Abstract
The Golgi apparatus is an essential component in the plant secretory pathway. The enrichment of Golgi membranes from plant tissue is fundamental to the study of this structurally complex organelle. The utilization of density centrifugation for the enrichment of Golgi membranes is still the most widely employed isolation technique. Generally, the procedure requires optimization depending on the plant tissue being employed. Here we provide a detailed enrichment procedure that has previously been used to characterize cell wall biosynthetic complexes from wheat seedlings. We also outline several downstream analyses procedures, including nucleoside diphosphatase assays, immunoblotting, and finally localization of putative Golgi proteins by fluorescent tags.
Keywords:
Density gradient centrifugation; Golgi apparatus; Nucleoside diphosphatase; Subcellular localization.
MeSH terms
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Acid Anhydride Hydrolases / chemistry
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Agrobacterium tumefaciens / genetics
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Agrobacterium tumefaciens / metabolism
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Blotting, Western
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Carrier Proteins / chemistry
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Carrier Proteins / isolation & purification*
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Cell Fractionation / instrumentation
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Cell Fractionation / methods*
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Centrifugation, Density Gradient / instrumentation
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Centrifugation, Density Gradient / methods
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Culture Media / chemistry
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Electroporation / methods
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Enzyme Assays
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Fluorescent Dyes / chemistry
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Golgi Apparatus / chemistry*
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Intracellular Membranes / chemistry
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Microsomes / chemistry
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Plant Proteins / chemistry
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Plant Proteins / isolation & purification*
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Seedlings / chemistry*
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Seedlings / growth & development
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Seeds / growth & development
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Sucrose / chemistry
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Transformation, Genetic
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Triticum / chemistry*
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Triticum / growth & development
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Ultracentrifugation / instrumentation
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Ultracentrifugation / methods
Substances
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Carrier Proteins
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Culture Media
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Fluorescent Dyes
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Plant Proteins
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Sucrose
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Acid Anhydride Hydrolases
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nucleoside-diphosphatase