Starch gel electrophoresis of certain enzymes from five species of Fomes

The electrophoretic banding patterns of five enzymes (esterase, catechol oxidase, peroxidase, alkaline phosphatase, and acid phosphatase) from eleven isolates of five species of the wood-rotting basidiomycete, Fomes, were subjected to mathematical analysis in order to examine taxonomic relationships. Generally, greater similarity was observed between the banding patterns for isolates of the same species than between those for isolates of different species. The experimental and analytical technique used in this study could be an aid for the determination of taxonomic relationships among these and other fungi.     

Measurement of high-molecular-weight hyaluronan in solid tissue using agarose gel electrophoresis

The size of hyaluronan in solid tissue was measured using a combination of agarose gel electrophoresis and a radiometric assay. Radiolabeled hyaluronan binding proteins, used in the radiometric assay, were also used to detect hyaluronan after transfer to a nylon membrane following gel electrophoresis. Lane intensity on the autoradiograph was linearly related to the amount applied to the gel between 10 and 100ng. The intensity was independent of the hyaluronan molecular weight for standards with molecular weights equal to or greater than 790,000. The radiometric assay was used to measure hyaluronan irrespective of size, while gel electrophoresis was used to measure hyaluronan with molecular weights greater than 0.79x10(6) or 4x10(6). Deferoxamine was used to inhibit depolymerization during the digestion of tissue samples with protease. The molecular weight pattern was similar for skin, skeletal muscle, heart, lung, small intestine, and large intestine despite large differences in hyaluronan content. For all tissues, 58% of the hyaluronan had a molecular weight greater than 4million. All tissues showed an absence of hyaluronan with a molecular weight below 790,000. The procedure can be used to study changes in hyaluronan size in tissue during inflammation and other pathological states.     

Starch gel electrophoresis: an effective method for separation of pathogenic and nonpathogenic Naegleria strains

Isoenzyme electrophoresis of 7 different enzyme systems was used to compare 24 strains of Naegleria fowleri and 6 strains of N. gruberi. The 30 strains could be grouped into 4 distinct categories based upon zymogram patterns. No interstrain band variation in all enzyme systems was demonstrated in pathogenic strains of N. fowleri. Three nonpathogenic high temperature-tolerant strains of Naegleria had similar zymograms. Four of the 5 remaining nonpathogenic Naegleria strains had no interstrain band variation. Based upon zymograms, the 22 pathogenic strains constitute a homogenous species. Similarly the high temperature-tolerant nonpathogenic strains formed a cohesive group. The remaining nonpathogenic strains could be separated into 2 groups.     

Quantitation of chick tissue zinc-metallothionein by gel electrophoresis and silver stain enhancement

Discontinuous gradient gel electrophoretic systems were developed to quantitate zinc-metallothionein (Zn-MT) in chick tissues, liver and pancreas. Gels were stained with Coomassie Blue initially, then enhanced by silver stain. At least 4 micrograms of Zn-MT could be detected after Coomassie Blue stain, and 1 microgram Zn-MT detected following silver stain enhancement. Significant linearity (correlation coefficient = 0.99) of a standard curve was established in the Coomassie Blue stained gels. The results of our experiment suggest that electrophoretic analysis is a simple and feasible method for the quantitation and identification of Zn-MT in chick tissues.     

Polyacrylamide gel electrophoresis and immunoblotting of proteins extracted from paraffin-embedded tissue sections

We show in this communication that polyacrylamide gel electrophoresis (PAGE) and immunoblotting of proteins can be performed using one to two 5-7 micron paraffin sections of tissues fixed in non-cross-linking fixatives (acetone, alcohol, or modified Carnoy's solution). Proteins for study were extracted from paraffin sections of mouse foot pad and liver. The presence of unaltered keratin polypeptides in tissues fixed with either acetone or alcohol was demonstrated in gels stained with Coomassie brilliant blue. The preservation of their antigenic determinants was demonstrated with immunoblotting. Furthermore, the immunoreactivity of soluble proteins, such as albumin, remained unaltered in immunoblots obtained from paraffin-embedded mouse liver sections. These data indicate that tissues embedded and stored in paraffin are useful for the above-mentioned biochemical and immunological studies and may therefore be an important technique for diagnostic pathology or retrospective studies.     

