Abstract：
:As proteins within cells are spatially organized according to their role, knowledge about protein localization gives insight into protein function. Here, we describe the LOPIT technique (localization of organelle proteins by isotope tagging) developed for the simultaneous and confident determination of the steady-state distribution of hundreds of integral membrane proteins within organelles. The technique uses a partial membrane fractionation strategy in conjunction with quantitative proteomics. Localization of proteins is achieved by measuring their distribution pattern across the density gradient using amine-reactive isotope tagging and comparing these patterns with those of known organelle residents. LOPIT relies on the assumption that proteins belonging to the same organelle will co-fractionate. Multivariate statistical tools are then used to group proteins according to the similarities in their distributions, and hence localization without complete centrifugal separation is achieved. The protocol requires approximately 3 weeks to complete and can be applied in a high-throughput manner to material from many varied sources.
journal_name
Nat Protocjournal_title
Nature protocolsauthors
Sadowski PG,Dunkley TP,Shadforth IP,Dupree P,Bessant C,Griffin JL,Lilley KSdoi
10.1038/nprot.2006.254subject
Has Abstractpub_date
2006-01-01 00:00:00pages
1778-89issue
4eissn
1754-2189issn
1750-2799pii
nprot.2006.254journal_volume
1pub_type
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