A novel and inexpensive source of allophycocyanin for multicolor flow cytometry.
Allophycocyanin (APC) belongs to a family of phycobiliproteins that are well suited as fluorescent reagents for flow cytometric analysis, since they have a broad excitation spectrum, a large Stoke's shift and they fluoresce with a high quantum yield. The widespread use of APC has been limited by the availability of raw material and high cost of the purified phycobiliprotein. We have assessed the suitability of dry, powdered Spirulina platensis, available at health food stores, as an inexpensive source of APC. APC was extracted from Spirulina platensis by overnight treatment with lysozyme, followed by ammonium sulfate precipitation. APC was then separated from phycocyanin (the only other major phycobiliprotein in Spirulina) by elution of bound material from an hydroxylapatite column using an increasing continuous phosphate gradient. APC isolated in this manner retained its normal trimeric structure. The absorbance and fluorescence excitation and emission spectra of the purified phycobiliproteins were identical to those previously shown for C-PC and APC. APC can be stored concentrated at 4 degrees C, frozen at -70 degrees C, or as a saturated ammonium sulfate precipitate, with no subunit dissociation or change in spectral properties. Moreover, APC has been conjugated to monoclonal and polyclonal antibodies for use in multicolor FACS analysis, with the conjugated antibody activity remaining stable for at least 2 years. Thus, this procedure is a simple, cost-effective method for preparing reagents for multicolor immunofluorescence and flow cytometry.
Jung
T M
TM
Department of Pathology, University of Iowa, College of Medicine, Iowa City 52242.
Dailey
M O
MO
D000458:Cyanobacteria / Q000032:analysis*; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D010798:Phycocyanin / Q000302:isolation & purification; D010860:Pigments, Biological
D010860:Pigments, Biological; C002497:allophycocyanin; D010798:Phycocyanin