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

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


Anyaporn Sawasdichai

Hsin-Tien Chen

Nazefah Abdul Hamid

Padma-Sheela Jayaraman

Kevin Gaston


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.

Journal Article Type Article
Publication Date Jul 23, 2010
Journal Journal of Visualized Experiments
Publisher Journal of Visualized Experiments
Peer Reviewed Peer Reviewed
Volume 41
Article Number e1958
APA6 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, doi:10.3791/1958
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