-goat IgG-HRP (SC-2056; Santa Cruz Biotechnology, USA) in PBS-T containing 2.5% milk for 1 hour at RT with agitation and subsequently washed with TBS-T for 3 times 5 minutes before reaction development using enhanced ECL reagent (Thermo Fisher Scientific, USA). Results were developed in the dark room for 10 seconds with CL-XPosureTM Film (Thermo Fisher Scientific, USA).
3
Western Blot Analysis of Key Signaling Proteins
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Samples were harvested with RIPA buffer (Vazyme) and processed with a lysis buffer containing phenylmethanesulfonyl fluoride (PMSF; Amresco) at 4°C. BCA protein assay kit (Beyotime Institute of Biotechnology, China) was used to measure protein concentration according to the manufacturer's protocol. Proteins were separated by 12% SDS-PAGE gels, then transferred to PVDF membranes (Bio-Rad, Calif) and blocked in 5% nonfat milk for 2 h. Subsequently the membranes were botted with specific primary antibodies against Ser9phospho GSK-3β (Ser9; 1:1,000, Cell Signaling), total GSK-3β (1:1,000, Cell Signaling), cleaved caspase-3 (1:1,000, Cell Signaling), HDAC3 (1:1,000, Cell Signaling), and GAPDH (1:1,000, Abcam) overnight at 4°C. The next day, membranes were washed 3 times with PBS-T for 10 min followed by incubation with the corresponding HRP-conjugated secondary antibodies (1:8,000, Abcam) for 1 h at 37°C. Membranes were then washed with PBS-T 3 times. The bands were visualized with enhanced ECL reagent (Thermo Scientific) and analyzed using Quantity One software. GAPDH was used as a control.
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