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Ribsowitches are putative drug targets as they often regulate the expression of essential bacterial genes. This finding necessitates the development of suitable assays, at best high-throughput (HT) compatible, which allow the screening of compound libraries for riboswitch activation. Here, we describe a HT-compatible fluorescence-based screening assay employing a minimal core motif of the Bacillus subtilis glmS riboswitch and the metabolite-induced self-cleavage assay using the full-length glmS ribozyme of Staphylococcus aureus for the identification of artificial molecules activating this regulatory RNA.

Drug screening is an essential and widely used technique for drug discovery in various biomedical fields notably in oncology. Here we describe a functional screening assay based on the bioluminescence detection of a secreted luciferase for monitoring cell viability of cancer cells in a high-throughput format. This assay allows the screening of large libraries comprising thousands of compounds and the identification of potential anticancer molecules in a rapid, facile, and cost-effective manner.

The identification of novel substrates of cytochrome P450 enzymes by high-throughput screening assays is of utmost importance to further increase the scope of these enzymes for future applications. Most screens are either confined to individual substrate analogues or hampered by low throughput due to elaborate analysis techniques. Here we describe a general high-throughput screening assay that interrogates the activity of P450 enzymes with the aid of catalase as a reporter enzyme.

As a model for teratogenicity research, zebrafish are gaining popularity and creditability. Zebrafish embryos have been proven to be a highly valuable tool in genetics and developmental biology research and have advanced our understanding of a number of known developmental toxicants. It has yet to be determined conclusively how reliably a zebrafish embryo screening assay predicts what will happen in mammalian models, but results from initial assessments have been encouraging. Here we have presented procedures for the basic care of a zebrafish colony to support embryo production, embryo collection and culturing, and teratogenicity experiments.

Understanding the molecular mechanisms of Alzheimer's disease (AD) is a challenging endeavor, namely, due to the fact that the disease only occurs in the central nervous system of elderly humans. Thus, model systems simply do not accurately portray this cellular landscape. While we cannot ask many mechanistic questions using the human brain as our test subject, cell culture techniques that have emerged at least provide us with the ability to pose fundamental biochemical questions, which may ultimately lead to translational outcomes for AD. In particular, the intracellular microtubule-associated protein tau that accumulates in AD is found in normal cells. Manipulating these cells may allow us to address basic questions about tau biology that would provide novel therapeutic strategies down the road. Here, we describe several techniques to explore tau cell biology using the Odyssey(®) Infrared Imaging system (Odyssey system) from LI-COR Biosciences. We provide a detailed protocol on how to perform a scalable drug screening assay called the In-Cell Western and follow-up these screens with standard Western analysis to confirm whether "hits" are valid by more traditional means. We provide some tips on where mistakes are most likely to occur, and we interpret our standard Western data, providing some estimation as to the composition of the banding pattern that is typical for this enigmatic protein.

HTS remains at the core of the drug discovery process, and so it is critical to design and implement HTS assays in a comprehensive fashion involving scientists from the disciplines of biology, chemistry, engineering, and informatics. This requires careful consideration of many options and variables, starting with the choice of screening strategy and ending with the discovery of lead compounds. At every step in this process, there are decisions to be made that can greatly impact the outcome of the HTS effort, to the point of making it a success or a failure. Although specific guidelines should be established to ensure that the screening assay reaches an acceptable level of quality, many choices require pragmatism and the ability to compromise opposing forces.

It is important to shorten the window period after acute HIV infection in which infected individuals are still antibody-negative, especially in blood donors. Newly developed fourth-generation assays detect antibodies to HIV-1, including subtype O, and to HIV-2 and, simultaneously, p24 antigen of HIV-1. To evaluate this assay for daily routine work we compared it with different third-generation assays using sera from uninfected patients and patients with known HIV infection. The most interesting sera are those drawn during seroconversion from freshly infected patients. Whenever we encounter such a patient with acute HIV infection we store the serum in aliquots at -20 degrees C. Thus, we were able to establish our own seroconversion panel and use it in our laboratory for evaluation of new assays. The new test was shown to be able to detect all chronically HIV-infected individuals and four of six patients during seroconversion although in two of these patients conventional assays for HIV antibodies were still negative. The rate of unspecific reactivities was slightly higher as compared with third-generation assays.

Protein-protein interactions are instrumental in virtually all biological processes and their understanding will shed light on designing novel and effective drugs for therapeutic interventions targeting the pathways in which they function. Protein-protein interactions have been studied using many genetic and biochemical methods, most recently, affinity capillary electrophoresis (ACE). We used ACE as a high-throughput screening assay to establish and define binding interactions between a therapeutic target protein and chemical entities from natural product or synthetic chemical libraries. Furthermore, ACE has demonstrated its value in the measurement of binding constants, the estimation of kinetic rate constants, and the determination of the stoichiometry of protein-protein interactions. Herein, we will describe qualitatively several assay formats using ACE for detecting protein-protein interactions, and discuss their advantages and limitations.