A versatile polyacrylamide gel electrophoresis based sulfotransferase assay

Background  

Sulfotransferases are a large group of enzymes that regulate the biological activity or availability of a wide spectrum of substrates through sulfation with the sulfur donor 3'-phosphoadenosine-5'-phosphosulfate (PAPS). These enzymes are known to be difficult to assay. A convenient assay is needed in order to better understand these enzymes.     

A micro-method for the assay of polyadenylate-containing ribonucleic acid by gel electrophoresis.

A micro-method for the determination of the electrophoretic profile of the various poly(A)-containing RNA species in a RNA sample was developed. The method is simple to carry out and allows for great reproducibility. It combines the resolving power of electrophoresis in agarose with the specificity of binding of poly(A) to poly(U)-containing glass-fibre filters. It consists of the following steps. (1) The molecules in an RNA sample are first separated according to their molecular weight by electrophoresis in agarose, at low ionic strength. 2. The molecules thus seperated are then submitted to a second electrophoresis in 'binding buffer' in a direction perpendicular to the first one. In the course of this electrophoresis the poly(A)-containing RNA species are seperated from other RNA species as they bind to a poly(U)-containing glass-fibre filter which is placed across the electrophoresis path. The method was used to determine the electrophoretic profile of Rhynchosciara salivary-gland messenger RNA. It was found that the population of messenger RNA in the gland is dominated by forms moving as 18 and 158 S peaks, but there is also polydisperse RNA with slower mobility.     

A denaturing gradient gel electrophoresis assay for sensitive detection of p53 mutations

p53 is a tumor suppressor gene located on 17p, a region of the human genome frequently deleted in tumors. Mutation of the p53 gene is an important step leading to development of many forms of human cancer. To simplify the analysis of tumors for p53 point mutations, we describe a GC-clamped denaturing gradient gel assay for detecting single-base substitutions within highly conserved regions of the p53 gene. This assay alows for efficient screening of tumors for single-base substitutions within the p53 gene and can be used to facilitate sequence analysis of p53 point mutations.     

A gel electrophoresis assay for the simultaneous determination of topoisomerase I inhibition and DNA intercalation

The DNA maintenance enzyme, topoisomerase I, is thought to play crucial roles in all living cells and for this reason inhibitors of this enzyme have been much studied. In this paper we describe a gel electrophoresis method capable of characterizing and quantifying inhibition of topoisomerase I by selected compounds. Inhibitors of topoisomerase I are often associated with intercalative binding to DNA and the method can simultaneously determine intercalative binding (as DNA unwinding) except in the cases where inhibition is prohibitively strong. The method uses closed circular (plasmid) DNA and can separate single-strand nicked, linearized (double-strand nicked), fully relaxed, partially relaxed (topoisomers), and supercoiled forms of the plasmid so that topoisomerase-dependent DNA cleavage (poisoning) can also be determined. By quantifying poisoning, inhibition, and intercalation simultaneously and separately in relation to reference compounds it is possible to make quantitative determinations of these phenomena for comparative purposes. Data for the topoisomerase I inhibitor, luteolin, are presented.     

Single cell gel electrophoresis assay: methodology and applications

The single cell gel electrophoresis or Comet assay is a sensitive, reliable, and rapid method for DNA double- and single-strand breaks, alkali-labile sites and delayed repair site detection, in eukariotic individual cells. Given its overall characteristics, this method has been widely used over the past few years in several different areas. In this paper we review the studies published to date about the principles, the basic methodology with currently used variations. We also explore the applications of this assay in: genotoxicology, clinical area, DNA repair studies, environmental biomonitoring and human monitoring.     

Single cell gel electrophoresis assay: a new technique for human biomonitoring studies

Human biomonitoring using the single cell gel electrophoresis (SCGE) or comet assay is a novel approach for the assessment of genetic damage in exposed populations. This assay enables the detection of various forms of DNA damage in individual cells with ease and speed and is, therefore, well suited to the analysis of a large group in a population. Here, application of SCGE assay in the identification of dietary protective factors, in clinical studies and in monitoring the risk of DNA damage resulting from occupational, environmental or lifestyle exposures is reviewed. Also, the comparative sensitivity of SCGE assay and conventional cytogenetic tests to detect genetic damage is discussed. Finally, strengths and shortcomings of the SCGE assay are addressed.     

