In situ subcellular fractionation of adherent and non-adherent mammalian cells

Sawasdichai, Anyaporn; Chen, Hsin-Tien; Abdul Hamid, Nazefah; Jayaraman, Padma-Sheela; Gaston, Kevin

Abstract

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, many proteins are present as a freely diffusing population in free exchange with a sub-population that is tightly associated with a particular subcellular domain or structure. In situ subcellular fractionation allows the visualization of protein compartmentalization and can also reveal protein sub-populations that localize to specific structures. For example, removal of soluble cytoplasmic proteins and loosely held nuclear proteins can reveal the stable association of some transcription factors with chromatin. Subsequent digestion of DNA can in some cases reveal association with the network of proteins and RNAs that is collectively termed the nuclear scaffold or nuclear matrix. Here we describe the steps required during the in situ fractionation of adherent and non-adherent mammalian cells on microscope coverslips. Protein visualization can be achieved using specific antibodies or fluorescent fusion proteins and fluorescence microscopy. Antibodies and/or fluorescent dyes that act as markers for specific compartments or structures allow protein localization to be mapped in detail. In situ fractionation can also be combined with western blotting to compare the amounts of protein present in each fraction. This simple biochemical approach can reveal associations that would otherwise remain undetected.

Citation

Sawasdichai, A., Chen, H., Abdul Hamid, N., Jayaraman, P., & Gaston, K. (2010). In situ subcellular fractionation of adherent and non-adherent mammalian cells. Journal of Visualized Experiments, 41, Article e1958. https://doi.org/10.3791/1958

• Journal Article Type: Article
• Acceptance Date: May 1, 2010
• Publication Date: Jul 23, 2010
• Deposit Date: Oct 31, 2018
• Journal: Journal of Visualized Experiments
• Publisher: Journal of Visualized Experiments
• Peer Reviewed: Peer Reviewed
• Volume: 41
• Article Number: e1958
• DOI: https://doi.org/10.3791/1958
• Public URL: https://nottingham-repository.worktribe.com/output/1037650
• Publisher URL: https://www.jove.com/video/1958/in-situ-subcellular-fractionation-adherent-non-adherent-mammalian