Abstract
Isolation of a protein/complex is important for its biochemical and structural characterization with mechanistic insights. TAP (tandem affinity purification) strategy allows rapid isolation of cellular proteins/complexes with a high level of purity. This methodology involves an immuno-affinity-based purification followed by a conformation-based isolation to obtain a highly homogeneous protein/complex. Here, we describe the TAP-mediated isolation of endogenous FACT (facilitates chromatin transcription; a heterodimer), an essential histone chaperone associated with BER (base excision repair). However, it is not clearly understood how FACT regulates BER. Such knowledge would advance our understanding of BER with implications in disease pathogenesis, since BER is an evolutionarily conserved process that is linked to various diseases including ageing, neurodegenerative disorders, and cancers. Using isolated FACT by TAP methodology, one can study the mechanisms of action of FACT in BER. Further, isolated FACT can be used for studies in other DNA transactions such as transcription and replication, as FACT is involved in these processes. Furthermore, TAP-mediated isolation strategy can be combined with mass spectrometry to identify the protein interaction partners of FACT.
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Acknowledgements
We thank Zhiguo Zhang for the yeast strain (TAP-tagged Pob3). The work in the Bhaumik laboratory was supported by the grants from the National Institutes of Health (GM088798-03). AK and SG were supported by the predoctoral fellowship of the American Heart Association and the doctoral fellowship of Southern Illinois University, respectively.
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Kaja, A., Barman, P., Guha, S., Bhaumik, S.R. (2023). Tandem Affinity Purification and Mass-Spectrometric Analysis of FACT and Associated Proteins. In: Bhakat, K.K., Hazra, T.K. (eds) Base Excision Repair Pathway. Methods in Molecular Biology, vol 2701. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3373-1_14
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DOI: https://doi.org/10.1007/978-1-0716-3373-1_14
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