AI Article Synopsis
- The text discusses three methods for isolating 26S proteasomes, which are important for studying how their activity and composition change in various conditions, including diseases and drug responses.
- The first method involves using affinity tags on proteasome subunits for advanced techniques like cryo-electron microscopy and mass spectrometry to analyze structure and composition.
- The second method relies on the proteasome's natural affinity for a ubiquitin-like domain for purification without tagged subunits, while the third method uses differential centrifugation to concentrate proteasomes based on their large size, preserving their functional associations.
Article Abstract
Rapid, gentle isolation of 26S proteasomes from cells or tissues is an essential step for studies of the changes in proteasome activity and composition that can occur under different physiological or pathological conditions and in response to pharmacological agents. We present here three different approaches to affinity purify or to prepare proteasome-rich cell fractions. The first method uses affinity tags fused to proteasome subunits and has been useful in several cell lines for studies of proteasome structure by cryo-electron microscopy and composition by mass spectrometry. A second method uses the proteasome's affinity for a ubiquitin-like (UBL) domain and can be used to purify these particles from any cell or tissue. This method does not require expression of a tagged subunit and has proven to be very useful to investigate how proteasomal activity changes in different physiological states (e.g., fasting or aging), with neurodegenerative diseases, and with drugs or hormones that cause subunit phosphorylation. A third, simple method that is based on the 26S proteasome's high molecular weight (about 2.5 MDa) concentrates these particles greatly by differential centrifugation. This method maintains the association of proteasomes with ubiquitin (Ub) conjugates and many other loosely associated regulatory proteins and is useful to study changes in proteasome composition under different conditions.
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| http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6427908 | PMC |
| http://dx.doi.org/10.1007/978-1-4939-8706-1_18 | DOI Listing |
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