生物化学技术

Studies in many areas of biology are hampered by the complexity of the system being studied. This suggests that such areas of study could benefit from the development and application of new and more powerful analytical tools. Traditionally, investigators have chosen analytical methods t ...

Nitric oxide and superoxide rapidly combine to form a toxic reaction product, peroxynitrite anion (ONOO−) (1,2). The oxidant reactivity of peroxynitrite is mediated by an intermediate with the biological activity of the hydroxyl radical. However, this product does not appear to be the hydr ...

Uric acid (UA), (7,9-dihydro-1H-purine-2, 6, 8 (3H)-trione), is the principal end product of purine metabolism (1); therefore, determinations of UA in biological samples can serve as a marker in detection of disorders associated with purine metabolism, such as gout, Lesch-Nyhan syndrome (2,3), ...

As the primary fat-soluble antioxidant in human tissues, α-tocopherol absorption and metabolism have been the focus of active investigation, as reviewed recently (1,2). Although the concentration of any metabolite measured in plasma or tissue at a given time can be used as an indicator of the s ...

Vitamin E is comprised of two homologous series of tocochromanols, termed “tocopherols” and “tocotrienols.” They are structurally related, having a common chromanol ring, but distinguished by their side chains. Tocopherols have a saturated phytyl tail, whereas the tocotrienols po ...

β-Carotene is the major precursor of vitamin A (retinol) and is found mainly in fruits and vegetables. In humans, 30–40% of β-carotene is absorbed via the intestine as an intact molecule and then incorporated into chylomicrons; however, the majority of β-carotene (60–70%) is metabolized to retin ...

Cells, tissues, and organs require energy to function normally. Some of them are metabolic providers, and some others are users. Body fluids act as transporters and distributors of metabolic “fuels” between cells and maintain relatively constant conditions through homeostasis. The r ...

The isolation of proteins from plants for two-dimentional electrophoresis (2-DE) is complicated by the plants’ hard cell walls, high frequency of modified N-termini, and insolubility of membrane proteins. Some of these difficulties are not limited to plant proteins, but are also rather c ...

Accurate quantitative and qualitative comparison of resolved proteins by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) requires that identical amounts of sample be loaded on each gel. Use of trichloroacetic acid (TCA) or organic solvent precipitation proto ...

The in vivo radioactive labeling of proteins is used to enhance the sensitivity of detection and to quantitate the abundance individual proteins. To compare the quantity and identity of resolved proteins using 2-D gels, identical amounts of sample must be loaded. It is therefore a necessity to m ...

There are several advantages to the use of carrier ampholytes for running IEF separations. Tube gels using carrier ampholytes are easy to prepare and do not require sophisticated gradient casting equipment. Ampholyte mixtures can be simply blended and optimized for wide or more limited pH r ...

Two-dimensional electrophoresis (2-DE) is potentially the most powerful technique known for resolving complex protein mixtures. Proteins can be purified to homogeneity in one step in most cases. The applications for 2-DE are numerous and diverse. They include the analysis of protein p ...

Nonequilibrium pH gel electrophoresis (NEpHGE) is a technique developed to resolve proteins with extremely basic isoelectric points (pH 7.5–11.0) (1,2). These proteins are difficult to resolve using standard IEF, because the presence of urea in IEF gels has a buffering effect and prevents ...

Several formats—small, standard, and large—can be employed to separate proteins by two-dimensional (2-D) protein electrophoresis. The number of proteins in the sample as well as the degree of resolution needed determine the particular format used. The small format is preferred when few p ...

Two-dimensional electrophoresis (2-DE) of proteins is used for several purposes, such as resolving a distinct group of proteins (e.g., serum proteins), revealing the heterogeneity of a particular protein (e.g., actin, transferrin), purifying a protein, or testing the purity of a protein ga ...

Dried gel strips containing immobilized pH gradients (IPG) were commercially introduced in 1991 (Pharmacia Biotech, Immobiline� DryStrip gels). Their adoption for the first dimension of 2-D electrophoresis has produced significant improvements over the classical O’Farrell ...

One of the main requirements for a 2-D protocol is reproducibility of spot position, and, indeed, the technique of isoelectric focusing on immobilized pH gradients (IPGs) is ideally suited to provide highly reproducible 1-d separations. IPGs are obtained through the copolymerization of ...

Two-dimensional electrophoresis (2-D) of proteins used to be an art practiced by a few researchers, and their worldwide meetings could be held in a side room of a medium-sized hotel. With the rapidly growing volume of sequence data produced by the genome projects and the development of new mass spect ...

Immobilized pH †adients_~AIPGs~B_for_isoelectric_focusing_~AIEF~B_were_introduced_in_1982_~A1~B._After_experiencing_initial_problems_in_handling_IPGs~E_a_basic_protocol_of_two~Fdimensional_electrophoresis_~A2~FDE~B_with_IPGs____________in_the_first_dimension~E_followed_by_horizontal_or_vertical_SDS_elect_...

Compared to classical two-dimensional electrophoresis (2-DE) with carrier ampholytes (1,2), the advent of 2-DE with immobilized pH gradients (IPG-Dalt) (3) has produced significant improvements in 2-D electrophoretic separation with respect to: