Biomek fx laboratory automation workstation

Screening was performed on
a Beckman Coulter Biomek FX Laboratory Automation Workstation (Fullerton,
CA, USA) integrated with a Beckman Coulter DTX880 plate reader with
UV/Vis capability. The screening assay was run in 384-well plates
containing a 60 μL volume of 50 mM sodium phosphate, 100 mM
sodium chloride buffer (pH 7.0), HPA (1 μg mL^–1), and the commercially available HPA substrate 2-chloro-4-nitrophenyl
α-maltotrioside (1 mM; K_M = 3.6
mM). Triton X-100 (0.01%) was included to minimize detection of nonspecific
inhibitors. The natural product extract samples were present as DMSO
solutions that contained 5 mg mL^–1 of dried methanolic
extract, which were tested at a dilution of 60 nL in a final assay
volume of 60 μL. HPA was found to be unaffected by the addition
of this small amount of DMSO (0.1%) and Triton X-100 (0.01%). The
integrity of the assay was tested through the use of two test plates
run as the first and last plate of each batch and which contained
a serial dilution of the known HPA inhibitor acarbose. Validation
of hits from the primary screen was performed manually on a UV/Vis
spectrophotometer. Among the hits examined in this secondary screen,
the natural extract from the sea anemone Stoichactis helianthus proved to have the highest activity against HPA. A more detailed
account of the screening methodology used can be found in Tarling
et al. 2008.^{17 (link)}