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., a method for quantitative protein determination in a microliter volume when neither the volume nor the protein content in that volume are known, and methods for the determination of glycoproteins, amino acids, and sugars in the picomole range.

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. Activation of the protein kinase A or calmodulin pathway do not result in an increased OL process production. Furthermore, TPA induced proliferation in a subpopulation of OL. We conclude that the stimulation of Pk-C is of utmost importance for OL regeneration.

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. This was made possible by taking advantage of the mutual solubility/insolubility of carrier ampholytes/proteins in saturated ammonium sulfate solution. In addition, improvement of protein determination in denaturing electrophoresis sample buffer containing the anionic detergent sodium dodecyl sulfate and the reducing agent 2-mercaptoethanol was achieved. The assay covers a range of sensitivity from 40 ng to 20 micrograms of protein. The procedure is applicable to large numbers of samples.

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. Through selection of two different counter ions--the characteristic feature of the present ionic system--the stacking limits of a multiphasic buffer system can be further widened, thus making it applicable to gel electrophoresis of a larger spectrum of rapidly migrating species, such as sodium dodecyl sulfate-proteins/peptides and nucleic acids, than has been possible previously. Highly sensitive detection methods for proteins as well as for polypeptides down to approximately Mr 1000 are described. Dansylated proteins/peptides were detected by their fluorescence either directly within the gel or following electroblotting into anion-exchange or polyvinylidene difluoride membranes. The latter procedure resulted in detection sensitivities of approximately 1 ng. Nondansylated proteins/peptides were either detected within the gel by colloidal Coomassie staining or by electroblotting into polyvinylidene difluoride membranes, followed by colloidal gold staining. Prior to both staining procedures the proteins/peptides were pretreated with glutardialdehyde in the presence of borate at near neutral pH values to generate protein/peptide polymers of poor solubility. For a given pH the efficiency of the latter procedure was significantly influenced by the nature of the buffer ion used in the fixation buffer. In contrast to conventional fixation procedures even small polypeptides (Mr 1000) were immobilized and approximately 15 ng and 0.75 ng could be detected after colloidal Coomassie and colloidal gold staining, 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. gel staining, mechanical, optical and electronic processing, as well as all the steps of data processing, can be viewed as communication channels. The factors affecting both the relevant and irrelevant part of the total information passed through these channels are discussed in the consistent frame provided by information theory. This view leads to a unifying context for analyzing the performance of quantitative densitometers.

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. Depending on local protein concentration, the dye binding to the same amount of a given protein differs considerably. Since the stainability of proteins using colloidal staining procedures, as with Coomassie Brilliant Blue dyes, is time-dependent and, in addition, also dependent on the pore size of a given polyacrylamide gel used for PAGE, calibration curves for quantitative determinations have to be prepared in polyacrylamide gels of the same composition as used for PAGE. Staining conditions also have to be identical for calibration gels and gels under analysis. If, however, a set of calibration curves is prepared for different staining times, it is possible to calculate a generalized calibration curve, allowing for quantitative evaluation with flexible staining time. Furthermore, and in consequence of the implications due to particle density, quantitative determination via densitometry is only possible by determining the protein amount of each single measuring point (pixel) via its absorbance on the basis of a calibration curve. Since the particle density is inherent in a calibration curve, the final summation of the protein amount per pixel will give values close to reality.

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. Repeated runs of the same sample reveal an essentially constant protein spot pattern. The time period between surgery and tissue preparation did not influence the number of protein spots when less than 15 h was involved. The protein spot number is always lower in synovial fluid than in either synovial tissue or serum in RA and non-RA patients. The mean value for the number of spots is 68 for the inflamed tissue irrespective of the cause of arthritis (RA and non-RA group taken together) and 47 for the control group. This difference is significant. We were able to definitely identify 7 spots in the tissue extract. We did not find RA-specific protein spots in either serum, synovial fluid or tissue extracts from the synovial membrane. The only significant difference between RA patients and either non-RA or control group patients concerning the protein spot pattern is the increased size of the immunoglobulin spot (mainly IgG) in RA. In addition, we discuss possible reasons for failure of the 2D electrophoresis technique to detect disease-specific protein patterns.

