生物化学技术

Mitochondria are key organelles that play a crucial role in cellular homeostasis. Dysfunction of these organelles is associated with a wide range of human diseases.Therefore, mapping the different components of mitochondria would provide invaluable information to gain further ...

Mitochondria are complex organelles, and their proteomics analysis requires a combination of techniques. The emphasis in this chapter is made first on mitochondria preparation from cultured mammalian cells, then on the separation of the mitochondrial proteins with two-dimensi ...

The mitochondrial oxidative phosphorylation involves five multimeric complexes imbedded in the inner membrane: complex I (Nicotinamide Adenine Dinucleotide (NADH) quinone oxidoreductase), II (succinate dehydrogenase), III (ubiquinol cytochrome c oxido reductase or ...

Because of its many connections to other cell systems, the nuclear envelope (NE)is essentially impossible to purify to homogeneity. To circumvent these problems, we developed a subtractive proteomics approach in which the fraction of interest and a fraction known to contaminate the fra ...

We describe here a method for analyzing a rat liver nuclear-insoluble protein fraction to determine candidate proteins participating in nuclear architecture formation. Rat liver nuclei are purified by sucrose density gradient centrifugation. The purified nuclei are treated w ...

Proteomics provides a powerful tool to characterize the protein content of an organelle. However, identifications obtained through mass spectrometry and database searching only make sense if the organelle sample is not heavily cross-contaminated. Besides the proteomic analy ...

Organelles are biochemically distinct subcellular compartments that perform specific functions within a cell. These roles are regulated by the complement of proteins associated with each organelle. Thus, a comprehensive understanding of an organelle’s proteome is necessary ...

The knowledge of the localization of proteins to a particular subcellular structure or organelle is an important step towards assigning function to proteins predicted by genome-sequencing projects that have yet to be characterized. Moreover, the localization of novel proteins to o ...

Organelle protein profiles have traditionally been analyzed by subcellular fractionation of a specific organelle followed by the identification of the protein components of specific fractions containing the target organelle(s) using mass spectrometry (MS). However, beca ...

Genome-wide transcriptional profiling provides a rich source of data for the validation and annotation of organelle proteomic data. Organelle biogenesis is in most cases accompanied by upregulation of genes encoding organelle-specific proteins. Consequently, identific ...

Since the first descriptions of electrophoresis in small diameter tubes in the 1970s and 1980s (1,2), capillary electrophoresis (CE) has been recognized for its potential to replace slab-gel electrophoresis for the analysis of nucleic acids (3,4). In particular, the availability of com ...

For decades electrophoresis, together with chromatography and centrifugation, has been one of the most important tools in biochemistry and biology. Size selective separations using synthetic polyacrylamide sieving gels (SDS-PAGE) (1), development of the two dimensional ele ...

Among the various techniques developed for mutation detection, single-strand conformation polymorphism (SSCP) analysis has become the most popular method for the screening of unknown mutation in small stretches of DNA. Its widespread use is related to its simplicity and low cost. SSCP ...

Capillary electrophoresis (CE) is an efficient and fast microseparation technique that is well suited for the separation of nucleic acids. CE is routinely used for the separation of double-stranded DNA fragments; single-stranded DNA fragments and polymerase chain reaction (PCR) pr ...

Single-strand DNA conformational polymorphism (SSCP) makes use of sequencedependent folding and structural conformation assumed by ssDNA for detection of small sequence changes or point mutations in DNA fragments. The technique was developed using polyacrylamide gel elec ...

The most fundamental difference between two individuals is DNA variation at the nucleotide level. This DNA variation, often referred to as a single nucleotide polymorphism or SNP, can occur approximately once every several hundred base pairs. The ability to accurately determine the DNA s ...

In recent years, the need for techniques capable of detecting and identifying point mutants has increased dramatically in the fields of genomics, cancer research, and molecular diagnostics. The large arsenal of methods for mutation detection ranges from direct sequencing and array hy ...

Small alterations in DNA sequence of genomic DNA lead to many human diseases, such as cancer, diabetes, heart disease, atherosclerosis, cystic fibrosis, Alzheimer disease, Duchenne muscular dystrophy, and various thalassemias. These alterations in DNA sequence include many types ...

One of the most sensitive methods of detection for capillary electrophoresis (CE) is laser-induced fluorescence (LIF). The reader is referred to a recent review (1) for a more detailed description of the technique, but in brief, laser irradiation is used to excite the fluorescent molecule, and t ...

Mutation detection has become a very important chapter in medicine, following the development of molecular technology for the study of pathogenic mutations in human diseases. DNA technologies allow great advances in basic knowledge of the pathophysiology of disorders, adding new m ...