PMID- 34985695
OWN - NLM
STAT- MEDLINE
DCOM- 20220329
LR  - 20220422
IS  - 1940-6029 (Electronic)
IS  - 1064-3745 (Linking)
VI  - 2378
DP  - 2022
TI  - High-Throughput Analysis of Protein Turnover with Tandem Fluorescent Protein 
      Timers.
PG  - 85-100
LID - 10.1007/978-1-0716-1732-8_6 [doi]
AB  - Tandem fluorescent protein timers (tFTs) are versatile reporters of protein 
      dynamics. A tFT consists of two fluorescent proteins with different maturation 
      kinetics and provides a ratiometric readout of protein age, which can be 
      exploited to follow intracellular trafficking, inheritance and turnover of 
      tFT-tagged proteins. Here, we detail a protocol for high-throughput analysis of 
      protein turnover with tFTs in yeast using fluorescence measurements of ordered 
      colony arrays. We describe guidelines on optimization of experimental design with 
      regard to the layout of colony arrays, growth conditions, and instrument choice. 
      Combined with semi-automated genetic crossing using synthetic genetic array (SGA) 
      methodology and high-throughput protein tagging with SWAp-Tag (SWAT) libraries, 
      this approach can be used to compare protein turnover across the proteome and to 
      identify regulators of protein turnover genome-wide.
CI  - (c) 2022. The Author(s).
FAU - Fung, Jia Jun
AU  - Fung JJ
AD  - Institute of Molecular Biology (IMB), Mainz, Germany.
FAU - Blocher-Juarez, Karla
AU  - Blocher-Juarez K
AD  - Institute of Molecular Biology (IMB), Mainz, Germany.
FAU - Khmelinskii, Anton
AU  - Khmelinskii A
AD  - Institute of Molecular Biology (IMB), Mainz, Germany. a.khmelinskii@imb-mainz.de.
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - United States
TA  - Methods Mol Biol
JT  - Methods in molecular biology (Clifton, N.J.)
JID - 9214969
RN  - 0 (Proteome)
SB  - IM
MH  - Kinetics
MH  - Proteolysis
MH  - *Proteome/metabolism
MH  - *Saccharomyces cerevisiae/genetics/metabolism
OTO - NOTNLM
OT  - Fluorescent proteins
OT  - Protein degradation
OT  - Protein dynamics
OT  - Protein quality control
OT  - Protein turnover
OT  - Proteostasis
OT  - Tandem fluorescent protein timers
OT  - tFT
EDAT- 2022/01/06 06:00
MHDA- 2022/03/30 06:00
CRDT- 2022/01/05 12:27
PHST- 2022/01/05 12:27 [entrez]
PHST- 2022/01/06 06:00 [pubmed]
PHST- 2022/03/30 06:00 [medline]
AID - 10.1007/978-1-0716-1732-8_6 [doi]
PST - ppublish
SO  - Methods Mol Biol. 2022;2378:85-100. doi: 10.1007/978-1-0716-1732-8_6.