A flow-cytometry-based method to simplify the analysis and quantification of protein association to chromatin in mammalian cells

By J. Forment, Stephen P. Jackson. 2015

77 citations recorded by the source corpus.

Protein accumulation on chromatin has traditionally been studied using immunofluorescence microscopy or biochemical cellular fractionation followed by western immunoblot analysis. As a way to improve the reproducibility of this kind of analysis, to make it easier to quantify and to allow a streamlined application in high-throughput screens, we recently combined a classical immunofluorescence microscopy detection technique with flow cytometry. In addition to the features described above, and by combining it with detection of both DNA content and DNA replication, this method allows unequivocal and direct assignment of cell cycle distribution of protein association to chromatin without the need for cell culture synchronization. Furthermore, it is relatively quick (takes no more than a working day from sample collection to quantification), requires less starting material compared with standard biochemical fractionation methods and overcomes the need for flat, adherent cell types that are required for immunofluorescence microscopy.

Subfields: Molecular Biology

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In Molecular Biology

1. Johannes C. Walter
2. Masato T. Kanemaki
3. Sachiko Muramatsu

Citations in the corpus

• In silico protein interaction screening uncovers DONSON’s role in replication initiation (2023, methodology): “S4C), all DONSON residues except those interacting with POLE2 (65-72) were deleted.”