Cells were washed with pre-cooling PBS and then loading buffer (20 mM Tris-HCl (pH 8.0), 137 mM NaCl, 10% glycerol, 1% NP-40, 2 mM EDTA) was added to lysate cells. Then Cocktail buffer (a mixture of protease inhibitor and phospholipase inhibitor; Roche, USA) and cells were incubated on ice for 20 min and centrifuged at 4 °C, 12,000 rpm for 15 min. The supernatant was collected and mixed with 5 mg/ml dimethyl suberimidate (Fluka, USA) and incubated at room temperature for 30 min. Then samples were mixed with isometric SDS loading buffer and subjected to SDS-PAGE for WB.
Copyright and License information: The Author(s) ©2019 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
Do you have any questions about this protocol?
Post your question to gather feedback from the community. We will also invite the authors of this
article to respond.
Ask a
question
Post a Question
