MadSci Network: Botany

Re: water soluble pigment extraction

Area: Botany
Posted By: karen Culver-Rymsza, Grad student oceanography
Date: Wed Jun 26 13:40:05 1996


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

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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