Isolation and immobilization of various plastid subtypes by magnetic immunoabsorption

Antibodies have been prepared which immuno-localize to the outer membrane of the pea chloroplast envelope and cause agglutination of isolated chloroplasts. This antisera is immunoreactive with a variety of plastid forms from both monocotyledonous and dicotyledonous plants. Whether such antibodies might be effectively used for isolation and immobilization of plastids from whole cell lysates has been tested. A system has been developed for immunolabeling various forms of higher plant plastids with biotinylated antibody and streptavidin magnetic nano-particles followed by separation of the plastids in a 0.6 Tesla high gradient magnetic field. Using this magnetic immunoabsorption procedure it has been possible to achieve a high degree of positive enrichment for chromoplasts, amyloplasts, and chloroplasts from whole cell lysates of several plant species. The integrity of these plastids has been examined by in organellar protein synthesis, ¹⁴C-ADP-glucose uptake, flow cytometry, in vitro synthesized precursor import and FITC-cationized ferritin staining of the plastid envelope. Western blot analysis showed significant enrichment for amyloplasts from cytosolic sucrose synthase in maize endosperm. Magnetic immunoabsorption of subcellular structures from whole cell lysates is a new method that may be useful in the in vitro analysis of many different cellular compartments from a wide range of organisms.