A method for easily customizable gradient gel electrophoresis

Gradient polyacrylamide gel electrophoresis is a powerful tool for the resolution of polypeptides by relative mobility. Here, we present a simplified method for generating polyacrylamide gradient gels for routine analysis without the need for specialized mixing equipment. The method allows for easily customizable gradients which can be optimized for specific polypeptide resolution requirements. Moreover, the method eliminates the possibility of buffer cross contamination in mixing equipment, and the time and resources saved with this method in place of traditional gradient mixing, or the purchase of pre-cast gels, are noteworthy given the frequency with which many labs use gradient gel SDS-PAGE.     

A method for horizontal polyacrylamide slab gel electrophoresis

We present a simplified method of preparation of polyacrylamide gels which is totally analogous to the procedure now widely used to pour and run horizontal agarose gels. The acrylamide is poured into an open air gel mold consisting of a glass plate with a masking tape border and a comb. It is subsequently run in a submarine horizontal electrophoresis apparatus. The electrophoretic mobility and resolution of DNA fragments obtained in such gels are identical to results obtained with gels poured and run in the vertical configuration. Numerous advantages of horizontal polyacrylamide gel electrophoresis are discussed.     

A single-pour horizontal gel electrophoresis apparatus

An apparatus is presented which allows rapid gel casting and employs non-platinum electrodes.     

A computer program for displaying two-dimensional gel electrophoresis data

A computer program is described which facilitates comparison between the pattern of spots seen on different two-dimensional polyacrylamide electrophoresis gels. Essentially, the position of each spot is replotted on a graph by the computer using its molecular weight and isoelectric point as coordinates. An intensity factor is also assigned to each point by the operator which will determine the size and shape of the final plotted spot on the computer drawn figure. The resulting plot makes it more feasible to compare patterns of spots between independently run two-dimensional electrophoresis gels.     

A high resolution PAS stain for polyacrylamide gel electrophoresis

An improved PAS method for the detection of glycoproteins after electrophoresis on acrylamide or urea-acrylamide gels is described. Stronger oxidizing conditions and a more controlled washing for the removal of periodic acid resulted in both increased staining intensity and band resolution. The method will not stain protein and it appears that a 2–3 μg sample of bound or precipitable carbohydrate would easily be detected. Mucopolysaccharides were not detectable by this method.     

A preparative gel electrophoresis apparatus for large scale separations

The annular shape used in the apparatus described here gives gels with a larger surface area than can be supported by gels of conventional geometry. Additional advantages are efficient cooling and small fraction volume.     

Single cell gel electrophoresis assay (comet assay): its importance in human biology

The estimation of genetic instability by direct extent of DNA damage and repair is an important aspect of studies on mutagenesis, carcinogenesis, aging and evolution. Different methods have been introduced from time to time in an effort to meet this need. Single cell gel electrophoresis (SCGE) assay is a new, simple and sensitive method of evaluating DNA damage and repair at individual cell level. This assay can be performed on extremely small number of cells and results can be obtained within a relatively short time. The SCGE assay has the potential to play an important role not only in the understanding of some of the fundamental aspects of human biology but also can be helpful in many practical ways. For reprint requests.     

Modification of commerical and custom-built slab gel electrophoresis units for two-dimensional polyacrylamide gel electrophoresis

Many commercial and custom-built slab gel electrophoresis units can be modified to function as two-dimensional polyacrylamide gel electrophoresis units with the insertion of Plexiglas adapters. These adapters can be made for about $50 a pair and can be used for either temporary or permanent modification of the slab gel units. The physical dimensions of the adapters can be varied to permit great flexibility in the diameter of cylinder gels and the thickness of slab gels that can be run together. For example, proteins from 6-mm cylinder gels can be easily separated on 1-mm slab gels, which can then be dried for autoradiography.