. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. SYMBIOTIC PLASTID GENES IN SLUGS 37 researchers have attempted LHC immunoprecipitations be- cause of the pitfalls involved in precipitating inner-mem- brane proteins (Anderson and Blobel, 1983). Instead, other protocols have been designed using mild detergents to ex- tract intact photosystem holocomplexes from the thyla- koids. followed by protein separation on sucrose density gradients (Fawley and Grossman, 1986; Buchel and Wil- helm, 1993: Wolfe end., 1994; Schmid et 1997). These isolations require large amounts of st
. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. SYMBIOTIC PLASTID GENES IN SLUGS 37 researchers have attempted LHC immunoprecipitations be- cause of the pitfalls involved in precipitating inner-mem- brane proteins (Anderson and Blobel, 1983). Instead, other protocols have been designed using mild detergents to ex- tract intact photosystem holocomplexes from the thyla- koids. followed by protein separation on sucrose density gradients (Fawley and Grossman, 1986; Buchel and Wil- helm, 1993: Wolfe end., 1994; Schmid et 1997). These isolations require large amounts of starting material (Schmid et 1997) that greatly exceed what is available to us in the slugs. So, instead, we used the LHCI antibody to demonstrate that LHCI had incorporated radioactivity. Following the procedure described above, the protein A Sepharose beads were reacted with anti-LHCI and then with a radiolabeled plastid protein extract. The antigen-anti- body-protein-A Sepharose bead complexes were repeatedly washed by centrifugation until the radioactivity in the su- pernatant was reduced to background. The washed antigen- antibody-protein-A Sepharose bead complexes were resus- pended in optifluor (Packard), and radioactivity was determined by a scintillation counter. Controls for nonspe- cific binding to protein-A Sepharose beads were conducted with the same procedure, but without the addition of the LHCI antibody. Counts per minute resulting from nonspe- cific binding were subtracted from experimental values for each inhibitor treatment and controls, and the final data were converted to cpm (jug chlorophyll)"' (ju,g protein)"1. The normalized data were averaged and expressed in terms of percent of control for each inhibitor. Results Plastid protein synthesis and identification The Coomassie-stained SDS-PAGE gels of protein ex- tracts from isolated slug plastids were similar to controls regardless of the inhibitor present, either CHX or CAP, indicating no diff
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Keywords: ., bookauthorlilliefrankrat, booksubjectbiology, booksubjectzoology
