Improve Research Reproducibility A Bio-protocol resource
Concise Method
tooltip

Proteinase K protection assay

LW Lisa Wetzel
SB Stéphane Blanchard
SR Sowmya Rama
VB Viola Beier
AK Anna Kaufmann
TW Thomas Wollert
request Request a Protocol
ask Ask a question
Favorite

GFP-LC3C transfected cells were harvested and resuspended in homogenization buffer (HB: 20 mM Tris-HCl pH 8.0, 140 mM NaCl, 250 mM sucrose, 1 mM EDTA) followed by mechanical lysis by passing the suspension 15 times through a 27 gauge syringe. The nuclear fraction was separated by centrifuging at 7700 × g for 5 min. The supernatant was subjected to ultracentrifugation at 100,000 × g for 30 min. The corresponding pellet was resuspended in HB buffer and incubated with Proteinase K at a final concentration of 100 µg/ml in the presence or absence of Triton X-100 (0.5%) for the indicated times. The reaction was stopped by addition of 1 mM phenylmehtylsulfonyl fluoride and analyzed by immunoblotting.

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.

0/150

tip Tips for asking effective questions

+ Description

Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images.

post Post a Question
0 Q&A