Methods in molecular biology
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Application of bioluminescence resonance energy transfer (BRET) assay has been of special value in measuring dynamic events such as protein-protein interactions (PPIs) in vitro or in vivo. It was only in the late 1990s the BRET assay using RLuc-YFP was introduced for biological research showing its use in determining interaction of two proteins involved in circadian rhythm. Several inherent attributes such as rapid and fairly sensitive ratiometric measurements, assessment of PPI irrespective of protein location in cellular compartment, and cost-effectiveness consenting to high-throughput assay development make BRET a popular genetic reporter-based assay for PPI studies. ⋯ In recent years, BRET-related research has become significantly versatile in the assay format and its applicability by adopting the assay on multiple detection devices such as small-animal optical imaging platform or bioluminescence microscope. Beyond the scope of quantitative measurement of PPIs and protein dimerization, molecular optical imaging applications based on BRET assays have broadened its scope for screening of pharmacological compounds by unifying in vitro, live cell, and in vivo animal/plant measurement all on one platform. Taking examples from the literature, this chapter contributes to in-depth methodological details on how to perform in vitro and in vivo BRET experiments, and illustrates its advantages as a single-format assay.
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Unique from other brain disorders, traumatic brain injury (TBI) generally results from a discrete biomechanical event that induces rapid head movement. The large size and high organization of the human brain makes it particularly vulnerable to traumatic injury from rotational accelerations that can cause dynamic deformation of the brain tissue. Therefore, replicating the injury biomechanics of human TBI in animal models presents a substantial challenge, particularly with regard to addressing brain size and injury parameters. ⋯ Through a range of head rotational kinematics, this model can produce functional and neuropathological changes across the spectrum from concussion to severe TBI. Notably, however, the model is very difficult to employ, requiring a highly skilled team for medical management, biomechanics, neurological recovery, and specialized outcome measures including neuromonitoring, neurophysiology, neuroimaging, and neuropathology. Nonetheless, while challenging, this clinically relevant model has proven valuable for identifying mechanisms of acute and progressive neuropathologies as well as for the evaluation of noninvasive diagnostic techniques and potential neuroprotective treatments following TBI.
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Protein phosphorylation plays an essential role in the regulation of various cellular functions. Dysregulation of phosphorylation is implicated in the pathogenesis of certain cancers, diabetes, cardiovascular diseases, and central nervous system disorders. As a result, protein kinases have become potential drug targets for treating a wide variety of diseases. ⋯ However, identification of bona fide kinase substrates has remained challenging, necessitating the development of new methods and techniques. The kinase assay linked phosphoproteomics (KALIP) approach integrates in vitro kinase assays with global phosphoproteomics experiments to identify the direct substrates of protein kinases. This strategy has demonstrated outstanding sensitivity and a low false-positive rate for kinase substrate screening.
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Protein kinases are widely considered to be invaluable target enzymes for drug discovery and for diagnosing diseases and assessing their prognosis. Effective analytical techniques for measuring the activities of cellular protein kinases are therefore required for studies in the field of phosphoproteomics. We have recently developed a highly sensitive microarray-based technique for tracing the activities of protein kinases. ⋯ In this chapter, we describe a standard protocol for detecting phosphopeptides by biotin-labeled Phos-tag. We also describe a microarray system for high-throughput profiling of intracellular protein kinase activities. The Phos-tag-based method is expected to be useful in the rapid detection of the complex range of phosphorylation reactions involved in cellular signaling events, and it has potential applications in high-throughput screening of kinase activators or inhibitors.
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Phosphoproteomics relies on methods for efficient purification and sequencing of phosphopeptides from highly complex biological systems, especially when using low amounts of starting material. Current methods for phosphopeptide enrichment, e.g., Immobilized Metal ion Affinity Chromatography and titanium dioxide chromatography provide varying degrees of selectivity and specificity for phosphopeptide enrichment. ⋯ The method relies on the initial enrichment and separation of mono- and multi-phosphorylated peptides using Immobilized Metal ion Affinity Chromatography and a subsequent enrichment of the mono-phosphorylated peptides using titanium dioxide chromatography. The two separate phosphopeptide fractions are then subsequently analyzed by mass spectrometric methods optimized for mono-phosphorylated and multi-phosphorylated peptides, respectively, resulting in improved identification of especially multi-phosphorylated peptides from a minimum amount of starting material.