Table 2.

Useful reagents for monitoring cellular ubiquitination using microscopy

Application	Reagent	Advantages	Disadvantages
Degradative proteasomal functions of Ub in biological processes	GFP‐tagged wild‐type ubiquitin and mutants	Robust and applicable in intact animals	Transfection required Potential influence on Ub homeostasis Limited to degradative functions of the ubiquitin/proteasome system
	GFP‐tagged physiological Ub substrates	Pathways often characterized in detail	Transfection required Limited to degradative functions of the ubiquitin/proteasome system
	Degron‐tagged nanobodies against GFP	Target flexibility	Transfection required Limited to degradative functions of the ubiquitin/proteasome system
Visualization of Ub chains, substrates and processes in cellular structures and processes	Ectopic expression of tagged Ub	Powerful in combination with biochemistry Ubiquitin mutants for chain characterization	Transfection required Competition with endogenous Ub Potential influence on Ub homeostasis Tags at Ub might interfere with function Ectopic expression of Ub mutants might influence native chain patterns N‐terminal tagging interferes with linear chain formation
	Ub chain sensors	Chain selective Applicable in living cells Compatible with BiFC Potential influences of sensors of process of interest can be used to discover novel chain functions	Transfection required Prone to influencing process of interest by interfering with endogenous chain recognition Prone to high background signals Target binding potentially susceptible to post‐translational modification of Ub chains
	Ub antibodies	Easy to use in immunofluorescence Characterized in detail Compatible with library screening and affinity maturation and optimization	Post‐fixation Only available against selected Ub chain targets
	Ub affimers	Versatile, especially in combination with library screening Small and physiochemically robust Compatible with genetic tagging and ectopic expression	Post‐fixation
	Ub(Dha) derivatives	Promising reagents for imaging Ub flux Compatible with live‐cell imaging and in principle with FRAP/FRET	Electroporation required Optimization required