A simple and general plant tissue extraction procedure for two-dimensional gel electrophoresis

A fast and simple extraction procedure of plant tissue for two-dimensional gel electrophoresis is presented. The procedure is especially useful for the extraction of plant cell suspension cultures, callus and other plant tissues having a high content of phenol oxidases, polysaccharides, polynucleotides, terpenoids and other substances interfering with isoelectric focusing. Due to the speed of the extraction procedure (about 20 min), large numbers of samples containing only milligram amounts of tissue can be easily processed. The simplicity of the method makes it particularly suitable for the extraction of radiolabeled tissues (³⁵S, ³²P). This method is perfectly compatible with silver staining, autoradiography and Western blotting analysis.Abbreviations DTT dithiothreitol - IEF isoelectric focusing - SDS sodium dodecyl sulfate - TEMED N,N,N,N-tetramethylethylenediamine     

Identification and quantification of GC-rich oligodeoxynucleotides in tissue extracts by capillary gel electrophoresis

Capillary gel electrophoresis (CGE) is a widely used method for quantification of oligonucleotide-based drugs, such as CpG oligodeoxynucleotides (CpG ODN), aptamers and small interfering ribonucleic acids (siRNAs) that allows accurate quantification of parent compound as well as metabolites. Stable secondary structure formation of these molecules frequently prevents analysis by conventional CGE methods and impedes pharmacokinetic assessment. Herein, we describe development of a CGE method for identification and quantification of complex mixtures of secondary structure forming GC-rich ODN in biological samples at dose levels of 0.5mg/kg and above. Samples containing GC-rich CpG ODN and metabolite markers were treated by solid-phase-extraction (SPE) and subsequently analyzed by CGE using a 50cm neutrally coated capillary at 60 degrees C together with a 7M urea buffer system containing 30% dimethylsulfoxide (DMSO). Peak resolutions >or=1 were typically achieved, enabling pharmacokinetic assessment of secondary structure forming oligonucleotides in biological samples that hitherto were unsusceptible to quantitative analysis.     

Developmental appearance of proteins identified by two-dimensional gel electrophoresis in mouse gonadal tissue

Gonadal protein patterns of the mouse were studied during fetal development by two-dimensional gel electrophoresis. Fetal mice at days 8.5, 10.5, 12.5, and 14.5 post-coitum were analyzed for male or female specific proteins. Although no sex specific proteins were found, several proteins were found which were expressed in significantly different amounts in the two sexes at about the time of gonadal differentiation. Hence, quantitative differences, rather than qualitative ones, could be related to the initiation of testis or ovary development.     

Capillary affinity gel electrophoresis

Capillary affinity gel electrophoresis is a new technique for the recognition of the specific DNA base and/or sequence. This technology is also applicable to the characterization of binding properties of DNA-based drugs, chiral separation, and the selective separation of antibody mimetics using imprinted polymers. This article reviews the present state of studies on the capillary affinity gel electrophoresis, including the principle, theory, methods, and applications of this technology. The great potential of capillary affinity gel electrophoresis for the detection of the mutation onDNA is illustrated.     

Two-dimensional difference gel electrophoresis

Two-dimensional difference gel electrophoresis (2D DIGE) is a modified form of 2D electrophoresis (2DE) that allows one to compare two or three protein samples simultaneously on the same gel. The proteins in each sample are covalently tagged with different color fluorescent dyes that are designed to have no effect on the relative migration of proteins during electrophoresis. Proteins that are common to the samples appear as 'spots' with a fixed ratio of fluorescent signals, whereas proteins that differ between the samples have different fluorescence ratios. With the appropriate imaging system, DIGE is capable of reliably detecting as little as 0.5 fmol of protein, and protein differences down to +/- 15%, over a >10,000-fold protein concentration range. DIGE combined with digital image analysis therefore greatly improves the statistical assessment of proteome variation. Here we describe a protocol for conducting DIGE experiments, which takes 2-3 d to complete.     

Continuous preparative gel electrophoresis

An apparatus is described for continuous electrophoresis in polyacrylamide gel. Experiments may be run for 10 days or longer. Protein loads may be 1 g per day or more, and there are no obvious obstacles to scaling up. A revised classification of electrophoretic processes is required.     

Two-dimensional agarose gel electrophoresis without gel manipulation

The apparatus and procedure to perform two-dimensional agarose gel electrophoresis without manipulating the gel used for the first electrophoresis (first-dimension gel) have been developed. The procedure is less complex, less damaging to first-dimension gels, and more precise than procedures that require manipulation of the first-dimension gel. When combined with gel-embedding techniques, the procedure presented can be used to perform the second electrophoresis in a gel different from the first-dimension gel. A first-dimension gel too dilute to be manipulated and a more concentrated gel for the second electrophoresis have been used to separate DNA open circles from a mixture of variable-length linear DNAs.