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. A polypeptide comprising residues 1-14 was not found. According to these results the log Mr versus Rf plot used for calibration must be revised. For the separation of low molecular weight polypeptides and peptides a new gel system based on the theory of multiphasic zone electrophoresis combined with a modified Coomassie staining procedure is reported.

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. Due to the well-known sex-specific differences in L-ethionine metabolism female rats were much more affected by chronic administration of L-ethionine. The underlying mechanism of enzyme inhibition by ethionine could be disturbed protein synthesis and impaired protein phosphorylation, which was suggested by pronounced decreases in ATP content in liver. In the high dosage group depletions mainly of the branched-chain amino acids and lysine occurred in serum and brain, whereas the concentrations of methionine and tryptophan were increased. Tyrosine tended to be decreased in the course of hyperphenylalaninemia. Hyperphenylalaninemia and other resulting amino acid imbalances obviously impaired brain development during the early postnatal period. Concomitantly with reductions in protein concentrations, the activity of cathepsin D, a major intralysosomal acid proteinase, was increased in brain, suggesting also higher protein catabolism in brain. Side effects of this treatment, however, were higher mortality, loss of body weight, and a general impression of delayed development, resembling a state of undernutrition to some extent. These obvious side effects of ethionine limit the usefulness of ethionine as a suitable model for classic phenylketonuria in suckling rats.

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.

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. Protein profiles of the different cell types were obtained by electrophoresis of the extracts and subsequent densitometry of the stained protein bands. About one-third of the protein of palisade parenchyma and of spongy parenchyma was Rubisco large subunit. Using chlorophyll (Chl):protein ratios previously obtained, we calculate mesophyll contained ca. 22 millimoles Rubisco per mole Chl. In contrast, guard-cell protoplast preparations were calculated to contain from 0.7 to 2.2 millimoles Rubisco per mole Chl. The upper end of this range is an overestimate resulting from contamination by mesophyll and to the method of peak integration. Extracts of excised guard cells were calculated to contain 0.05 to 0.17 millimole Rubisco per mole Chl. We conclude that Rubisco is absent, or virtually so, in guard cells of V. faba.

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. The signal-to-background ratio decreases by 10-30% depending on the smoothness of the support.

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.

[Microanalysis, a simple method of fast and reliable diagnosis]. / Mikroanalytik, ein einfacher Weg zur schnellen und sicheren Diagnose.

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. The method has been applied to staining of polyacrylamide gels after sodium dodecyl sulfate-electrophoresis and isoelectric focusing in carrier ampholyte-generated pH gradients.

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. The labeled lipids were studied by fluorography and scintillation counting. [(14)C]acetate was incorporated in decreasing order into neutral lipids, phosphatidylcholine, ethanolamine phosphatides, galactocerebrosides, phosphatidylinositol, phosphatidylserine, sulfatides and sphingomyelin. From the [(14)C]acetate incorporated into ethanolamine and choline phosphatides, 71.6 and 14.8%, respectively, were found in plasmalogens. Among neutral lipids, [(14)C]acetate labeled not only cholesterol but also large amounts of triglycerides. No cholesterol esters were synthesized. [(3)H]galactose primarily labeled galactocerebrosides, sulfatides, and monogalactosyl diglyceride. [(35)S]sulfate incorporation was restricted to sulfatides. Together with our previous results concerning proteins, these data show that: (1) oligodendrocytes remain highly differentiated in long-term cultures; (2) they are able to synthesize the major components of myelin; (3) they synthesize surprisingly high amounts of triglycerides and of monogalactosyl diglyceride, a marker for myelination.

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. As shown for 14C-labelled phenylalanine and tyrosine, the method allows the simultaneous determination of the specific activity of 22 amino acids. About 10(-13) mol of an amino acid with a specific activity of less than 5 GBq/mmol can be detected and measured by this method.