1) 1 cm3 of the upper 0–5 cm section of each core was removed an

1). 1 cm3 of the upper 0–5 cm section of each core was removed and stored frozen into 15 ml centrifuges tubes until the later analysis. ELISA and protein phosphatase 1 inhibition assay (PPIA 1) are the most sensitive methods widely used for determination of microcystin (Adamovsky et al., 2007, Amorim and Vasconcelos, 1999, Babica et al., 2006, Kankaanpaa et al., 2007, Msagati et al., 2006, Nicholson et al., 2007, Sipia et al.,

2006 and Yu et al., 2002). ELISA is often advised for the analyses of cyanobacterial toxins when their concentrations are lower than high-performance liquid chromatography (HPLC) detection limit (Mazur-Marzec et al., 2006). However, occasionally ELISA can give false positive results, therefore to confirm the occurrence of microcystin in samples PPIA was additionally employed. Mussels and sediment samples were lyophilized (TEGA, Germany) and then extracted using 30 ml PI3K inhibitor of pure methanol per 1 g mussel and sediment dry weight. Extracts were disrupted by sonication (5 min) and then centrifuged for 15 min at 10,000 rpm 20 °C. The solvents were removed by rotary evaporation and the residue was re-dissolved in 1 ml of MiliQ water. After that samples were vortexed for 1 min and then centrifuged

for 15 min at 12,000 rpm 20 °C. Later on, the samples Androgen Receptor Antagonist screening library were subjected to solid phase extraction on Sep-Pak Vac C18 cartridges (200 mg, Waters, Massachusetts, USA). Chlorophyll a was extracted by adding 80% ethanol to sediment samples ( Jespersen and Christoffersen, 1987). After 24 h samples were centrifuged and obtained supernatant analyzed spectrophotometrically according to Lorenzen (1967).

Extracts of mussels and sediments were diluted in MilliQ water (10–5,000 times) and analyzed by enzyme-linked immunosorbent assay (ELISA). The ELISA test was performed using 3-mercaptopyruvate sulfurtransferase the EnviroGuard kit (Strategic Diagnostics, Newark, DE, USA), according to the manufacturers’ instructions. The same extracts were also analyzed by colorimetric protein phosphatase 1 inhibition assay (PPIA). The PPIA was carried out on a 96-well microplate according to the method described by Rapala et al. (2002). Bovine serum albumin (BSA) was obtained from Sigma–Aldrich (St. Louis, MO, USA). Dithiothreitol (DTT), MgCl2·6H2O, MnCl2·4H2O, Na2SO4, p-nitrophenyl phosphate (p-NNP – the substrate), tris-(hydroxymethyl)-aminomethane (Tris) were of analytical grade. The substrate and enzyme buffers were prepared immediately before the test. Catalytic subunits (2.5 U) of commercially available enzyme (PP1; New England Biolabs, USA) were diluted in 1.5 ml of the enzyme buffer. Subsequently 10 μl of standard solutions or sample were added to the well and mixed with 10 μl of PP1 in buffer. After 5 min incubation, 200 μl of p-NPP in buffer solution was added to each well. The content of the wells was mixed by swirling the plate sideways. After 2-h incubation at 37 °C, the absorbance of the solutions was measured.

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