生物化学技术

Nontraumatic, simple, and reproducible procedures for the introduction of nonpermeant molecules into adherent mammalian cells byin situ electroporation are described. Ctells are grown on a glass slide, half of which is coated with electrically conductive, optically transpar ...

Jet injection can be used to introduce genes into the cells of differentiated tissues of living animals and organ cultures. When a solution of plasmid DNA is jet injected into a selected tissue or organ, cells lying in or near the path of the jet injection are transfected with the DNA and the introduced gene ...

High-temperature gas chromatography and gas chromatography-inass spectrometry for the analyses of oligosaccharides derived from glycoproteins or glycosphingolipids has been developed. Pcrmethylatcd oligosaccharides with up to about 12 sugar residues and masses up to ...

This article describes the direct sequencing of PCR-amplified DNA, a technique that bypasses the problem of replication errors sometimes associated with other PCR procedures. The direct sequencing procedure produces an “average sequence” of all the copies of the target. Any miscopi ...

The authors describe an efficient method for generating large deletions (200 nts) of precise length using the PCR-based method of gene splicing by overlap extension (1). This method is technically simple and less time consuming than conventional loop-out mutagenesis techniques requ ...

Isoprenylated proteins are involved in signal transduction, control of cell growth and differentiation, organization of the nuclear lamina and cytoskeleton, and vesicle sorting. The isoprenoid moiety facilitates the interaction of these proteins with membranes and/or other ...

Lectin affinity electrophoresis is a powerful technique to investigate the interaction between a lectin and its ligand. Affinity electrophoresis results from the reduced mobility of a charged species owing to its interaction with an immobile species. In this protocol, a two-dimensi ...

Purification of DNA fragments from acrylamide or agarose gels is a commonly used technique in the molecular biology laboratory. This article describes a rapid, efficient, and inexpensive method of purifying DNA fractions from an agarose gel. The purified DNA is suitable for use in a wide range of ...

Irradiation and fusion gene transfer (IFGT) is a technique that spans the gap between the limitations of molecular methods and somatic-cell genetics, allowing the separation of DNA fragments between 0.25 and 30 Mb in size. In conjunction with genetic linkage analysis and physical mapping t ...

The high-resolution separation achievable with capillary electrophoresis has been applied successfully to the analysis of glycoproteins. Inherent in the implementation of this technology for glycoprotein analysis is the use of specific buffer additives. Bifunctional ca ...

A procedure for isolation of polymerase chain reaction (PCR) fragments from an automated system for DNA fragment analysis is described. The technique is used for screening of unknown mutations or polymorphisms. Fragments are isolated during the analysis from the system by an automated u ...

We describe the use of UV-crosslinking in combination with SDS-PAGE to determine the approximate molecular weight of DNA-binding proteins. A 5-bromo-2′-deoxyuridine (5-BrdU)-substituted, radioactively labeled double-stranded oligonucleotide representing the prot ...

This work describes protocols for the production of single-chain antibody and T-cell receptor fragments inE. coli. A choice of methods is given for the purification of the recombinant fragments that rely on the use of either immunoaffinity or metal chelate affinity chromatography. The TCR ...

This article describes the development of cell-free transcription systems from the cultured cells and fat body tissues of a Lepidpteran insect, the silkworm,Bombyx mori. Detailed protocols are provided for the culture of aB. mori cell line, rearing larvae, preparation of whole cell as well ...

The introduction of binary plasmids intoAgrobacterium hosts forAgrobacterium-mediated transformation of plants is most readily achieved by electroporation. However, occasionally, no transformed colonies are recovered and the transformation program is delayed. Po ...

An inexpensive monophasic reagent has been developed for the extraction of total RNA from cells or tissues. The main ingredients of the reagent arePhenol,Isoamyl alcohol,Guanidinium isothiocyanate, andBeta-mercaptoethanol (PIG-B). The quality and yield of RNA obtained by this r ...

A simple method for removing contaminating genomic DNA from an RNA preparation is presented. The method involves digestion of the RNA with RNase-free DNase I at room temperature followed by inactivation of the enzyme at 65�C in presence of EDTA. This method produces an RNA sample that is negative for ...

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is one of the most powerful separation techniques for complex protein solutions. The proteins are first separated according to their isoelectric point, driven by an electric field across a pH gradient. The pH gradient ne ...

An in vitro transcription assay of drug-DNA interactions has been described and is based largely on the stablelac UV5-initiated transcription complex. This system utilizes a synchronized population of radiolabeled nascent RNA 10 nucleotides long. Reaction of this initiated tran ...

The position of unsaturation, chain branching, and other structural features of fatty acids are not often apparent from the mass spectra of common derivatives such as methyl esters because of factors such as charge location at the carboxy termiunus and migration of double bonds. The spectra of ...