An understanding of endomembrane processes and their biological implication is important for understanding plant growth and development. Endosomes are a heterogeneous collection of organelles in the sorting and delivery of internalized material from cell surface and the transport of materials from Golgi to lysosome or vacuole. In plant cells, two clearly defined endosomal compartments have been identified: the trans-Golgi network (early endosome equivalent) and the multivesicular body (late endosome equivalent). Traditionally, methods for isolation and purification of these two compartments are tedious and time-consuming involving sucrose gradient centrifugation. In some cases, sucrose gradient isolation is coupled with immunoaffinity purification and larger amount of starting materials are usually required (10-50 g). We have developed this kit using a spin-column-based format coupling with selective precipitation for enrichment of endosomal compartment. This approach is easy and rapid and only small amount of starting material is required.
Detection of Enriched Endosomes from N. benthmiana by Western blotting. The plant leaf that carries GFP-tagged ara6 (early endosome marker) was processed as described in the protocol and probed with anti-ara6 and anti-histone antibodies. Lane 1, Total tissue lysate; lane 2, Supernatant from step 4 of the protocol; lane 3, Enriched endosome fraction.
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