Proteins were extracted from 100 mg of main spike tissue immediately after sampling. Sampled tissue was ground on ice in 3 volumes of a protein extraction buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.25% TritonX-100, 1mM EDTA, 10 mM NaF, 1 mM Na3VO3, 0.25% NP-40, 1 mM PMSF, 1 × protease inhibitor EDTA-free (Roche Diagnostics GmbH, Mannheim, Germany), 10 µM MG132), centrifuged and frozen in the presence of loading buffer (0.35 M Tris-HCl (pH 6.8), 10.28% (w/v) SDS, 36% (v/v) glycerol, 5% beta-mercaptoethanol, 0.012% (w/v) bromophenol blue). Protein concentration was measured with a Bradford assay (Bradford Reagent, Bio-Rad) and 40 µg of proteins were run on 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel. Proteins were transferred to an Immobilon-P transfer membrane (Millipore) with Trans-Blot SD Semi-Dry Transfer Cell (Bio-Rad, Hercules, CA, USA) for 1 h. The membrane was blocked in 3% milk in phosphate-buffered saline (PBS). Proteins were detected and visualized with a PHY0960S Agrisera antibody (1:1,000), an AS:09602 Goat anti-Rabbit IgG (H&L) Agrisera secondary antibody (1:25,000), and Amersham ECL (Cytiva) reagents.
Amersham ecl
Manufactured by Cytiva
Sourced in Japan, United States
The Amersham ECL is a chemiluminescent detection system used for the identification and quantification of proteins in Western blotting applications. It provides a sensitive and reliable method for detecting low-abundance proteins.
Automatically generated - may contain errors
Lab products found in correlation
Immobilon p transfer membrane, by Merck Group (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
Trans blot sd semi dry transfer cell, by Bio-Rad (1 mentions)
Skim milk, by BD (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Las 3000, by Fujifilm (1 mentions)
Amersham hyperfilm ecl, by GE Healthcare (1 mentions)
Mouse monoclonal anti gfp antibody, by Roche (1 mentions)
4 protocols using amersham ecl
1
Protein Extraction and Detection from Spike Tissue
2
Optimized SDS-PAGE and Western Blotting Protocol
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Sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting were performed as previously described,16 (link),17 (link)) with minor modifications. Briefly, the protein samples were mixed with sample buffer (62.5 mM Tris-HCl, pH 6.8, 5.0% sucrose, 2.0% SDS, 0.1 M DTT, containing with 0.002% bromophenol blue) and loaded onto 10.5% SDS-PAGE. Each sample was transferred to a PVDF membrane (pore size: 0.45 µm; Merck Millipore, Tokyo, Japan). The membranes were incubated with 5.0% skim milk (Becton, Dickinson and Company, Franklin Lakes, NJ) in 20 mM Tris-HCl (pH 7.5) containing 150 mM NaCl and 0.1% Tween 20 (5.0% skim milk in T-TBS) for 60 min at room temperature to block non-specific reactions. Thereafter, membranes were incubated with IVIg (Glovenin® or Venilon®) as the primary antibody (1:100–500) diluted in blocking buffer or T-TBS for 60 min, washed three times with T-TBS, and incubated with HRP-conjugated anti-human IgG antibodies in T-TBS for 30 min. After washing with T-TBS, immunoreactivity was detected using Amersham ECL (Cytiva, Tokyo, Japan). Immunoreactive signals were captured using an LAS-3000 (Fujifilm, Tokyo, Japan). The size markers were aligned with the positive bands. The analyzed membranes were stained with 0.1% amido black 10B to visualize loaded total protein. All antibodies used are listed in Table 1 .
3
Yeast Cell Lysis and Immunoblotting
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Yeast cells were grown to log phase in SD media at 30 °C and 10 OD600/ml equivalents of cells were harvested and stored at –80 °C. Cells were thawed and lysed by vortexing in 100 μl of Thorner buffer (8 M Urea, 5% SDS, 40 mM Tris-Cl (pH 6.4), 1% beta-mercaptoethanol and 0.4 mg/ml bromophenol blue) with ~100 μl of acid-washed glass beads/sample at 70 °C for 5 min. Lysates were centrifuged at 14,000 RPM for 30 s and separated on 8% SDS-PAGE gels followed by western blotting with monoclonal mouse anti-GFP antibodies (11–814–460-001; Roche) or monoclonal mouse anti-PGK1 antibodies (AB_2532235, 22C5D8, Invitrogen), and secondary polyclonal goat anti-mouse antibodies conjugated to horseradish peroxidase (115–035-146; Jackson ImmunoResearch Laboratories). Blots were developed with Amersham ECL (RPN2232, Cytiva) chemiluminescent western blot detection reagent and exposed using Amersham Hyperfilm ECL (GE Healthcare).
4
Protein Extraction and Western Blot Analysis
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Proteins from mouse hepatic tissues were extracted and lysed in RIPA buffer as described previously [22 (link)]. The proteins were separated in a 5–20% SDS gradient gel electrophoresis and were further transferred to a nitrocellulose membrane via a wet transfer system. Saturation of membranes was done with polyvinyl alcohol (PVA) or TBS-Tween 0.1%-milk-5%. Membranes were then washed with TBS-Tween 0.1%, and incubated with the appropriate primary antibody overnight, diluted in TBS-Tween 0.1%-milk-5% or in TBS-Tween 0.1%-BSA-3–5% following the manufacturer’s instructions. After overnight incubation at 4 °C, membranes were washed with TBS-Tween 0.1% and incubated with the secondary antibody conjugated with horseradish peroxidase (HRP) for 1 h at room temperature. Detection of proteins was done using a chemiluminescent detection reagent (Amersham™ ECL, catalog number: RPN2232, Cytiva Life Sciences, Marlborough, MA, USA). Blots were imaged with the Syngene PXi technology, and the bands were quantified using the GeneTools from Syngene software (4.3.9.0, Synoptics, Cambridge, UK). Antibodies used in this study and corresponding dilutions are listed in Table S2 .
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