Isolation of Ubiquitinated Proteins to High Purity from In Vivo Samples.
Ubiquitination pathways are widely used within eukaryotic cells. The complexity of ubiquitin signaling gives rise to a number of problems in the study of specific pathways. One problem is that not all processes regulated by ubiquitin are shared among the different cells of an organism (e.g., neurotransmitter release is only carried out in neuronal cells). Moreover, these processes are often highly temporally dynamic. It is essential therefore to use the right system for each biological question, so that we can characterize pathways specifically in the tissue or cells of interest. However, low stoichiometry, and the unstable nature of many ubiquitin conjugates, presents a technical barrier to studying this modification in vivo. Here, we describe two approaches to isolate ubiquitinated proteins to high purity. The first one favors isolation of the whole mixture of ubiquitinated material from a given tissue or cell type, generating a survey of the ubiquitome landscape for a specific condition. The second one favors the isolation of just one specific protein, in order to facilitate the characterization of its ubiquitinated fraction. In both cases, highly stringent denaturing buffers are used to minimize the presence of contaminating material in the sample.
Biokimika eta Biologia Molekularra Saila, Zientzia eta Teknologia Fakultatea, University of the Basque Country (UPV/EHU), Leioa, 48940, Spain.
Juanma
J
Ramirez
Department of Genetics, University of Cambridge, Cambridge, CB2 3EH, UK.|Department of Cell Biology, Harvard Medical School, Boston, MA, 02115, USA.
Mingwei
M
Min
CIC bioGUNE, Derio, 48160, Spain.
Rosa
R
Barrio
Department of Genetics, University of Cambridge, Cambridge, CB2 3EH, UK. acl34@cam.ac.uk.
Catherine
C
Lindon
Biokimika eta Biologia Molekularra Saila, Zientzia eta Teknologia Fakultatea, University of the Basque Country (UPV/EHU), Leioa, 48940, Spain. ugo.mayor@ehu.eus.|Ikerbasque, Basque Foundation for Science, Bilbao, 48013, Spain. ugo.mayor@ehu.eus.
Ugo
U
Mayor
D000818:Animals; D006801:Humans; D020543:Proteome / Q000302:isolation & purification / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D057149:Ubiquitinated Proteins / Q000302:isolation & purification / Q000378:metabolism*; D054875:Ubiquitination
D020543:Proteome; D057149:Ubiquitinated Proteins
Denaturing conditions; Isolation; Substrates; Ubiquitination