Table 1.

Ubiquitin-independent degradation assays

Assay	Strengths	Weaknesses
Absence of ubiquitination	Can be a visually compelling, companion experiment.	Ubiquitinated intermediates are short-lived and challenging to isolate, so negative results are difficult to interpret without extensive positive controls.
Unstable in ats20 cells	Most direct and reliable assay when proper ubiquitin-dependent controls are also analyzed.	Positive controls such as p53 only show ubiquitin- dependent degradation impairment but not necessarilya complete inhibition; Possibility that specific degradation events depend more highly on Ube 1L2 as opposed to the ts Ube 1 protein.
Immune to bDN ubiquitin	Direct and reliable assay when proper ubiquitin- dependent controls are also analyzed.	Transfection based assay in which it is difficult to achieve high-level inhibition even of positive controls.
Unstable lysine-less mutant	Simple assay for a subset of small proteins but that requires inhibitors to confirm proteasome- mediated degradation.	Larger proteins require multiple lysine mutations that may affect overall structure; Must be conscious of N- terminal-mediated ubiquitin-dependent degradation.
In vitro degradation	Defined assay favored by protein biochemists.	Inability to confirm that processes that can occur in vitro actually do occur In vitro; Technically demanding assay that requires all necessary reaction components (which may or may not be known).

^ats20: temperature sensitive

^bDN: dominant negative