MICROPLATE READERS
well plates (Figure 2) that was suitable with automation for screening of large compound libraries in days. In another example related to
targeted protein degradation, a NanoBRET-based approach was used to look at the dose- dependent ubiquitination of BRD4. Bromodomain-containing protein 4 is a transcriptional regulator implicated in cancer biology and infl ammation. Ubiquitination is a crucial step in the action of PROTACs, small molecules that help target unwanted proteins to the ubiquitin-proteasome system. In this case, PROTAC ARV-771 brought a BRD4-labeled HiBiT (a subunit of the NanoLuc luciferase) into proximity with the E3 ubiquitin ligase. Ubiquitin tags on the target protein earmarked it for degradation by the proteasome. Here it was possible to measure the live cell kinetics of ternary complex formation with the E3 ligase as well as the eff iciency with which the target protein is ubiquitinated, crucial information to confi rm mode of action and to probe ways to improve drug eff icacy (Figure 3).
fl uorescently labelled peptide ligand that binds to LC3A. p62 LIR peptide, a peptide ligand of LC3A, was labelled with a Cy5 fl uorophore to act as a tracer molecule. The addition of competing ligands of LC3A to the LC3A-tracer complex results in a displacement of tracer and a reduction of the fl uorescence polarisation signal (Figure 1). Miniaturisation of this assay allowed for library screening in 1536-
WHAT MICROPLATE READERS BRING TO STUDIES OF PROTEIN-PROTEIN INTERACTIONS Many applications in the laboratory
need to be performed at scale and sensitivity and speed are crucial. High- throughput screening assays need to be compatible with automation to accelerate measurements but must also deliver the required sensitivity. Binding studies
Figure 4: The PHERAstar is a multimode microplate reader
for protein-protein interactions provide valuable information on reaction kinetics, dissociation constants and the stoichiometry of interactions. FRET, for example, can be used together with dye-labelled proteins to determine the stoichiometry of protein interactions. Awareness of stoichiometry can be crucial for example in calculations to quantify labelled biomolecules that assemble or are
Figure 2: Miniaturisation of the LC3A fl uorescence polarisation assay allowed for library screen in 1536 well plates
Figure 3: NanoBRET assay monitoring ternary complex formation in live cell kinetics
active in diff erent ratios (dimers, trimers, etc.). Studies on binding kinetics on BMG Labtech microplate readers are facilitated by the MARS (Measurement, Analysis & Reporting) data analysis software. Multimode microplate readers HER star FS
like theP A Xfrom BMG
Labtech off er many features that make them ideal platforms for research applications at scale (Figure 4). Users have fl exibility of choice since all commonly-used detection modes are available at the performance level required for screening of molecules. In addition, features such as on-board reagent injectors, simultaneous dual emission for the detection of two emission signals at the same time, and ultrafast sampling rates with detection times of up to 0.01s allow kinetic analysis of interactions in real time at high throughput. Collectively, these features provide the robust high performance needed for automated applications at scale in the modern research laboratory. ■
For more information visit
www.bmglabtech.com
www.scientistlive.com 25
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