Published: Vol 8, Iss 19, Oct 5, 2018 DOI: 10.21769/BioProtoc.3035 Views: 19327
Reviewed by: Pabitra Kumar SahooAnna La TorreAnonymous reviewer(s)
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Abstract
Shuttling of proteins between different cellular compartments controls their proteostasis and can contribute in some cases to regulate their activity. Biochemical analysis of chromatin-bound proteins, such as transcription factors, is often difficult because of their low yield and due to the interference from nucleic acids. This protocol describes a method to efficiently fractionate cells combined with a mechanical (i.e., sonication) or an enzymatic treatment (i.e., benzonase) that facilitates analysis of chromatin-bound protein extracts by Western blot analysis or by protein pull-down assays. This approach can be valuable to enrich a particular protein within a particular subcellular fraction either to study specific post-translational modification patterns or to identify specific protein-protein interactions.
Keywords: Chromatin-bound proteinsBackground
The activity and post-translational regulation of many chromatin-bound proteins are poorly studied due to technical difficulties in isolating them for biochemical analysis. This is, even more, the case with transcription factors, such as the basic Helix-loop-Helix (bHLH) transcription factors that often harbor scarce temporal and spatial pattern of expression in tissues or cellular models (Dennis et al., 2018). Protocol refinement helps to lift technical limitation when the amount of biological materials become a barrier to investigate molecular pathways (Gillotin and Guillemot, 2016). In our recent study, we endeavored to understand how proteolysis of the proneural bHLH transcription factor Ascl1 is regulated in cellular models of neuronal differentiation (Gillotin et al., 2018). For this, we adapted existing protocols for subcellular fractionation to obtain efficient separation of the cytoplasm, nuclear and chromatin fractions. To be able to perform biochemical analysis on the chromatin fraction, we either used sonication to shear nucleic acids or treated this fraction with benzonase to degrade nucleic acids. These steps were essential to perform Western blot analysis or protein pull-down assays (Figure 1). This approach successfully led to the analysis of Ascl1 ubiquitylation pattern in the cytoplasm and in the chromatin fractions (Gillotin et al., 2018) and can be extended to the study of other chromatin-bound proteins and to other post-translational modifications.
Figure 1. Workflow of the protocol. First, the collection of each subcellular fraction is performed by successive protein extractions with the buffer E1, then with the buffer E2 and with the buffer E3. Second, the chromatin fraction is either sonicated to shear the DNA or treated with benzonase to digest nucleic acids. Third, each fraction is biochemically analyzed for a protein of interest. The purity of each fraction should be controlled by Western blot for α-Tubulin, LaminB and Histone H3 depicting respectively the cytoplasmic fraction, the nuclear fraction and the chromatin fraction.
Materials and Reagents
Note: All stock solutions should be stored at room temperature except when indicated otherwise.
Equipment
Procedure
Notes:
For Western blot analysis:
For pull-down assays:
Data analysis
Representative data for the efficiency of the cellular fractionation and for ubiquitin pull-down assay are shown in Gillotin et al. (2018) (https://www.nature.com/articles/s41598-018-23056-4.pdf). To assess the efficiency of the cellular fractionation, it is recommended to analyze each fraction by Western blot analysis for α-Tubulin, LaminB and Histone H3 as representative of the cytoplasmic, nuclear and chromatin fractions respectively.
Recipes
Note: Prepare all the solutions fresh from stock solutions before starting and keep on ice. Each solution is complemented with protease inhibitor cocktail and adding specific inhibitors should be considered to study post-translational modifications (e.g., phosphatase inhibitor, DUBs inhibitor).
Acknowledgments
Illustrations have been modified from the Servier Medical Art service (Les Laboratoires Servier). This work was adapted in Anna Philpott’s laboratory (University of Cambridge) from protocols established in Jason Carroll's laboratory (University of Cambridge). The author is currently supported by funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 713140 (The Meso_Brain Project, www.mesobrain.eu).
Competing interests
The author declares no competing interest.
References
Article Information
Copyright
© 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Gillotin, S. (2018). Isolation of Chromatin-bound Proteins from Subcellular Fractions for Biochemical Analysis. Bio-protocol 8(19): e3035. DOI: 10.21769/BioProtoc.3035.
Category
Neuroscience > Cellular mechanisms > Protein isolation
Stem Cell > Pluripotent stem cell > Cell-based analysis
Biochemistry > Protein > Isolation and purification
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