Methods in molecular biology
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Protein functions rely on their ability to engage in specific protein-protein interactions and form complexes that are dynamically regulated by stimuli. Bioluminescence resonance energy transfer (BRET) is a highly sensitive technique, which allows monitoring of interaction between two proteins: one tagged with the luminescent donor Renilla luciferase, the other with a fluorescent acceptor such as YFP. ⋯ To this aim, we tag proteins of interest, transfect cells with these fusions, and use the high-sensitivity microscopy, combined with electron multiplying cooled charge-coupled device (EMCCD) cameras and improved bioluminescence probes. We thus achieve rapid acquisition of high-resolution BRET images and study the localization and dynamics of protein-protein interactions in individual live cells.
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Knowledge of novel antibiotic resistance genes aids in the understanding of how antibiotics function and how bacteria fight them. This knowledge also allows future generations of an antibiotic or antibiotic group to be altered to allow the greatest efficacy. The method described here is very simple in theory. ⋯ Any colony that grows will possess the antibiotic resistance gene and can be further examined. In actual practice, however, this technique can be complicated. The detailed protocol will need to be optimized for each bacterial strain, vector, and cell line chosen.
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Protein kinases (PKs) are widely recognized as valuable targets for disease diagnosis and drug discovery. For this reason, we have developed a sensitive peptide microarray for detecting intracellular PK activity. Peptides are immobilized on a glutaraldehyde-premodified high-amino terminal glass slide, by spotting 2 nL volumes of substrate peptide solutions with an automated microarray spotter. ⋯ The peptide microarray system involves simple peptide immobilization, requires low sample volumes and provides a high density array. Importantly, it provides high sensitivity for detecting PK activities in cell lysates. Thus, the peptide microarray system is expected to be useful for a high-throughput kinase assay to investigate intracellular kinase activity and has potential applications in disease diagnosis and drug discovery.
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Migraine is a high prevalence disorder which affects a significant proportion of the general population, especially women during their central and more productive time of the life, thus causing severe disability. The genetic basis of the disease is unknown and the mechanism is poorly understood. The possibility that following a perturbation in the central nervous system, and particularly in the brainstem, trigeminal neurons become hyperexcitable and produce an uncontrolled release of sensory neuropeptides which eventually results in arterial vasodilatation and neuronal sensitization, has been gaining credit from studies in experimental animals and humans. In particular, experimental and clinical data with antagonists of the calcitonin gene-related peptide (CGRP) propose this molecule and its receptor as a major target for migraine treatment.