生物化学技术

The ability of two-dimensional electrophoresis (2-DE), based on the original method developed more than 20 years ago (1), to separate simultaneously up to several thousand proteins using large-format gels (2) has made it the method of choice for the analysis of protein expression in complex bi ...

The profile of the two-dimensional (2-D) separation of the protein complement (proteome) of eukaryotic cells and tissues typically contains obvious “trains” of spots that differ in pI and/or apparent molecular mass. These are usually isoforms of the same protein and result from a variety of p ...

Prevalent methods for visualizing proteins resolved by two-dimensional (2-D) gel electrophoresis include autoradiography, silver staining, and Coomassie brilliant blue staining (1,2. The organic dye Coomassie brilliant blue is capable of detecting as little as 100 ng of protein, ...

Escherichia coli has been studied for many years by two-dimensional polyacrylamide gel electrophoresis (2-D gels) (1,2). This method has provided much information about the physiology of E. coli, particularly related to how the levels or synthesis rates of large numbers of proteins varied ...

Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) images, when used to analyze protein expression in large sets of biological samples, are best processed by computer. In this manner, one can detect and quantify even the faintest spots, quantitatively compare 2-D images am ...

A federated two-dimensional (2-D) database is a database containing 2-D data that are accessible on the World Wide Web (WWW) and that are dynamically linked to other similar databases through active hypertext links over the Internet (1,2). Federated 2-D databases respect five rules that can be f ...

In ref. 1, we described a computer-assisted visual method, Flicker, for comparing two two-dimensional (2-D) protein gel images across the Internet is described (see Note 5). This approach may be useful for comparing similar samples created in different laboratories to help putatively iden ...

Many laboratories have identified proteins on two-dimensional (2-D) maps, and have built a master gel and a related 2-D database containing data specific to the identified proteins. Several of these databases have been made available on the World Wide Web (WWW) (1), and many more will undoubtedly ...

Proteome analysis has been revolutionized by the marriage of two-dimensional (2-D) gel electrophoresis and mass spectrometry (1–4). Mass spectrometry in combination with database searching permits the large-scale identification of proteins, and 2-D gel electrophoresis all ...

Bacterial genomes range greatly in size, encoding from as few as 500 to more than 4000 proteins. The tools of bioinformatics have become more and more accurate at predicting open reading frames within stretches of DNA, and within this decade, the proteome complement of one or more genomes should be co ...

First devised in a time dominated by one-dimensional SDS-PAGE, immunoblotting (also dubbed “Western” blotting, ), is now widely used in conjunction with 2-D PAGE (electrofocusing/SDS-PAGE), whose diffusion is favored by its long-awaited standardization (3,4) and by the coming of age of pr ...

Two-dimensional electrophoresis (2-DE) is one of the most advanced techniques for direct separation of considerable numbers of proteins. Direct microsequence analysis of the amino (N)-terminal partial sequence of the electroblotted protein spots from the 2-DE gels and the homolo ...

Amino acid analysis is a powerful and sensitive technique for the determination of the amino acid composition of proteins. In the past, the automation of amino acid analysis was based on postcolumn ninhydrin analysis, e.g., the Beckman 6300. However, a demand for high sensitivity and high sample t ...

The yeast Saccharomyces cerevisiae became the first eukaryotic organism to have its entire genome sequenced (1). With the completion of the genome, over 6000 genes on 16 chromosomes were identified. Several laboratories are undertaking the task of identifying the yeast proteins on two- ...

For years, proteolytic digest and peptide purification were essential tools for the biochemist wishing to determine the primary structure of a protein. The strategies changed with the availability of a polypeptide gas-phase sequencer and the advent of DNA recombinant technology. Ho ...

Methods combining the high throughput and sensitivity of matrix-assisted laser desorption ionization mass spectometry (MALDI-MS) with polyacrylamide gel electrophoresis (PAGE) are rapidly gaining attention, but almost exclusively for applications eluting proteol ...

Polyacrylamide gel electrophoresis (PAGE) is probably the most general and widespread protein separation technique. This simple method is used in almost every molecular biology and biochemistry laboratory to monitor protein experiments, including consecutive stages of pr ...

Methods for the identification of proteins have advanced dramatically this decade through the introduction of mass spectrometric techniques and instrumentation sensitive enough to be applicable to biological systems (1). The two mass spectrometric techniques that have prov ...

Protein identification and analysis software performs a central role in the investigation of proteins from two-dimensional (2-D) gels. For protein identification, the user matches certain empirically gained information against a protein database to define a protein as already k ...

The sequencing of complete genomes is providing an enormous resource for understanding the biology of organisms (1). A complete genome sequence, however, will still leave many unanswered questions about the basic biology of the organism. Which open reading frames are authentic genes? Wh ...