Cells were harvested and homogenized in RIPA lysis buffer (10 mM Tris, pH 7.2, 150 mM NaCl, 1% deoxycholate, 1% TritonX-100, 0.1% SDS, 5 mM EDTA) containing complete protease inhibitor cocktail, phospho-stop (Roche Diagnostics, Meylan, France), DTT (Sigma-Aldrich) and vanadate (Thermo Fisher Scientific). Exosomes were centrifuged for 2 h at 100,000g at 4°C. The supernatants were discarded and the pellets were lysed by complemented RIPA lysis buffer (10 mM Tris, pH 7.2, 150 mM NaCl, 1% deoxycholate, 1% TritonX-100, 0.1% SDS, 5 mM EDTA), phospho-stop, (Roche Diagnostics) and vanadate (Thermo Fisher Scientific). After 30 min on ice, protein content was measured by the BCA assay. Equal amounts of soluble protein (15–25 µg) were denatured by heating at 95°C for 5 min, resolved in SDS-PAGE and transferred to PVDF membranes. The membranes were blocked in 5% non-fat dry milk in TBS-Tween for 1 h and probed initially with specific primary antibody followed by horseradish peroxidase-conjugated secondary antibody. Blotted protein bands were detected by chemiluminescence (Supersignal, Thermo Fisher Scientific) exposure on X-ray films (Kodak, Rochester, NY, USA).
Vanadate
Manufactured by Thermo Fisher Scientific
Sourced in France
Vanadate is a laboratory equipment product offered by Thermo Fisher Scientific. It is a chemical compound that serves as a source of the element vanadium, which is used in various scientific applications. The core function of vanadate is to provide a reliable and consistent supply of vanadium for researchers and scientists in their experiments and analyses.
Automatically generated - may contain errors
Lab products found in correlation
Dithiothreitol (dtt), by Merck Group (4 mentions)
Phosphostop, by Roche (3 mentions)
X ray film, by Kodak (2 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Protein assay, by Bio-Rad (1 mentions)
Precast sds page gel, by Thermo Fisher Scientific (1 mentions)
Bradford protein assay, by Bio-Rad (1 mentions)
Dithiothreitol (dtt), by Thermo Fisher Scientific (1 mentions)
Nupage, by Thermo Fisher Scientific (1 mentions)
Western lightning, by PerkinElmer (1 mentions)
Nef1002001pk, by PerkinElmer (1 mentions)
X ray film exposure, by Kodak (1 mentions)
4 protocols using vanadate
1
Exosome Protein Isolation and Western Blot Analysis
2
Exosome Protein Extraction and Analysis
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Cells were harvested and homogenized in RIPA lysis buffer (10 mM Tris, pH 7.2, 150 mM NaCl, 1% deoxycholate, 1% Triton, 0.1% SDS, 5 mM EDTA) containing complete protease inhibitor cocktail, phospho-stop (Roche Diagnostics, Meylan, France), dithiothreitol (Sigma Aldrich) and vanadate (Life technologies). The exosomes were centrifuged for 120 minutes at 120,000 g, 4°C. The supernatants were discarded and the pellets were lysed by complemented RIPA lysis buffer. After 1 hour on ice, the samples were sonicated and protein quantification was carried out with a Bio-Rad protein assay. Equal amounts of soluble protein (15–25 μg) were denaturated by heating at 95°C for 5 minutes, resolved in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes. The membranes were blocked in 5% non-fat dry milk in TBS-T for 1 hour and probed initially with specific primary antibody and horseradish peroxidase-conjugated secondary antibody. The protein bands were detected by chemiluminescence (Supersignal, Pierce) exposure on X-ray films (Kodak).
3
Western Blot Analysis of Cellular Proteins
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The cell pellets and exosomes were lysed in RIPA lysis buffer (10 mM Tris, pH 7.2, 150 mM NaCl, 1% deoxycholate, 1% Triton, 0.1% SDS, 5 mM EDTA) containing complete protease inhibitor cocktail, phospho-stop (Roche Diagnostics, Meylan, France), dithiothreitol (Sigma Aldrich, St. Louis, MO, USA) and vanadate (Life technologies, Carlsbad, CA, USA). Equal amounts of soluble protein (30 micrograms) were loaded in precast SDS-PAGE gels (Life technologies, Carlsbad, CA, USA) and transferred to PVDF membranes for probing with specific primary antibodies overnight at 4 °C and subsequently with horseradish peroxidase-conjugated secondary antibody. Detection of the protein bands was achieved using chemiluminescence (Life technologies, Carlsbad, CA, USA) and exposure on X-ray films (Kodak Rochester, NY, USA). The t-test was used for the comparisons of various measurements between control and experimental conditions as well as between densitometric data in Western blots. p value < 0.05 was considered statistically significant.
4
Western Blot Protein Expression Analysis
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Cells were harvested and homogenized in RIPA lysis buffer (10 mM Tris, pH 7.2, 150 mM NaCl, 1% deoxycholate, 1% Triton, 0.1% SDS, 5 mM EDTA) containing complete protease inhibitor cocktail, phospho-stop, (Roche Diagnostics, Meylan, France), dithiothreitol (Sigma Aldrich, D0632) and vanadate (Life Technologies, S5608). After 30minutes on ice, samples were sonicated and protein quantification was carried out using a Bio-Rad Bradford protein assay. Equal amounts of soluble proteins (15-25 μg) were loaded with LDS sample buffer (NuPAGE, Life Technologies) and dithiothreitol denaturated by boiling and resolved by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred in 20% methanol to a methanol-activated PVDF membrane (Perkin Elmer, NEF1002001PK). After blocking in 5% non-fat dry milk in TBS for 1h and probing with a specific primary antibody overnight and a horseradish peroxidase-conjugated secondary antibody for 1h, both in 5% non-fat dry milk in TBS, the protein bands were detected by Western Lightning (Perkin Elmer) and X-ray film exposure (Kodak). Protein loading was normalized by using anti-GAPDH or anti-actin antibodies.
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