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Measurement of Cholesterol in Membranes

Cholesterol is an amphipathic molecule of great importance in biology. It can be measured by enzymic and chemical methods, directly or indirectly following extraction from tissues or membranes. Before determination, cholesterol esters must be hydrolyzed using either chemical me ...

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Chemical Assays for Proteins

The choice of a protein assay for membranes and membrane proteins is dependent on a number of considerations: sensitivity, specificity (both with respect to variation between proteins and interference by nonprotein components), and simplicity.

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Analysis of Fatty Acids by Gas Liquid Chromatography

The fatty acids (FA) of animal, plant, and microbial origin are predominantly unbranched aliphatic chains with an even number of carbon atoms and a single carboxyl group. FA can be classified as saturated, monounsaturated, or polyunsaturated (Fig. 1). Saturates have all single carbon-to-c ...

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Separation and Analysis of Membrane Proteins by SDS-Polyacrylamide Gel Electrophoresis

Polyacrylamide gel electrophoresis (PAGE) in the presence of the anionic detergent, sodium dodecyl sulfate (SDS), is probably the most commonly used technique for the analysis of protein mixtures. SDS is a very effective solubilizing agent for a wide range of polypeptides, including mem ...

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The Identification of Subcellular Fractions from Mammalian Cells

The chapters in the two volumes of Biomembrane Protocols are broadly devoted to the methodologies involved in the structural and functional characterization of cell membranes. In many cases, a study of a particular membrane presupposes that it can be isolated by disruption of the cells and fr ...

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Quantification of Human Cytomegalovirus by Competitive PCR and Capillary Electrophoresis

Human cytomegalovirus (CMV) is a ubiquitous pathogen found in 40–100% of adults, and in about 1% of live births in the United States (1). It is the most common fetal and perinatal infectious organism; approx 10% of infected neonates are born with symptomatic congenital CMV disease, which is the most comm ...

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Enzyme Analysis: Cathepsin D as an Example

In enzyme analysis, capillary electrophoresis (CE) offers the ease of product separation from the substrate, with the ability to use expensive reagents in microvolumes. In CE, enzymes can be measured either as mass (when they are present in high concentration) by direct light absorbency, or by ...

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Myoglobin Analysis

Myoglobin represents the stores of oxygen in muscle tissues. Because of its relatively small mol wt, myoglobin is often used in electrophoretic techniques as a mol wt marker, and also as a test for separation efficiency in capillary electrophoresis (CE). The separation of myoglobin can be acco ...

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Analysis and Classification of Serum Cryoglobulins

Cryoglobulins (CG) are immunoglobulins that reversibly precipitate from serum in cold temperatures. They are classified into three types, based on the monoclonality of the γ-globulins present (1): Type I contains only monoclonal bands; type II contains mixed polyclonal immunoglo ...

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Immunosubtraction as a Means of Typing Monoclonal and Other Proteins in Serum and Urine

Serum and urine protein electrophoresis are used primarily to screen for the presence of monoclonal proteins found in conditions such as myeloma, non-Hodgkin’s lymphoma, macroglobulinemia, and so on. Having demonstrated the presence of an abnormal band, further testing is required to ...

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Electrophoresis of Cerebrospinal Fluid

Under normal circumstances, cerebrospinal fluid (CSF) is a clear and colorless fluid that is formed in the ventricles of the brain. It is in close proximity to the surface of both the brain and spinal cord, and, as a result, the analysis of CSF proteins and other constituents in samples taken by lumbar punct ...

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Study Origin of Germ Cells and Formation of New Primary Follicles in Adult Human and Rat Ovaries

The central thesis regarding the human ovaries is that, although primordial germ cells in embryonal ovaries are of extraovarian origin, those generated during the fetal period and in postnatal life are derived from the ovarian surface epithelium (OSE) bipotent cells. With the assistance ...

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Testicular Germ Cell Tumors in Mice: New Ways to Study a Genetically Complex Trait

Testicular germ cell tumors (TGCTs) are the most common cancer affecting young men. Although TGCTs are common and the genetic component of susceptibility is unusually strong, discovery of TGCT susceptibility genes in humans has been challenging. The 129/Sv inbred mouse strain is an impor ...

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Production of Knockdown Rats by Lentiviral Transduction of Embryos with Short Hairpin RNA Transgenes

The primary method for determining the function of a gene in rodents has been to make a knockout mouse through homologous recombination in embryonic stem cells. However, with the advent of RNA interference (RNAi) technology, new methods for studying gene function are now possible in a wide array of ...

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Deriving Mouse Spermatogonial Stem Cell Lines

In adult males, spermatogonial stem cells function to replenish developing gametes that are continuously released from the testes as mature spermatozoa. Because of their potential importance to research, medicine, industry, and conservation, numerous attempts have been made in t ...

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Isolating Highly Pure Rat Spermatogonial Stem Cells in Culture

Methods are detailed for isolating highly pure populations of spermatogonial stem cells from primary cultures of testis cells prepared from 22- to 24-day-old rats. The procedure is based on the principle that testicular somatic cells bind tightly to plastic and collagen matrices when cul ...

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Spermatogonial Stem Cell Transplantation, Testicular Function, and Restoration of Male Fertility in Mice

Mammalian spermatogonial stem cells, sometimes called male germline stem cells, are a small population of adult tissue-specific stem cells present in the testis. Formation of the spermatogonial stem cell population early in life and differentiation of spermatogonial stem cells in a ...

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Isolation of Mesenchymal Stem Cells from Murine Bone Marrow by Immunodepletion

Mesenchymal stem cells are typically enriched from bone marrow via their preferential attachment to tissue culture plastic. However, this isolation method has proven ineffective for murine MSCs because various hematopoietic cell lineages survive and/or proliferate on strom ...

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Gene Expression Analysis at the Single Cell Level Using the Human Bone Marrow Stromal Cell as a Model: Sample Preparation Methods

Recent advances in molecular technology, including gene expression microarray analysis, have allowed researchers to examine global patterns of gene expression at high resolution in populations of cultured cells or tissues. Although these techniques can be applied with great so ...

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Freezing Harvested hMSCs and Recovery of hMSCs from Frozen Vials for Subsequent Expansion, Analysis, and Experimentation

Human multipotential stromal cells (hMSCs) are easily isolated from bone marrow and can be expanded by up to 200-fold in culture. Cultures of hMSCs are heterogeneous mixtures of stem/progenitor cells and more mature cell types. The proportion of each cell type in a given culture depends on how the c ...

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