Optimization of intracellular microcystin-LR extraction for its analysis by protein phosphatase inhibition assay.
Microcystins are toxins produced by some strains of cyanobacteria. Several methods have been developed to allow the quantification of microcystins, which are mainly endotoxins. Among those methods, the protein phosphatase inhibition assay is a good candidate as a screening method because of its sensitivity, simplicity and specificity. In this work a method for intracellular microcystin extraction in field water samples and lab cyanobacterial cultures prior to their analysis by protein phosphatase inhibition assay has been optimized. Microcystin-LR and Microcystis aeruginosa PCC 7806 were used as reference microcystin and strain, respectively, in order to optimize the protocol. The protocol consists on filtering the sample through a nylon filter of 0.8 microm, filter extraction with methanol 80% 0.1% trifluoroacetic acid (TFA) 0.1% tween 20, extract centrifugation and supernatant dilution (1/20). The establishment of an extraction protocol was carried out determining the extraction volume, time of extraction and number of extractions. The advantages of the method developed in this work are basically its simplicity and avoiding the use of specific and expensive equipment.
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Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias and Instituto de Biocomputación y Física de Sistemas Complejos (BIFI), Universidad de Zaragoza, Pedro Cerbuna 12, 50009 Zaragoza, Spain. esevilla@unizar.es
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D017033:Anabaena / Q000166:cytology / Q000378:metabolism; D002734:Chlorophyll / Q000737:chemistry; D008387:Marine Toxins; D052998:Microcystins / Q000737:chemistry*; D046931:Microcystis / Q000166:cytology / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors*; D014871:Water Microbiology; D014874:Water Pollutants, Chemical
D008387:Marine Toxins; D052998:Microcystins; D014874:Water Pollutants, Chemical; D002734:Chlorophyll; D010749:Phosphoprotein Phosphatases; C057862:cyanoginosin LR