Luminescence Detection Assays
Luminescence techniques have revolutionized the study of biological systems by making it possible to visualize molecular events with exceptional sensitivity and selectivity. The development of fluorophores with enhanced photophysical properties has contributed to the growing success of luminescent approaches. For the purpose of creating intelligent chemical sensors that can track enzyme activity, these fluorophores can be included into receptor systems. Creative Biolabs provides luminescence detection assays to determinate kinase activities and screen kinase regulators for the development of new drugs.
Luminescence Detection Assays in Creative Biolabs
- ADP detection assay
After fluorescence-based detection proved to be successful for the majority of kinase assays, luminescence-based detection was introduced and adopted as a standard platform for biochemical and cell-based experiments. The idea is that the kinase process's unused ATP is depleted in the first phase, concurrently stopping the reaction, and the ADP that is produced as a byproduct of the kinase reaction is transformed into ATP in the second step. In turn, luciferin uses this freshly created ATP to create oxyluciferin, which releases a yellow-green photon and produces light when exposed to oxygen. The firefly luciferase enzyme, which is frequently employed in luminescence investigations, catalyzes this process.
- Amplified luminescent proximity homogenous assay
The idea of the amplified luminescent proximity homogenous test is the same as that of FRET. Donor beads activate acceptor beads when they approach, releasing light in the 520–620 nm region. Because of the hydrogel coating on these beads, nonspecific binding and self-aggregation are prevented. Because phthalocyanine, a photosensitizer, is present on the donor beads, ambient oxygen is transformed to singlet oxygen when the bead is excited at 680 nm. The energy carried by this singlet oxygen reaches acceptor beads that are close by, causing them to release light at 520–620 nm. There is no signal visible when the donor and acceptor beads are separated.
Fig.1. Amplified luminescent proximity homogenous assay.1
- Lanthanide-based sensors assay
Lanthanides have been employed as probes to measure a wide range of enzyme activity, mostly by identifying the tiny molecules involved in the catalytic process. One especially useful tactic for the development of lanthanide-based kinase sensors has been the creation of an efficient coordinating environment upon phosphorylation. The foundation of the probe design is the substitution of a serine residue for a crucial glutamic acid residue, which is necessary for tight metal binding in the calcium-binding loop of EF hands. A substantial loss of metal-binding affinity would follow from this substitution, but it might be regained after being phosphorylated by particular kinases and producing a chelating phosphoserine residue that reconstitutes the metal-binding site.
Fig.2. Lanthanide-based sensors assay.2
The following characteristics of an optimal luminescence detection assay for kinase activity should be met: a high selectivity for pyrophosphate or ATP; a low sensitivity to interference from metal ions, proteins, or other biophosphates; and a very fast fluorescence response to allow for kinetic measurements. Please contact us for more information about our luminescence detection assays for kinase drug discovery. Our knowledgeable staff provides the most polished solutions to guarantee our clients' research continues forward.
References
- Yanamandra, Mahesh, Sayan Mitra, and Archana Giri. "Development and application of PI3K assays for novel drug discovery." Expert Opinion on Drug Discovery 10.2 (2015): 171-186.
- Pazos, Elena, and M. Eugenio Vázquez. "Advances in lanthanide‐based luminescent peptide probes for monitoring the activity of kinase and phosphatase." Biotechnology Journal 9.2 (2014): 241-252.
