Takashi Manabe

An electroblotting apparatus for multiple replica technique and identification of human serum proteins on micro two-dimensional gels

A new apparatus for electrophoretic transfer of proteins from micro polyacrylamide slab gels has been developed. The apparatus enabled the easy changing of nitrocellulose sheets and was suited for obtaining multiple blots from a gel. Electrophoretic conditions were determined so that all of the blots obtained sequentially from one slab gel were successfully used to visualize specific proteins irrespective of their molecular weight. Combining the transfer technique with the technique of parallel micro two-dimensional electrophoresis, 20 blots could be obtained within 1 h of electroblotting time. The locations of 28 human serum proteins were determined simultaneously on these blots using commercial specific antisera.

Quantitative analysis of two-dimensional electropherograms with a television camera—microcomputer system

Abstract A system to quantitate dye-stained proteins on two-dimensional polyacrylamide gels employing a television camera for data acquisition and a microcomputer for data analysis is described. All the equipment comprising the system and most of the software are commercially available. Therefore, minimal knowledge of computer software is needed to construct the system. The system is handy, low cost and useful for practical quantitation of protein spots, although it is semi-quantitative compared with previously available system. The system is especially suited for the analysis of micro-two-dimensional gels since high resolution up to 30 μm is obtained.

Automated two-dimensional liquid chromatographic system for mapping proteins in highly complex mixtures

An automated two-dimensional liquid chromatographic system was developed for systematic protein separations which could serve for analytical mapping and preparative separations of proteins. The system applies the principles of the column-switching technique, and consists of two different columns connected in tandem through an electrical column switching valve, two pumping systems to operate each column independently and a system controller to perform sequential chromatography on the two columns. A protein mixture is applied to the first-dimensional anion-exchange column and is separated by stepwise elution with an increasing sodium chloride concentration. The eluent is introduced directly to the second-dimensional reversed-phase column, and is further separated by gradient elution with an increasing acetonitrile concentration. The two elution stages are synchronized by a computer program. By this system, very complex protein mixtures such as crude cerebellar extracts were resolved reproducibly into ca. 200 peaks within 12 h. The method can be used for the total analysis of proteins in various tissues and cells without complicated premanupulation of samples, and allows the simultaneous analysis of a protein isolated by chromatography. The isolated protein is most suitable for use in the strategy of protein and gene sequence analysis.

Capillary electrophoresis of nucleic acids with a fully automated apparatus

Abstract Nucleic acids with a wide range of molecular size, from mononucleotides to calf thymus DNAs, were subjected to capillary electrophoresis employing the conditions for isotachophoretic analysis of proteins; Ampholine was mixed with the sample solution and the electrolyte solutions for isotachophoresis were used. For reproducible analysis, electrophoresis was performed with a fully automated apparatus. Nucleotides were separated from bases owing to their high electrophoretic mobility. The chain length of oligonucleotides was not the major factor with regard to their electrophoretic mobility. Polynucleotides (above 10 3 bases) appeared as sharp UV peaks in the zone of the hydrogencarbonate ion and their UV peak width was increased by DNase digestion, suggesting heterogeneity of the product. The applicability of capillary electrophoresis to the separation of large-molecular-sized polynucleotides has been demonstrated.

Spectroscopic aspects of copper binding site in bovine serum amine oxidase

Amine oxidases are known to catalyze oxidative deaminations of amines by accepting two electrons from amines and transferring them to the molecular oxygen. They may be conveniently divided into two classes: copper~ont~~g amine oxidases and FADcontaining ones. The copper-enzymes such as bovine [l-3] and pig [4-71 plasma amine oxidases, pig kidney [8-lo] and pea seedling [l I] diamine oxidases, and fungal amine oxidase [ 12-141 have been known to contain type-2 coppers (non-blue and ESRactive Cu(I1)) [ 151. In spite of many spectroscopic investigations on the copper binding site of these oxidases, ligating groups and function of the copper ion in the enzymes are still unknown; copper(I1) was found to restore the activity of copperdepleted oxidase [ 1 ,111, but no change in the ESR spectrum has been observed even after anaerobic treatment of the enzyme with a substrate /2,8,14]. This paper describes the results of spectroscopic studies of bovine serum amine oxidase (SAO) as the first approach to the ligating groups around the copper ions.

Apparatus for coupled high-performance liquid chromatography and capillary electrophoresis in the analysis of complex protein mixtures

Abstract Apparatus for coupled high-performance gel permeation chromatography (HPLC) and capillary electrophoresis was constructed and applied to the analysis of complex protein mixtures. To avoid electric leakage to the HPLC system, an electromagnetic pinch valve was inserted in the line from the column outlet to the injection port of the capillary electrophoresis apparatus. All the procedures for chromatography and electrophoresis were automated with the aid of a system controller. The apparatus was successfully used for the automatic two-step separation of human serum proteins and water-soluble proteins in bovine brain. Proteins separated by gel permeation HPLC were further analysed by capillary electrophoresis according to their characteristic electrophoretic mobilities.

Gel permeation chromatography combined with capillary electrophoresis for microanalysis of proteins

A new type of apparatus for the analysis of complex protein mixtures, in which gel permeation chromatography was combined with capillary electrophoresis, was constructed. Proteins were separated according to their molecular size in the first step, then separated according to their electrophoretic mobility in the second step. The outlet of a microbore column was connected with the sample injection port of a capillary electrophoresis apparatus. All the procedures of chromatography and electrophoresis were automated with the aid of a system controller. Preliminary results on the separation of proteins are presented.

Two-dimensional separation system for analysis of proteins employing isoelectric focusing and high-performance liquid chromatography

Abstract A new type of separation system, which combined isoelectric focusing with high-performance liquid chromatography, was designed for the analysis and fractionation of serum proteins. For the first dimensional separation, carrier-free isoelectric focusing was used to separate proteins according to their electric charge. For the isoelectric focusing, an instrument which consisted of multiple chambers was deviced. For the second dimensional separation, high-performance gel permeation chromatography was used to separate proteins according to their molecular size. Human serum was subjected to analysis with this two-dimensional system, and separation of serum proteins according to their p I and molecular size was demonstrated.

Detection of the changes in protein distribution of rat plasma induced by carbon tetrachloride administration by means of two-dimensional electrophoresis

The changes in rat plasma protein distribution after carbon tetrachloride administration were examined using two-dimensional electrophoresis, utilizing isoelectric focusing in polyacrylamide gel in the first dimension and pore gradient polyacrylamide gel electrophoresis in the second dimension. Drastic changes in amount of protein were observed at more than 20 spot positions including those of transferrin, Gc-globulin and low-density lipoprotein. The time course of the changes was examined, and the most drastic changes were observed at 2 days after carbon tetrachloride administration.

Amino acid micronalysis of proteins extracted from spot of fixed, stained, two-dimensional gels

A method for the extraction of proteins from fixed, stained, two-dimensional polyacrylamide gels and subsequent amino acid microanalysis was described. Human serum proteins were separated by two-dimensional electrophoresis, the stained spots were punched out and the proteins in each piece of gel were extracted with 0.1 M sodium hydroxide–2% thiodiglycol. The extracted proteins were hydrolysed and applied to an amino acid analyser equipped with a fluorimeter for detection of the reaction products with o-phthaldialdehyde. By reducing the amount of background contaminants, amino acid analysis of 1 μg or less of extracted proteins became possible. The amino acid composition of the proteins was compared with reported compositions of serum proteins by calculating correlation coefficients, which we designated “similarity indices of amino acid composition”. These indices were useful for the identification of the extracted proteins.