Microelectrophoresis and auxilary micromethods.

In this retrospect of approximately 30 years of work with micromethods, some of them developed in our own laboratory, their principles and application to different separation problems are described, such as one- and two-dimensional microelectrophoresis in capillaries and microslab gels, isoelectric focusing in capillaries or microslab gels, microchromatography, microphotometry, and microfluorometry for qualitative and quantitative evaluation of separation patterns. In addition, some useful auxiliary methods are also described, e.g.

Protein kinase C stimulation enhances the process formation of adult oligodendrocytes and induces proliferation.

Oligodendrocytes (OL) isolated from adult pig brains regenerate their processes and form a network of fibers after 14-18 days in vitro (DIV). Stimulation of protein kinase C (Pk-C) by tumour promoters such as 12-O-tetradecanoylphorbol-13-acetate (TPA) produces at day 7 in vitro a similar network after only 20 hr. H-7, an inhibitor of Pk-C, as well as amiloride, which inhibits the subsequent Na+/H+ exchange, reversibly suppress this effect.

A dot-blot assay for quantitation of nanogram amounts of protein in the presence of carrier ampholytes and other possibly interfering substances.

A method for protein determination in one- and two-dimensional electrophoresis sample buffer is presented. Accurate quantitation of protein in two-dimensional electrophoresis sample buffer (9.5 M urea, 2% Nonidet P-40, 2% carrier ampholytes, and 5% 2-mercaptoethanol) required removal of carrier ampholytes prior to the assay.

A new multiphasic buffer system for sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins and peptides with molecular masses 100,000-1000, and their detection with picomolar sensitivity.

A novel multiphasic buffer system for high resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis of dansylated and nondansylated proteins/peptides in the relative molecular mass (Mr) range of 100,000-1000 is described. The system, based on Jovin's theory of multiphasic zone electrophoresis, allows complete stacking and destacking of proteins/peptides within the above Mr range. The buffer system uses Bicine and sulfate as trailing and leading ion, respectively, and Bistris and Tris as counter ions in the stacking and separating phase, respectively.

Quantitative densitometry from the point of view of information theory.

The process of quantitative densitometry is analyzed with methods developed in information theory. It is shown that the steps involved in densitometry, e.g.

Essential problems in quantification of proteins following colloidal staining with coomassie brilliant blue dyes in polyacrylamide gels, and their solution.

Quantitative determination of stained proteins following polyacrylamide gel electrophoresis (PAGE) is of increasing interest especially since computer-aided densitometers have become available as well as recipes for sensitive and background-free staining with Coomassie Brilliant Blue dyes. However, avoidance of separation artifacts is not the only essential prerequisite for quantitative evaluation. The local particle density of a protein in a given gel is of critical importance since it determines its stainability.

Two-dimensional electrophoresis of synovial tissue, synovial fluid and serum in patients with rheumatoid arthritis and related diseases.

Using the technique of two-dimensional (2D) electrophoresis with consecutive silver staining, we investigated samples of serum, synovial fluid and synovial tissue obtained from 19 patients suffering from rheumatoid arthritis (RA) or non-RA arthritis. From these experiments we have drawn the following conclusions. 2D electrophoresis of serum, synovial fluid and synovial tissue extracts taken from patients suffering from joint diseases is a reproducible method.

Application of a new electrophoresis technique (2D cryostat section electrophoresis) to synovial tissue of RA patients and comparison with immunohistochemical staining methods.

We describe a new two dimensional electrophoresis technique which is based on the combination of cryostat section technology and IEF- and SDS-gel electrophoresis. The optimal conditions for two dimensional cryostat section electrophoresis are investigated. The application of this technique to synovial membranes of patients suffering from rheumatoid arthritis and osteoarthritis is described.

Gas-phase sequencing after electroblotting on polyvinylidene difluoride membranes assigns correct molecular weights to myoglobin molecular weight markers.

Commercially available polypeptide marker kits containing peptides generated by cyanogen bromide cleavage of either horse heart myoglobin or sperm whale myoglobin have been investigated by sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE), followed by electroblotting on polyvinylidene difluoride membranes, and gas-phase sequencing. It could be shown that the molecular weights assigned to the SDS-PAGE bands by the companies are incorrect. Arranged in descending order, the marker kits are composed of the following polypeptide fragments from myoglobin: positions 1-153, 1-131, 56-153, 56-131, 1-55, and 132-153.

Biochemical and developmental features of experimental phenylketonuria induced by L-ethionine in suckling rats.

Suckling rats were injected subcutaneously with doses of L-ethionine (0.1 mumole/g body wt) at intervals of 12 hr. In the latter group, phenylalanine hydroxylase was effectively inhibited in vivo resulting in hyperphenylalaninemia and phenylketonuria.

Electrophoretic Assay for Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase in Guard Cells and Other Leaf Cells of Vicia faba L.

The ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) contents of guard cells and other cells of Vicia faba L. leaflet were determined. To prevent proteolysis, proteins of frozen protoplast preparations or of cells excised from freeze-dried leaf were extracted directly in a sodium-dodecyl-sulfate-containing solution, which was heated immediately after sample addition.

