生物化学技术

Two-dimensional gel electrophoresis (2-DE) is a useful proteomic tool to unravel some of organism gene expression complexities, and to study directly the gene products and several of their corresponding posttranslational modifications. A simple and universal sample applicat ...

2-D gel electrophoresis is one of the most effective techniques for high-resolution separation of complex protein mixtures. Recent developments in the field of protein mass spectrometry allow the rapid and highly sensitive identification of proteins, and thus amplify the power of pre ...

The reproducibility and resolving power of immobilized pH gradient (IPG) led two-dimensional gel electrophoresis (2-DE) technology to the heart of proteome projects. Despite the recent progress achieved in this field, 2-DE is still not widely used as a screening tool either in industry or in ...

After IEF-IPG (see Chapters 21–24), the IPG gel strips are equilibrated in the presence of SDS, DTT, urea, glycerol, and iodoacetamide, and placed onto the surface of a horizontal or on top of a vertical SDS gel (1–3) (see Chapter 26). For horizontal setups, the laboratory-made or ready-made SDS-PAGE gel (E ...

The second dimension of 2-D-PAGE uses denaturation and detergent, usually sodium dodecyl sulfate (SDS), to separate proteins by exploiting their molecular mass differences. During SDS-PAGE, all the proteins in the mixture have the same net charge per gram, and movement through the gel is bas ...

Several approaches can be used for identifying 2-DE resolved proteins, including microsequencing, immunoblotting with highly specific antibodies, and mass spectrometry-based technologies (for a review of the latter approaches, see ref. 1). In the case of immunoblotting, althou ...

The task of separating complex mixtures of proteins into individual components was revolutionized by the development of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Modifications of this technique employing gradient gels significantly impr ...

A major mechanism that cells use to regulate protein function is by phosphorylation and/or dephosphorylation of serine, threonine, and tyrosine residues. Phosphopeptide mapping of these phosphorylated residues allows investigation into the positive and negative regulato ...

The solubilization process for 2-D electrophoresis has to achieve four parallel goals: 1. Breaking macromolecular interactions in order to yield separate polypeptide chains: This in

Rapid, reliable, and cost-efficient methods of ribonucleic acid (RNA) oligonucleotide synthesis are in demand owing to an increasing awareness of critical structural, functional, and regulatory roles of RNA throughout biology. The most promising area of growth and development is in ...

This chapter enables the reader to carry out the solid-phase synthesis of ribonucleic acid (RNA) using β-cyanoethyl phosphoramidite chemistry combined with tert-butyldimethylsilyl protection of the ribose 2′-hydroxyl group. Phosphoramidite monomers are activated with 5- ...

Conditions for the enzymatic release, chemical derivatization, and analysis of oligosaccharides from the consensus glycosylation sites on antibodies are described. Release of the oligosaccharides is from the native protein. The APTS derivatives of the released oligosaccha ...

This chapter presents methods for capillary electrophoresis (CE) fingerprinting of proteins in a cell extract and in single cells. A custom-made CE instrument with laser-induced fluorescence (LIF) detection, used for the analyses, is described. Detailed procedures are given for: (1) on ...

Protein adsorption to capillary walls is one of the major complications in protein analyses with capillary electrophoresis (CE). Coating the capillary with different materials is used to reduce the adsorption. This chapter overviews different approaches used for capillary coat ...

Autoradiography is used to visualize and quantitate radiolabeled proteins that are resolved by 2-D protein gel electrophoresis. Proteins are commonly labeled in vivo with either 3H, 14C, 35S (low-energy (β-emitters), 32P (high-energy β-emitter), or 125I (high-energy γ-rays). During f ...

The detection of radiolabeled proteins is of fundamental importance in experimental biology. 35S- and 14C-labeled proteins can provide investigators with information about protein expression, synthesis, and degradation. Moreover, posttranslational modifications, s ...

Silver staining of polyacrylamide gels was introduced in 1979 by Switzer et al. (1) and rapidly gained popularity owing to its high sensitivity, ca. 100 times higher than staining with Coomassie blue. However, the first silver-staining protocols were not trouble-free. High backgrounds and ...

The ability of two-dimensional electrophoresis (2-DE) to separate complex mixtures of proteins, such as represented by cells, tissues, and even whole organisms, has been recognized for more than 20 yr (1). Using “standard”-format (around 20�20 cm) 2-D gels, the method is capable of routinely se ...

The identification of proteins using preparative gel electrophoresis and mass spectrometry requires reversible staining of relatively thick (1–1.5 mm) polyacrylamide gels. We have found that staining with colloidal Coomassie brilliant blue G-250 or negative staining with im ...

Electrophoretically separated proteins may be visualized using organic dyes, such as Ponceau red, Amido black, fast green, or most commonly Coomassie Brilliant Blue (1,2). Alternatively, sensitive detection methods have been devised using metal ions and colloids of gold, silver, cop ...