SVG-A cells were lysed in a buffer containing 50 mM Tris-HCl (pH 7.5), 0.3 M NaCl, 0.5% Triton X-100, and protease inhibitors (1x, Roche) for 20 min on ice, with intense vortexing at the beginning and end of the incubation. Lysates were cleared by centrifugation at 18000xg for 15 min at 4°C before transferring the supernatant to new tubes. The protein content in the lysates and in the purified EV fractions was measured in the presence of 0.2% SDS, using Pierce BCA protein assay kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. For each WB, 20 μg of lysates or 3 μg of EV pellets were loaded on 4%−12% NuPAGE Bis-Tris Protein Gels (Invitrogen) and ran under non-reducing conditions. Transfer was done using iBlot2 NC Transfer stacks (Invitrogen) prior to primary antibody incubation overnight at 4°C. Membranes were revealed by chemiluminescence using Clarity or Clarity Max Western ECL Blotting Substrates (Bio-Rad) and images were acquired using ChemiDoc Touch system (Bio-Rad).
Iblot2 nc transfer stacks
Manufactured by Thermo Fisher Scientific
The IBlot2 NC Transfer stacks are a laboratory equipment product designed for use in the transfer of proteins from polyacrylamide gels to nitrocellulose membranes. The stacks provide a standardized and efficient method for completing this critical step in western blotting workflows.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Chemidoc touch system, by Bio-Rad (2 mentions)
Clarity or clarity max western ecl blotting substrates, by Bio-Rad (2 mentions)
Nupage bis tris protein gel, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor, by Roche (2 mentions)
2 protocols using iblot2 nc transfer stacks
1
Protein Extraction and Western Blotting from SVG-A Cells
2
Protein Extraction and Western Blotting from SVG-A Cells
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SVG-A cells were lysed in a buffer containing 50 mM Tris-HCl (pH 7.5), 0.3 M NaCl, 0.5% Triton X-100, and protease inhibitors (1x, Roche) for 20 min on ice, with intense vortexing at the beginning and end of the incubation. Lysates were cleared by centrifugation at 18000xg for 15 min at 4°C before transferring the supernatant to new tubes. The protein content in the lysates and in the purified EV fractions was measured in the presence of 0.2% SDS, using Pierce BCA protein assay kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. For each WB, 20 μg of lysates or 3 μg of EV pellets were loaded on 4%−12% NuPAGE Bis-Tris Protein Gels (Invitrogen) and ran under non-reducing conditions. Transfer was done using iBlot2 NC Transfer stacks (Invitrogen) prior to primary antibody incubation overnight at 4°C. Membranes were revealed by chemiluminescence using Clarity or Clarity Max Western ECL Blotting Substrates (Bio-Rad) and images were acquired using ChemiDoc Touch system (Bio-Rad).
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