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Purification and proteomic analysis of plant plasma membranes.

Publiceringsår: 2008
Språk: Engelska
Sidor: 161-173
Publikation/Tidskrift/Serie: Methods in molecular biology (Clifton, N.J.)
Volym: 432
Dokumenttyp: Artikel
Förlag: Humana Press

Sammanfattning

All techniques needed for proteomic analyses of plant plasma membranes are described in detail, from isolation of plasma membranes to protein identification by mass spectrometry (MS). Plasma membranes are isolated by aqueous two-phase partitioning yielding vesicles with a cytoplasmic side-in orientation and a purity of about 95%. These vesicles are turned inside-out by treatment with Brij 58, which removes soluble contaminating proteins enclosed in the vesicles as well as loosely attached proteins. The final plasma membrane preparation thus retains all integral proteins and many peripheral proteins. Proteins are separated by one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and protein bands are excised and digested with trypsin. Peptides in tryptic digests are separated by nanoflow liquid chromatography and either fed directly into an ESI-MS or spotted onto matrix-assisted laser desorption ionization (MALDI) plates for analysis with MALDI-MS. Finally, data processing and database searching are used for protein identification to define a plasma membrane proteome.

Disputation

Nyckelord

  • Biology and Life Sciences
  • Chemistry

Övrigt

Published
Yes
  • ISSN: 1064-3745

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