You often need several pieces of information to really understand what is happening within a cell or population of cells. If your cells are not proliferating, are they dying? Or, are you seeing cytostasis? If they are dying, what is the mechanism? Is it apoptosis or necrosis? If you are seeing apoptosis, what is the pathway: intrinsic or extrinsic?
If you are measuring expression of a reporter gene and you see a decrease in expression, is that decrease due to transfection inefficiencies, cytotoxicity, or true down regulation of your reporter gene?
To investigate these multiple parameters, you can run assays in parallel, but that requires more sample, and sample isn’t always abundant.
Multiplexing assays allows you to obtain information about multiple parameters or events (e.g., reporter gene expression and cell viability; caspase-3 activity and cell viability) from a single sample. Multiplexing saves sample, saves time and gives you a more complete picture of the biology that is happening with your experimental sample.
A quick search of the PubMed database for “dual luciferase” quickly returns over 1,000 papers. The Dual-Luciferase® Reporter Assay is a powerful tool that allows researchers to ask a multitude of questions about gene control and expression in a system that itself could be normalized and internally controlled. For more than 15 years, firefly and Renilla luciferases have been valuable tools for researchers asking many different kinds of questions in the life sciences. In a recent webinar, Biologically Relevant Assays for Oncology: Harnessing the Power of Bioluminescence, Neal Cosby discussed how bioluminescent chemistries have formed the basis of a range of powerful assays and research tools for scientists who are asking questions about the deep and complex genetic and cellular story associated with cancer. Here we talk a bit of about bioluminescent chemistries, some of the newest bioluminescent tools available, and how some of these tools can be used to probe the deeper questions of cell biology, including cancer biology. Continue reading “Shining a Bright Light on Deep Questions in Biology with Bioluminescence”
If you could design the ideal kinase assay system what would it look like?
Would it be able to match, point for point, the results of the tried-and-true isotopic assay methods but not have any of the associated safety and waste disposal issues?
Would it avoid the use of specific antibodies?
Would it minimize false hits associated with many of the fluorescence-based assays?
Would it be affordable technology, adaptable to any laboratory’s throughput from 96-well to 1,536-well automated screening?
Would it be universal—able to assess the function of any kind of kinase (protein, lipid or sugar) that uses any kind of substrate?
Would it be able to detect low conversion rates (low-activity enzymes) with a high signal-to-background ratio?
Would you be able to use it with substrates that are multiphosphorylated?
If you answered “yes” to any of the above questions, you might want to take a look at the Promega Webinar “Enabling Kinase Research with a Luminescent ADP Detection Platform and Complete Kinase Enzyme Systems”, presented by Hicham Zegzouti, PhD, research scientist at Promega. Here he describes the ADP-Glo™ Assay platform, which meets these and several other criteria.
The precise molecular lesion that occurs with the Philadelphia Chromosome translocation—a rearrangement that creates a bcr-abl fusion in which the abl tyrosine kinase is constitutively active leading to the development of chronic myeloid leukemia is the first description of dysregulation of a kinase leading to a particular disease state. However, the human genome contains 518 protein kinases and many other atypical kinases, and one-third of all human proteins are phosphorylated. It is now estimated that over 400 human diseases are caused by dysregulation or mutation of kinases, making kinases a major target for drug discovery efforts. Continue reading “The Ideal Kinase Assay”