Table 1.

A comparison of the scope and limitations of experiments in cell extract with in vitro reconstituted and in-cell single-molecule systems.

Experimental consideration	In vitro reconstitution	In live cells (in cellulo)	In cell extract
Temperature of measurement	Can be independently varied	Confined to physiological temperatures	Similar to in vitro, can be independently varied within optimized range
Concentration of component	Can be independently varied	Can be altered by manipulating gene expression characteristics	Can be extended beyond in cellulo by doping in purified components
Purity of component	Highly pure, but involves time-consuming purification steps	Complex mixture	Similar to in cellulo, but affords local “pull-down” to enrich for specific components (51–54)
Macromolecular Crowding	Can be simulated	Has intrinsic crowding	Can be simulated
Equilibrium measurements	Long-term equilibration is possible	Non-equilibrium	Similar to in vitro
Experimentally tractable (long term imaging)	Oxygen scavenging systems can enhance imaging times	Short term imaging only	Similar to in vitro
Small molecule testing and/or exchange	Yes. Highly controllable	Possible, but limited within the constraints of maintaining cell viability	Similar to in vitro, but retaining much of the complexity of the cell