Methods in molecular biology
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With the appearance of a new generation of high-performance hybrid mass spectrometers, high accuracy (sub-parts-per-million) mass spectrometry is becoming increasingly available to a wider scientific community. Here we discuss the advantages of such mass spectrometric instrumentation in the global analysis of protein phosphorylation. We describe a detailed workflow for fractionation and enrichment of phosphopeptides from digests of whole cell/tissue lysates by strong cation exchange and TiO(2) chromatography and their subsequent measurement on an LTQ-Orbitrap mass spectrometer under several acquisition regimes.
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RNAi holds promise for neurodegenerative disorders caused by gain-of-function mutations. We and others have demonstrated proof-of-principle for viral-mediated RNAi in a mouse model of motor neuron disease. ⋯ This chapter describes the design, production, and titration of lentivirus and adeno-associated virus capable of mediating SOD1 knockdown in vivo. The delivery of the virus to the spinal cord directly, through intraspinal injection, or indirectly, through intramuscular injection, is also described, as well as the methods pertaining to the analysis of spinal cord transduction, SOD1 silencing, and determination of motor neuron protection.
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Molecular imaging offers many unique opportunities to study biological processes in intact organisms. Bioluminescence is the emission of light from biochemical reactions that occur within a living organism. Luciferase has been used as a reporter gene in transgenic mice but, until bioluminescence imaging was described, the detection of luciferase activity required either sectioning of the animal or excision of tissue and homogenization to measure enzyme activities in a conventional luminometer. ⋯ This imaging modality has proven to be a very powerful methodology to detect luciferase reporter activity in intact animal models. This form of optical imaging is low cost and noninvasive and facilitates real-time analysis of disease processes at the molecular level in living organisms. Bioluminescence provides a noninvasive method to monitor gene expression in vivo and has enormous potential to elucidate the pathobiology of lung diseases in intact mouse models, including models of inflammation/injury, infection, and cancer.
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Peptides scanned from whole protein sequences are the core information for many peptide bioinformatics research such as functional site prediction, protein structure identification, and protein function recognition. In these applications, we normally need to assign a peptide to one of the given categories using a computer model. ⋯ Among various machine learning approaches, including neural networks, peptide machines have demonstrated excellent performance in many applications. This chapter discusses the basic concepts of peptide classification, commonly used feature extraction methods, three peptide machines, and some important issues in peptide classification.
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There are various types of liposomes used for cancer therapy, but these can all be placed into three distinct categories based on the surface charge of vesicles: neutral, anionic and cationic. This chapter describes the more rigorous and easy methods used for liposome manufacture, with references, to aid the reader in preparing these formulations in-house.