Automatic evaluation of nucleic acid sequencing gel autoradiographs.

An algorithm for automatic evaluation of nucleic acid sequencing gel antoradiographs is described which is simple and fast, with a low level error rate.

Mirror densitometry for higher sensitivity and resolution.

In order to increase the pathway of the light inside a gel (or autoradiogram) during scanning, it is placed on top of a mirror and the densitometry is performed in a vertical reflection mode. As the light passes the gel a second time after being reflected by the mirror, the absorbance is nearly doubled. This increase in absorbance results in higher sensitivity and resolution.

Vertical remission densitometry for the evaluation of nontransparent samples.

Gels dried onto filter paper or their blots on membranes are not suitable for densitometric evaluations in the transmission mode because these samples are nearly nontransparent. By scanning in the remission mode, evaluation with good results is possible. Data acquisition by a computer-controlled high speed scanning photometer and digital image processing affords a spatial resolution and accuracy similar to that in the transmission mode.

[Microanalysis, a simple method of fast and reliable diagnosis].

Microanalytical methods are not widely used although they are easy to perform and reproducible results are obtained. For one analysis, for example microelectrophoresis of proteins, less than a microgram of a protein mixture is sufficient since the detection limit for a well separated protein band is in the lower nanogram range. Further, a great advantage of micromethods is their saving in time required for the analyses which is approximately only one tenth of the time necessary for the corresponding macromethod.

The effects of sensory stimulation on the free amino acid pool of the developing brain.

Developing chicks were subjected to three different paradigmata of sensory stimulation, and the effects on the free amino acid concentrations in the blood and in various brain regions were monitored. The free amino acid pool of individual brain regions was found to be affected in a treatment- and age-specific manner. The increased neuronal activity resulting from sensory stimulation seems to affect intrinsic factors involved in the regulation of the free amino acid pool, most likely via modulations of the rate of metabolization of individual amino acids and/or of the rate of synthesis and degradation of individual proteins in certain brain regions.

Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250.

An improved procedure for staining of proteins following separation in polyacrylamide gels is described which utilizes the colloidal properties of Coomassie Brilliant Blue G-250 and R-250. The new method is based on addition of 20% v/v methanol and higher concentrations of ammonium sulfate to the staining solution previously described. The method combines the advantage of much shorter staining time with high sensitivity, a clear background not requiring destaining, stepwise staining, and stable fixation after staining.

Lipid synthesis by oligodendrocytes from adult pig brain maintained in long-term culture.

Oligodendrocytes were isolated from adult pig brain and cultivated for 18-24 days. [(14)C]acetate, [(3)H]galactose or [(35)S]sulfate were added to the medium for an additional 24 h. Lipids were extracted and separated by high-performance thin-layer chromatography.

Determination of the specific radioactivity of amino acids by a combination of thin-layer chromatography and quantitative autoradiography.

A new method for the analysis of the specific activity of amino acids is described. The analysis is carried out by thin-layer chromatography of the dansylated amino acids, computerized fluorescence evaluation and activity measurement by quantitative autoradiography. Quantitative evaluation of the autoradiographs is achieved by careful calibration of the X-ray film blackening.

Acceleration of brain tubulin formation in the optic lobe of the chick embryo by light stimulation.

The effect of light exposure on the protein patterns of optic lobe and forebrain of the chick embryo was analysed by a high-resolution micro-two-dimensional polyacrylamide gel electrophoresis and computerized quantitation. Experiments were done on three groups of eggs: control group was incubated in the dark; simultaneously, in the same incubator, one group of eggs was illuminated by constant light, another by intermittent light (3 sec interval) from day 10 to day 16 of incubation. In embryos exposed to intermittent light the relative amount of tubulins was significantly increased in the optic lobe.

[Facilitating cervix dilatation of the non-pregnant uterus by intracervical administration of gels containing prostaglandin and calcium chloride].

In a prospective, randomised study, 50 non-pregnant patients were treated intracervically with 3 ml 5% tylose, 50 micrograms sulprostone, 100 micrograms sulprostone gel, 3 ml 2.5 mM or 9.0 mM calcium chloride gel in order to soften the cervix 12-14 hours before diagnostic curettage.

Regional protein patterns of the embryonic chick brain: developmental shifts and the effects of light stimulation.

The developmental changes in the regional protein patterns of the embryonic brain of chick were analysed by micro-two-dimensional polyacrylamide gel electrophoresis stained by a highly sensitive procedure with Coomassie Brilliant Blue G-250 that completely avoids background staining. More than 400 protein spots were resolved in a computerized plot. Many proteins of the various embryonic brain regions varied with age.

Fatty acid composition of myelin lipids from developing rat forebrain and spinal cord: influence of experimental hyperphenylalaninaemia.

The fatty acid composition of individual myelin lipids from rat forebrain and spinal cord was analysed at 20 and 30 days p.p. During this phase of rapid myelination the proportions of C 16:0 and C 18:0 fatty acids decreased whereas the relative amounts of long chain and unsaturated fatty acids increased in most lipid classes.