化学生物学实验技术

The application of conventional enzymological methods to the study of hairpin and hammerhead ribozymes has led to valuable insights into the mechanisms by which these small RNAs catalyze phosphodiester cleavage and ligation reactions. Here, protocols are presented for measuring ...

RNA interference is the process that double-stranded RNA (dsRNA) induces the homology-dependent degradation of cognate mRNA mediated by 21- to 23-nt small interfering RNA (siRNA). Successful application of RNAi in functional genomics and proteomics, cancer gene therapy, and virus p ...

The ability to regulate endogenous gene expression by cleaving mRNA is important in basic and applied biological research. This is now driven predominantly by small interfering RNAs (siRNAs) as they induce sequence-specific gene silencing. However, the major obstacle to the use of siRN ...

Suppression by double-stranded RNA (dsRNA) of expression of a target gene is known as RNA interference (RNAi). Tobacco BY-2 cell suspension has been used as a model cultured plant cell, because it is possible to produce populations of tobacco BY-2 cell suspensions that are uniform and divide sync ...

In this chapter, phage-display peptide technology has been used to select peptides that internalize into breast-cancer cells. The used biopanning procedure provides information on the best peptide to be used. When one of the selected peptides was conjugated to an antisense oligonucle ...

This chapter provides a basic overview of most of the oligonucleotide delivery systems available for an in vitro use. Two major classes are described: systems that act through an endocytosis process (e.g., lipid-based vectors, nanoparticles, and polycations) and systems that by-pass this ...

Antiretroviral drug therapy can effectively reduce the viral load, and is associated with a degree of immune reconstitution in human immunodeficiency virus (HIV)-infected patients. However, the presence of a latent viral reservoir, the development of drug resistance, drug toxici ...

The implementation of a hematopoietic progenitor-cell gene-therapy program involves the performance of laboratory procedures and compliance with the current code of Good Manufacturing Practices. This chapter explains the multiple laboratory steps used in our recent Phase I g ...

Conventional analysis of nucleic acid reactions (cleavages, ligations) is performed with a (radioactive) labeled substrate, and reaction products (one aliqout for each time-point) are analyzed by gel electrophoresis followed by detection (X-ray film + densitometer or phospho ...

The photoreceptor phosphodiesterase (PDE6) is the central effector of visual transduction in vertebrate retinal photoreceptors. Distinct isozymes of PDE6 exist in rods and cones. Mammalian retina serves as an abundant source of tissue for PDE6 purification. Methods are described ...

This chapter covers the possible solutions to problems that may be encountered in routine operation. Further details may be available in specific instrument manuals or by contacting appropriate service specialists. The most likely problem areas are:

Commercial instruments have been available since 1988, and systems are currently available from over 20 suppliers. Recent survey papers have considered the relative performance and features of these commercial systems (1,2). Systems are available in both single bench-top units or in m ...

This section will describe the fundamental theory, equations, and definitions necessary to comprehend the concepts involved in capillary electrophoresis (CE). This is not an exhaustive treatment, but is considered sufficient to comprehend and appreciate the principles of CE. More ...

This chapter is intended to act as a brief guide to the operations required to perform a series of CE analyses and subsequent care of the capillary.

The CE instrument is controlled by a PC. The instrument settings are defined on the PC in method files. Settings, such as temperature, autosampler vial positions, injection parameters, and rinse cycles, are defined in the steps within the method. A typical method is given below. The exact settings f ...

Early reports of quantitative analysis by CE on homemade systems indicated that peak area precisions of ∼5% relative standard deviations (RSD) could be expected. The advent of reliable commercial instruments and improved methodology can now enable RSD figures of 1–2% or better to be routin ...

The options for conducting quantitative analysis are similar to those adopted in HPLC (1). The output format is similar to an HPLC chromatogram, i.e., a plot of UV absorbance vs time. Therefore, HPLC data handling and peak integration packages are generally applicable to CE. Main component assay m ...

This chapter provides some possible starting points for method development and further optimization. A later subsection in this chapter presents some starting points and guidelines for method development of chiral separations. A more extensive treatment of chiral separations is ...

The criteria for validation applied to CE methods (1–3) are similar to those employed for other quantitative separative techniques, such as HPLC (4). The extent of tests applied will vary according to the nature of the application and the extent of intended usage. Generally, such aspects as accur ...

The injection volumes involved in CE are very small (in the order of 1–10 nL). In addition, the area of capillary employed for on-column detection may be only 50 � 200 �m. Both these factors influence the detector sensitivity to a large extent. CE is less sensitive when directly compared to HPLC with typical i ...