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Mitochondrial Proteome: Toward the Detection and Profiling of Disease Associated Alterations

Existing at the heart of cellular energy metabolism, the mitochondrion is uniquely positioned to have a major impact on human disease processes. Examples of mitochondrial impact on human pathology abound and include etiologies ranging from inborn errors of metabolism to the site of acti ...

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Mass Spectrometry-Based Biomarker Discovery

Discovery of candidate biomarkers within the entire proteome is one of the most important and challenging goals in proteomic research. Mass spectrometry-based proteomic is a modern and promising technology for semiquantitative and qualitative assessment of proteins, enabli ...

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Serum Low-Molecular-Weight Protein Fractionation for Biomarker Discovery

Protein biomarkers provide the key diagnostic information for the detection of disease, risk of disease progression, and a patient’s likely response to drug therapy. Potential biomarkers exist in biofluids, such as serum, urine, and cerebrospinal fluid. Unfortunately, discoveri ...

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Reverse-Phase Protein Microarrays

Cancer is the consequence of intra- and extracellular signaling network deregulation that derives from alteration of genetic and proteomic cellular homeostasis. Mapping the individual molecular circuitry of a patient’s tumor cells is the starting point for rational personali ...

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Tissue Microarrays as a Tool in the Discovery and Validation of Predictive Biomarkers

The tissue microarray (TMA) is the embodiment of high-throughput pathology. The platform combines tens to hundreds of tissue samples on a single microscope slide for interrogation with routine molecular pathology tools. TMAs have enabled the rapid and cost-effective screening of bi ...

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Antibody Microarrays: Analysis of Cystic Fibrosis

Cystic fibrosis (CF) is the most common autosomal recessive disease in the USA and Europe, whose life-limiting phenotype is manifest on epithelial cells throughout the body. The principal cause of morbidity and mortality is a massively proinflammatory condition in the lung. The mutation ...

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Laser Capture Microdissection: ArcturusXT Infrared Capture and UV Cutting Methods

Laser capture microdissection (LCM) is a technique that allows the precise procurement of enriched cell populations from a heterogeneous tissue under direct microscopic visualization. LCM can be used to harvest the cells of interest directly or can be used to isolate specific cells by abl ...

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Antibody Validation by Western Blotting

Validation of antibodies is an integral part of translational research, particularly for biomarker discovery. Validation is essential to show the specificity of the reagent (antibody) and to confirm the identity of the protein biomarker, prior to implementing the biomarker in clini ...

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Measurement and Manipulation of [Ca2+]i in Suspensions of Platelets and Cell Cultures

The importance of cytosolic calcium (Ca2+) elevation in the regulation of platelet functions (shape change, aggregation, and secretion) has been widely acknowledged. Therefore its concentration must be tightly regulated, with most of it being sequestrated in intracellular orga ...

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Isolation and Analysis of Platelet Lipid Rafts

The cell membrane can no longer be viewed as a homogenous fluid bilayer; instead, it is now known to contain discrete lateral microdomains with characteristic subsets of lipids and proteins. One such microdomain, termed the “lipid raft,” has received much attention over the past few years. Seve ...

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Phosphoinositides: Lipid Kinases and Phosphatases

Phosphoinositides (PIs) are a family of eight quantitatively minor membrane lipids playing important roles in the control of a variety of intracellular signaling mechanisms in eukaryotic cells (1–4). The metabolism of these peculiar lipids is highly controlled by a set of enzymes such as k ...

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The Study of Serine-Threonine Kinases

Serine-threonine kinases play crucial roles in activating and inhibitory signaling pathways that control platelet activation. Diversity in isozyme composition, catalytic and regulatory subunits, and subcellular localization provide a multitude of signaling propert ...

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Protein Tyrosine Phosphatases

The reversible covalent modification of proteins by the addition and removal of a phosphate group is an important theme in signal transduction in mammalian cells. There is a large superfamily of protein kinases that catalyse the addition of a phosphate group to hydroxyl residues on the side ch ...

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Study of Tyrosine Kinases and Protein Tyrosine Phosphorylation

In recent years, our increased understanding of the complex signal transduction mechanisms that regulate cellular function has fueled huge advances in all aspects of biomedical science and cell biology. Platelet and megakaryocyte function is no exception to this. In the last 10 yr our und ...

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Inhibitors of Cellular Signaling Targets: Designs and Limitations

Kinases carry out the reversible phosphorylation of proteins and lipids, and are responsible for direct or indirect control of almost every signaling pathway in cells, leading to responses such as proliferation, differentiation, metabolism, transport, and gene expression. It is es ...

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Pharmacological Approaches to Studying Platelet Function: An Overview

Although pharmacology may be strictly viewed as the study of drugs (see, for example, the British Pharmacological Society Web site at http://www.bps.ac.uk/BPS.html), for many of us it also involves the use of drugs to study receptors. Indeed, the fundamental principle behind a pharmacologic ...

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In Vitro Changes of Platelet Parameters: Lessons From Blood Banking

Platelets, the smallest cellular components of blood, are critically involved at each step of the hemostatic response, from the initial sealing of damaged endothelium to supporting coagulation reactions, and finally, in the retraction of the fibrin clot that enhances fibrinolysis a ...

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Manipulation of Gene Expression in Megakaryocytes

The process of megakaryocyte generation in the bone marrow and subsequent differentiation leading to platelet production is little understood. Known as megakaryo-poiesis, it involves a number of unique biological features, including an increase in the nuclear DNA content (endor ...

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Signaling Receptors on Platelets and Megakaryocytes

Although the body of knowledge is far from complete, much has been learned about the receptors that enable circulating platelets to become activated at sites of vascular injury. A variety of approaches have been used to acquire this information, many of which are described elsewhere in this book. ...

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Positional Cloning: Complex Cardiovascular Traits

Cardiovascular traits represent the quintessential complex trait derived from the confluence of numerous genetic and environmental risk factors. Therefore, positional cloning of cardiovascular genes contributing to the common forms of cardiovascular disease is much more ...

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