Protocol for Nucleo-Cytoplasmic Fractionation of Mammalian Cells to Study Protein Translocation via Immunoblotting

Authors

Adhikarimayum Lakhikumar Sharma, Thomas Jefferson UniversityFollow
Eddy Zhu, Thomas Jefferson University
Rithika Padyala, Thomas Jefferson University
Kavya Parikh, Thomas Jefferson UniversityFollow
Mudit Tyagi, Thomas Jefferson UniversityFollow

Document Type

Article

Publication Date

12-19-2025

Comments

This article is the author's final published version in STAR Protocols, Volume 6, Issue 4, December 2025, Article Number 104265.

The published version is available at https://doi.org/10.1016/j.xpro.2025.104265. Copyright © 2025 The Authors.

Abstract

Studying protein translocation is essential for understanding protein function and regulatory mechanisms, including the activity of transcription factors. Here, we present a protocol for isolating high-quality cytoplasmic and nuclear protein fractions from mammalian cells. We describe steps for separating cytoplasmic proteins, washing nuclei, and extracting nuclear proteins. We then detail procedures for quantifying proteins and analyzing their distribution by immunoblotting within a single immunoblot. This protocol is applicable to both suspension and adherent cell types. For complete details on the use and execution of this protocol, please refer to Sharma et al.

Recommended Citation

Sharma, Adhikarimayum Lakhikumar; Zhu, Eddy; Padyala, Rithika; Parikh, Kavya; and Tyagi, Mudit, "Protocol for Nucleo-Cytoplasmic Fractionation of Mammalian Cells to Study Protein Translocation via Immunoblotting" (2025). Center for Translational Medicine Faculty Papers. Paper 142.
https://jdc.jefferson.edu/transmedfp/142

Creative Commons License

This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.

PubMed ID

41379621

Language

English