Cells were lysed with lysis buffer (10 mM Hepes, 50 mM NaCl, 1 mM EDTA, 10% glycerol, 1% Triton, 30 mM NaF, and 1 mM Na3VO4) in the presence of protease inhibitor cocktail tablet (Sigma-Aldrich). For immunoprecipitation, lysates were incubated with Protein G Sepharose beads and the antibody overnight. Samples were boiled and separated to Tris-glycine SDS–PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were incubated with the primary antibody and HRP-conjugated secondary antibodies (GE Healthcare). Immunoblots were visualized using the Amersham ECL Prime Western blotting detection reagent (GE Healthcare) and Bio-Rad ChemiDoc touch. The band intensity was measured by Image Lab software.
Amersham ecl prime western
Manufactured by GE Healthcare
The Amersham ECL Prime Western Blotting Detection Reagent is a chemiluminescent substrate used for the detection of proteins in Western blot analysis. It is designed to provide high-sensitivity detection of target proteins labeled with horseradish peroxidase (HRP)-conjugated antibodies.
Automatically generated - may contain errors
Lab products found in correlation
Amersham ecl prime, by Cytiva (3 mentions)
Hrp conjugated secondary antibody, by GE Healthcare (1 mentions)
Protease inhibitor cocktail tablet, by Merck Group (1 mentions)
Chemidoc touch, by Bio-Rad (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Amersham imager 600, by GE Healthcare (1 mentions)
Amersham imager, by Cytiva (1 mentions)
3 protocols using amersham ecl prime western
1
Immunoprecipitation and Western Blot Analysis
2
Western Blot Analysis of Protein Expression
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Western blot analysis was performed as described previously [34 (link)]. Briefly, cells were washed three times with PBS, lysed in lysis buffer (50 mM Tris–HCl [pH 6.8], 1% sodium dodecyl sulfate, 10% glycerol), and stored at − 70 °C until use. Equal amounts of total proteins were separated by SDS–polyacrylamide gel electrophoresis, transferred to a polyvinylidene fluoride membrane (Millipore), and proteins were immunodetected using the appropriate primary antibody followed by incubation with horseradish peroxidase-conjugated secondary antibody. Amersham ECL Prime western blotting Detection Reagent (GE Healthcare Life Sciences) was used to visualize antibody-antigen complexes. All used antibodies are listed in Table S2 .
3
Western Blot Analysis of Leaflet Proteins
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Proteins extracted from leaflet tissue (extracted in 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 5 mM EDTA, 0.1% (v/v) Triton X-100, 1× protease inhibitor followed by centrifugation at 12,000×g, at 4°C for 10 minutes, and the supernatant was transferred to a clean tube) or purified from E. coli expression were separated by SDS-PAGE and detected by WB using a 1:2,000 dilution of the α-TD2 antibody or a 1:200 dilution of the α-pAdi3 antibody with a 1:3,000 dilution of an α-rabbit-HRP antibody (ThermoFisher). WB detection was conducted using Amersham ECL prime western blot detection reagent (GE Healthcare) and the chemiluminescent signal was detected using an Amersham Imager 600 (GE Healthcare).
Identification of TD2 as the protein detected by the α-pAdi3 antibody using 2D SDS-PAGE and mass spectrometry
Identification of TD2 as the protein detected by the α-pAdi3 antibody using 2D SDS-PAGE and mass spectrometry
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