Table 1.

Peptide tags that have been used to label the N-terminus of ubiquitin for affinity purification of ubiquitinated proteins.

#	Tags	Amino acid sequence	Resins or antibodies for purification	Advantages	Disadvantages	Examples
1	Hexahistidine (His6)	HHHHHH	Ni-NTA, TALON	Can be used under both native and denaturing conditions Can tolerate low percentage of organic solvent during the washing step Can retain protein activity under native conditions Anti-tag antibodies are available for biochemical verification	Can bind to histidine-rich proteins Not compatible with EDTA and high concentrations of dithiothreitol (up to 10 mM), TCEP (up to 20 mM), or mercaptoethanol (up to 20 mM)	(Jeon et al., 2007; Kirkpatrick et al., 2005; Meierhofer et al., 2008; Peng and Cheng, 2005; Peng et al., 2003)
2	Biotin acceptor peptide (BAP)	GLNDIFEAQ KIEWHE	Streptavidin, Neutravidin, Monomeric avidin	Can be used with stringent denaturing conditions Can tolerate organic solvent during the washing step Anti-tag antibodies are available for biochemical verification	Requires coexpression of BirA in cells Requires exogenous free biotin Difficult to completely elute tagged proteins from beads Resin cannot be reused Small amount of endogenous biotinylated proteins are also purified	(Meierhofer et al., 2008; Tagwerker et al., 2006)
3	Strep	WSHPQFEK	Strep-Tactin	Elution using mild conditions, such as low concentrations of desthiobiotin or biotin Resin can be reused when desthiobiotin is used to elute tagged proteins Anti-Strep antibody is available for biochemical validation Can allow protein activity to be retained	It is typically used under native conditions with low concentrations of detergent Interacting partners may also be pulled down during the purification	(Danielsen et al., 2011)
4	FLAG	DYKDDDDK	Anti-FLAG antibody	High quality antibodies available for purification Mild elution conditions with competitor peptides to reduce non-specific binding Resin can be reused for several time	Antibody is relative expensive Cannot be used under denaturing or reducing conditions Interacting partners may also be pulled down during the purification	(Argenzio et al., 2011; Kim et al., 2011a; Oshikawa et al., 2012)

^*A common advantage is that all the tags are very small and their presence at the N-terminus of ubiquitin does not significantly affect the ubiquitination pathways.

^**A common disadvantage is that free ubiquitin is purified simultaneously.