Linked Life Data

20431533

Source:http://linkedlifedata.com/resource/pubmed/id/20431533

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
5
pubmed:dateCreated
2010-4-30
pubmed:abstractText
The post-translational modification of proteins with ubiquitin and ubiquitin-like proteins (Ubl) is vital to many cellular functions, and thus the identification of Ubl targets is key to understanding their function. In most cases, only a small proportion of the cellular pool of proteins is found conjugated to a particular Ubl, making identification of Ubl targets technically challenging. For the purposes of proteomic analyses, we have developed a protocol for the large-scale purification of Ubl-linked proteins that minimizes sample contamination with noncovalent interactors and prevents the cleavage of Ubl-substrate bonds catalyzed by Ubl-specific proteases. This is achieved by introducing a denaturing lysis step (in the presence of sodium dodecyl sulfate and alkylating agents that irreversibly inhibit Ubl proteases) before TAP (tandem affinity purification) that allows for efficient purification of putative Ubl-specific substrates in a form suitable for proteomic analysis. The timescale from cell lysis to purified protein sample is 5-6 d.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:issn
1750-2799
pubmed:author
pubmed:issnType
Electronic
pubmed:volume
5
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
873-82
pubmed:meshHeading
pubmed:year
2010
pubmed:articleTitle
High-stringency tandem affinity purification of proteins conjugated to ubiquitin-like moieties.
pubmed:affiliation
Welcome Trust Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee, Dundee, Scotland, UK. f.golebiowski@dundee.ac.uk
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't