生物化学技术

This chapter presents an integrated and modular microsystem providing rapid analyses of low femtomole of in-gel digests for proteomics applications. Enhancement of sample throughput is facilitated using an autosampler, a microfabricated device comprising a large (2.4-μL tot ...

Complex proteome samples require efficient separation and detection methods in order to characterize their protein components. On-line combination of capillary isoelectric focusing (CIEF) with electrospray ionization (ESI) mass spectrometry (MS) is shown as an effective m ...

Separation in capillary electrophoresis (CE) is based on the movement of charged compounds inside a background electrolyte under an applied potential. Because the mechanism of separation of CE differs from that of conventional high-performance liquid chromatography (HPLC), wh ...

A novel technique, frontal analysis continuous capillary electrophoresis (FACCE), has been described as an effective way to study protein-polyelectrolyte binding. FACCE involves continuous sampling, integrating sample injection and separation into one process that prov ...

The recently developed whole-column-imaging detection technique for capillary electrophoresis (CE) and capillary isoelectric focusing (CIEF), a commercial whole-column-imaged CIEF instrument and its standard operation protocol are introduced. Furthermore, new de ...

This chapter provides an overview of protein charge ladders—collections of protein derivatives that differ in charge—and capillary electrophoresis (CE). The combination of charge ladders and CE is a useful biophysical tool for measuring the net charge of proteins and the role of electr ...

A screening method is described for determining whether a drug or small solute has significant interactions at the two major binding sites on human serum albumin (HSA). This method uses affinity capillary electrophoresis (ACE) to perform a mobility shift assay, where the solute of interest is ...

Afffinity capillary electrophoresis (ACE) is a new analytical technique that has been shown to be an efficient and accurate tool in studying biomolecular noncovalent interactions and determining binding and dissociation constants of formed complexes. ACE uses as its basis the chan ...

A method for determining the purity of recombinant carboxypeptidase B utilizing CE-SDS has been developed and validated for use in a manufacturing quality control laboratory. The method was optimized, prior to validation, to allow for the lowest limit of quantitation. The method was vali ...

Capillary electrophoresis (CE) became a versatile technique for analysis of biological macromolecules. We have applied capillary zone electrophoresis (CZE) and SDS-gel CE for the characterization of recombinant proteins during development of major bioprocessing steps, i ...

Capillary electrophoresis (CE) is experiencing increased use in the field of separation science. Part of its growing popularity of capillary electrophoresis can be attributed to the high efficiency of the separations achievable with the technique, making it an attractive tool for bi ...

Surfactants such as didodecyldimethyl ammonium bromide (DDAB) and 1,2-dilauroyl-sn-phosphatidylcholine (DLPC) form bilayers at the walls of bare silica capillaries. Once formed, these bilayers are stable in the absence of surfactant in the buffer. DDAB provides a cationic bilay ...

Amino acid chromatography is used to detect both primary disorders of amino acid metabolism (e.g., maple syrup urine disease) and disorders of renal tubular reabsorption (e.g., cystinuria) (see Notes 1–4). In most patients with disorders in the former group, the abnormal amino acids are clearly ...

In classical, ninhydrin-based amino acid analysis (1,2), the ion-exchange matrix used for separation becomes contaminated upon consecutive analyzes of extremely lipid-rich samples; in our experience, already after approx 20–30 samples. Therefore, in analysis of lipid-rich mat ...

The separation and determination of O-phosphoamino acids has been carried out by thin-layer chromatography (1–8, thin-layer electrophoresis (1,6,7,9–14), gel electrophoresis (15), amino acid analyzer (16–18), high-performance liquid chromatography (HPLC) (19–33), capill ...

The determination of sulfur amino acids, glutathione (GSH), and related aminothiols has been carried out by isotachophoresis (1), amino acid analyzer (AAA) (2,3), gas chromatography (GC) (4–8), high-performance liquid chromatography (HPLC) (9–32), GC-mass spectrometry (GC-MS) (33, ...

Over the last two decades, various studies have shown that moderate and persistent hyperhomocysteinemia is implicated in the development of atherosclerosis, which is responsible for 50% of all mortality and morbidity in Western countries. Considering that the most traditional risk ...

The Maillard reaction, popularly known as nonenzymatic glycosylation (NEG) or glycation, is a complex chemical reaction and occurs in vivo between reactive aldose or ketose sugars and protein-bound free amino groups (1). NEG has been implicated in diabetic or age-related complicatio ...

Amino acid analysis of proteins is one of the best and most accurate methodologies to quantify proteins. Ideally, samples should be pure prior to hydrolysis, however, they are often not only at low concentration, but also in buffers, salts, and/or detergents. Sample contaminants contribute to ...

Although lacking the speed and sensitivity of more widely heralded techniques such as mass spectrometry, amino acid analysis remains an indispensable tool in a complete biotechnology laboratory responsible for the analysis of protein pharmaceuticals.