Purification and proteomic analysis of plant plasma membranes.

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Purification and proteomic analysis of plant plasma membranes. / Alexandersson, Erik; Gustavsson, Niklas; Bernfur, Katja; Karlsson, Adine; Kjellbom, Per; Larsson, Christer.

I: Methods in Molecular Biology, Vol. 432, 2008, s. 161-173.

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T1 - Purification and proteomic analysis of plant plasma membranes.

AU - Alexandersson, Erik

AU - Gustavsson, Niklas

AU - Bernfur, Katja

AU - Karlsson, Adine

AU - Kjellbom, Per

AU - Larsson, Christer

PY - 2008

Y1 - 2008

N2 - 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.

AB - 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.

U2 - 10.1007/978-1-59745-028-7_11

DO - 10.1007/978-1-59745-028-7_11

M3 - Article

VL - 432

SP - 161

EP - 173

JO - Methods in Molecular Biology

JF - Methods in Molecular Biology

SN - 1940-6029

ER -