Improve Research Reproducibility A Bio-protocol resource
Concise Method
tooltip

Cell culture, co-immunoprecipitation, and protein degradation assay

RC Run-Ze Chen
XC Xuebo Cheng
YT Yuexi Tan
TC Tien-Chien Chang
HL Hailong Lv
YJ Yichang Jia
request Request a Protocol
ask Ask a question
Favorite

HEK293T cells were maintained in DMEM culture medium supplemented with 10% (v/v) FBS, 50 U/mL penicillin, and 50 μg/mL streptomycin in a humidified incubator with 5% CO2 at 37 °C. Cells were lysed with lysis buffer (20 mM Tris-HCl, pH 7.5, 1 mM EDTA, 150 mM NaCl, 0.5% NP-40) supplemented with protease inhibitor cocktail and PMSF. The cell lysis was centrifuged at 10,000 × g for ten minutes. One tenth the volume of the supernatants was pipetted as input samples. The remaining supernatants were incubated with M2 Magnetic Beads (M8823, Sigma) overnight by the standard protocol. For protein degradation assay, 10 μM MG132 was added to the culture medium just before plasmid transfection and then incubated for 16–24 hours.

Copyright and License information: The Author(s) ©2020
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.

post Post a Question
0 Q&A