Method for the Purification of Endogenous Unanchored Polyubiquitin Chains.
Unanchored polyubiquitin chains are endogenous non-substrate linked ubiquitin polymers which have emerging roles in the control of cellular physiology. We describe an affinity purification method based on an isolated ubiquitin-binding domain, the ZnF_UBP domain of the deubiquitinating enzyme USP5, which permits the selective purification of mixtures of endogenous unanchored polyubiquitin chains that are amenable to downstream molecular analyses. Further, we present methods for detection of unanchored polyubiquitin chains in purified fractions.
School of Life Sciences, Queen's Medical Centre, University of Nottingham, Nottingham, NG7 2UH, UK.
Daniel
D
Scott
Institute of Cell Biology, University of Edinburgh, Edinburgh, UK.
Jo
J
Strachan
School of Life Sciences, Queen's Medical Centre, University of Nottingham, Nottingham, NG7 2UH, UK.
Varun Gopala
VG
Krishna
School of Life Sciences, Queen's Medical Centre, University of Nottingham, Nottingham, NG7 2UH, UK.
Barry
B
Shaw
School of Life Sciences, Queen's Medical Centre, University of Nottingham, Nottingham, NG7 2UH, UK.
David J
DJ
Tooth
School of Chemistry, University of Nottingham, University Park, Nottingham, NG7 2RD, UK.|Centre for Biomolecular Sciences, University of Nottingham, University Park, Nottingham, NG7 2RD, UK.
Mark S
MS
Searle
School of Chemistry, University of Nottingham, University Park, Nottingham, NG7 2RD, UK.
Neil J
NJ
Oldham
School of Life Sciences, Queen's Medical Centre, University of Nottingham, Nottingham, NG7 2UH, UK. robert.layfield@nottingham.ac.uk.
Rob
R
Layfield
D000818:Animals; D006801:Humans; D025821:Polyubiquitin / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D040901:Proteomics / Q000379:methods; D025801:Ubiquitin / Q000302:isolation & purification / Q000378:metabolism*; D064570:Ubiquitin-Specific Proteases / Q000302:isolation & purification / Q000378:metabolism; D057149:Ubiquitinated Proteins / Q000302:isolation & purification / Q000378:metabolism; D054875:Ubiquitination
D025801:Ubiquitin; D057149:Ubiquitinated Proteins; D025821:Polyubiquitin; D064570:Ubiquitin-Specific Proteases
Free polyubiquitin; USP5; Ubiquitin; Ubiquitin-binding domain; Unanchored polyubiquitin; ZnF_UBP domain