PHERAstar FSX

Cullin-RING E3 ubiquitin ligases (CRLs) are activated by neddylation and inactivated by de-neddylation that is accomplished by the COP9 signalosome (CSN). Trapping CRLs in their inactive state is reported to elevate tumor suppressors. Therefore, inhibition of CSN is a potential modality for cancer treatment.

Here, we show the development of a high-throughput de-neddylation assay for a CRL substrate that was used to find inhibitors of CSN. The fluorescence polarization assay was optimized using a fluorophore with comparatively long fluorescence lifetime.

Performing the assay in a 384 well plate and reading it on a PHERAstar^® microplate reader provided an assay window of 130 mP. The reader's capability of simultaneous detection of the two polarization channels accelerated the measurement and enabled kinetic reads. Hence, the assay is applicable for both CSN inhibitor finding and characterization.

Metallic nanoparticles became subject of intensive research because of their potential antibiotic properties. Nanoparticles such as silver, gold or zinc oxide particles are easily and cost-effectively synthesized by blending metal salts with plant extracts that reduce the metal. Different extracts, varying in the plant or the part of the plant used for the extract, are currently investigated in regard to their capacity to form nanoparticles and their antimicrobial efficacy. The formation of nanoparticles can be verified by UV-Vis spectroscopy due to surface plasmon resonance of the particles that lead to a characteristic spectrum defined by the underlying metal and particle size. Subsequent analysis of nanoparticles on microbial growth is typically tested by methods based on absorbance changes.

Here, we present how the spectrometer-based BMG LABTECH instruments are used to quickly confirm Ag and ZnO nanoparticle formation and their inhibitory effect on the diarrhea-causing bacteria Vibrio cholerae and enterotoxic Escherichia coli (ETEC).

The cell membrane is a bilayer of phospholipids with embedded proteins. It contains cholesterol that determines the membrane's fluidity, permeability and activity of membrane proteins. Changes in membrane cholesterol are implicated in diseases such as Alzheimer's and cancer, demanding its investigation.

NR12S is a fluorophore that exhibits emission maxima at 560 and 630 nm dependent on the liquid order of the membrane. Incorporation of the dye in a liquid ordered phase (increased cholesterol) shifts the emission to 560 nm, whereas in a liquid disordered phase (lower cholesterol) it emits at 630 nm. Hence, the 560/630 ratio correlates with the membrane cholesterol content.

Inhibition of cholesterol synthesis by lovastatin or incubation with methyl-§-cyclodextrin decreased membrane cholesterol in hematological cells and was reported by decreases in the 560/630 ratio. Both emissions were conveniently measured on the filter-based FLUOstar^® Omega microplate reader and could be analyzed with one-click using the MARS analysis software.

Due to their role in signal transmission from the outside to the inside of a cell, G-protein coupled receptors are prominent drug targets. Signaling from a stimulated Gq-coupled receptor activates Phospholipase C (PLC) and results in production of the second messengers PIP₂, DAG and finally in the release of Ca²⁺.

Montana Molecular developed genetically encoded biosensors that decrease (downward) or increase (upward) fluorescence in presence of the specific Gq associated second messengers. They are available in red and green to enable multiplexing.

The sensors were expressed in HEK293 cells and the acetylcholine receptor was activated by Carbachol. Generation of DAG, PIP₂ and Ca²⁺ was reliably reported by the biosensors and the CLARIOstar^® proved a suitable detector: filter-based and monochromator-based measurements were sensitive and fast enough to detect changes of up to two second messengers upon carbachol injection in one well, necessary for unambiguous identification of Gq signaling.

Measuring the protein concentration of liquid samples is a routine analysis in many life science laboratories. Accurate quantification is often a critical step for subsequent analyses such as protein characterization or western blots. Absorbance-based methods are well-established, easy to handle and cheap. They can be performed in microplates, allowing for high sample numbers processed at a time and low reagent volumes.

Spectrometer-based BMG LABTECH readers capture absorbance spectra or absorbance at discrete wavelengths from 220-1000 nm in less than a second per well and therefore can easily read these assays. The most common methods absorbance at 280 nm, Bradford, Bicin-Choninic Acid (BCA) and Lowry assay are presented here.