|MadSci Network: Botany|
Charlotte, I am drawing from 2 references, one of which is quite dated, but contain protocols for pigment extraction. These are fairly involved extractions and much too involved to type in completely. Get these references from the local academic library or through library loan. Newer references may be useful for HPLC separation. Siegelman, H.W. & J. H. Kycia. Algal Biliproteins. IN: Handbook of Phycological Methods. Hellebust & Craigie (eds) Cambridge Univ. Press. 1973 Kremer, B.P. Electrophoretic separation and spectral characterization of algal phycobiliproteins. IN: Experimental Phycology - A laboratory manual. Lobban, Chapman & Kremer (eds) Cambridge U Press 1988. From Kremer: These are just the very basics, the references have complete instructions. Extraction: Homogenize by grinding 20 g Fresh or 5 g freeze dried algae (preferably under N2). Extract on COLD 0.1 M phosphate buffer, pH 6.9, w/ 1mM EDTA, 1 mM Dithiothreitol) stirring continuously, overnight in COLD (refrigerator). Ultrasonic cell disruption prior to extraction may improve yield. Purification: Filter suspension through cheesecloth and paper filters Centrifuge to remove particulates. Fractionate by adding increasing amounts of ammonium sulfate (~2 g/100 ml/min) to salt out the phycobiliproteins (PCBs)while stirring and COLD. At approx. 30% saturation, chlorophyll-protein will precipitate. Remove this contaminant. At 30-60% PCBs will precipitate. Keep this fraction. The procedure for further handling of the PCBs, resuspension, purification and separation are in the references, and too long to be reprinted here. They involve methanol and/or chloroform cleaning of the extracts, and removal of the bound proteins. The older reference has information on chromatography (silica gel) separation, and the newer reference has info on electrophoretic separation. Both contain steps for spectral characterization.
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