Methods in molecular biology
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The posttranslational modification of proteins is important for the regulation of enzymatic activity, protein half-life, and interaction with other molecules. One of the best understood posttranslational modifications is the reversible phosphorylation of proteins at serine, threonine, or tyrosine residues. ⋯ Furthermore, phosphoproteome analyses are incompatible with long organelle isolation procedures prior to analysis, because of the highly dynamic nature of regulatory phosphorylations. In this chapter, we provide a detailed step-by-step overview of the complex experimental setup required for successful chloroplast phosphoproteome analysis, report our experience with existing methods, and comment on their application in the field.
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Since the first fungal genome was sequenced in 1996, sequencing technologies have advanced dramatically. In recent years, it has become possible to cost-effectively generate vast amounts of DNA sequence data using a number of cell- and electrophoresis-free sequencing technologies, commonly known as "next" or "second" generation. In this chapter, we present a brief overview of next-generation sequencers that are commercially available now. Their potential applications in fungal genomics studies are discussed.
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Understanding the signaling pathways governing pluripotency and self-renewal is a prerequisite for better controlling stem cell differentiation to specific fates. Reversible protein phosphorylation is one of the most important posttranslational modifications regulating signaling pathways in biological processes. Global analysis of dynamic changes in protein phosphorylation is, therefore, key to understanding signaling at the system level. ⋯ Our method combines the use of strong cation exchange (SCX) and titanium dioxide (TiO(2)) for phosphopeptide enrichment, high-resolution MS for peptide and protein identification, and stable isotope labeling by amino acids in cell culture (SILAC) for quantification. This approach allows us to identify thousands of phosphorylation sites and profile their relative abundance during differentiation. This systems-biology-based approach provides new insights into how human pluripotent stem cells exit the pluripotent state.
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For certain applications, particularly experiments involving high-resolution imaging, it is necessary to culture cells on glass slides or cover glasses. This chapter describes techniques for successfully growing human embryonic stem cells (hESCs) on glass surfaces under three different conditions - serum-containing, serum-free, and following single-cell dissociation. It is anticipated that these techniques will extrapolate to other types of pluripotent stem cells such as induced pluripotent stem cells (iPSCs) and embryonic germ cells (EGCs).
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Human embryonic stem cells (hESCs) are pluripotent cells derived from the embryo at the blastocyst stage. Their embryonic origin confers upon them the capacity to proliferate indefinitely in vitro while maintaining the capacity to differentiate into a large variety of cell types. ⋯ Consequently, the possibility to expand hESCs in serum-free and in feeder-free culture conditions is becoming a major challenge to deliver the clinical promises of hESCs. Here, we describe the basic principles of growing hESCs in a chemically defined medium (CDM) devoid of serum and feeders.