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PubMed1988

Two-dimensional electrophoresis of cereal prolamins: applications to biochemical and genetic analyses.

Three complementary two-dimensional systems for the analysis of cereal prolamins are described. These are electrophoresis at pH 3.1 followed by electrophoresis at pH 9.2, isoelectric focusing (IEF) followed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and SDS-PAGE under non-reducing conditions followed by SDS-PAGE with reduction. They together give information on the pIs, Mrs and charge properties of the individual prolamin components, and on their interactions to form oligomers stabilized by inter-chain disulphide bonds. The three systems are then compared for their effectiveness in analysing unreduced prolamin I fractions from wheat and rye, and the IEF/SDS-PAGE system for analysing reduced and pyridylethylated prolamin fractions from all the major cereals. Finally, applications of the systems in biochemical and genetic studies are discussed and illustrated with three examples: analysis of the structural relationships of the S-rich prolamins (B hordeins and gamma-type hordeins) of barley, determination of the interactions of wheat and rye prolamin subunits in a 2RS/2BL translocation line, and the mapping of genes for alpha-type prolamins in the wild grass Haynaldia villosa.

Chromosome Mapping↗

PubMed1988

The use of two-dimensional gel electrophoresis in the analysis of organ-specific maize proteins.

Two-dimensional gel electrophoresis with non-equilibrium pH gradient electrophoresis in the first dimension and sodium dodecyl sulfate-polyacrylamide gels in the second dimension has been used for the analysis of organ-specific proteins in maize. The method has been used to study the whole protein pattern of developing organs and adult leaves as well as protein patterns of in vitro translation. Examples of two-dimensional immunoblotting and in vitro translation of endosperm-specific proteins are also shown. Two-dimensional gel electrophoresis appears as an essential analytical step in the identification of organ-specific proteins and for the detection of the protein products related to organ-specific genes identified by other means.

Chloroplasts↗

PubMed1989

Improved separation of alpha chains of collagen type I, type III, and type V by noninterrupted electrophoresis using thioglycolic acid as a negatively charged reducer.

Improved separation of alpha chains of collagen type I (alpha 1 [I]2 alpha 2[I]), type III(alpha 1[III]3), and type V (alpha 1[V]alpha 3[V])was achieved by noninterrupted sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a negatively charged reducer, thioglycolic acid. The thioglycolic acid, added to the running buffer of the cathodic reservoir, in the middle of electrophoresis quickly migrated in the gel anode, reducing interchain disulfide linkages in collagen type III and dissociating it into its alpha chain monomer, alpha 1[III], without an interruption of electrophoresis. The alpha chain, alpha 1[III], migrated more slowly than the alpha 1 [I] and alpha 2[I] chains of collagen type I, resulting in an excellent separation of alpha 1[III] from alpha 1[I]. The mobility of alpha 1[III] could be controlled by varying the time of thioglycolic acid addition to the running buffer. This enabled us not only to separate alpha 1[III] from alpha 1[I] and alpha 1[V], but also to precisely quantitate these alpha chains, even at low protein loading of mixed samples.

Collagen↗

PubMedDate not supplied

Measurement of protein-DNA interaction parameters by electrophoresis mobility shift assay.

Native gel electrophoresis (mobility shift) assays may be used to obtain quantitative information about the site distribution, equilibria and kinetics of protein-DNA interactions. These applications depend on the ability of the electrophoretic system to resolve the reaction components, and on their stabilities during the separation process. Factors which affect the lifetimes and mobilities of protein-DNA complexes during electrophoresis include reaction and electrophoresis buffer composition, pH, and ionic strength; the presence of low molecular weight effectors and enzymatic substrates; the nature and concentration of the gel matrix; the temperature; the molecular weights of protein and DNA; the stoichiometric ratios of their complexes; and the possibility of conformational and configurational isomerization of reaction components. We discuss how these factors influence the acquisition of quantitative data from electrophoretic patterns and band intensities, and present formulas for the estimation of equilibrium constants and rate constants for prototypical DNA-protein interactions.

Binding, Competitive↗

PubMed1989

Gel electrophoresis of native actin and the actin-deoxyribonuclease I complex.

Electrophoresis of monomeric actin (G-actin) on 8-25% acrylamide Pharmacia PhastGels was carried out using gels and agarose buffer strips preequilibrated in buffer containing adenosine triphosphate (ATP), calcium ions (Ca2+) and dithiothreitol. On these gels G-actin ran as a sharp band at an apparent molecular mass of 45 kDa relative to standard proteins which is slightly greater than its actual molecular mass of 42 kDa. Electrophoresis in the absence of these solutes led to denaturation and aggregation of the protein, as reflected by a long streak. Filamentous actin (F-actin) did not enter the gel. The actin monomer-binding protein, deoxyribonuclease I, (DNase I) forms a binary complex with G-actin. The purity and apparent molecular mass 74 kDa of this complex were determined by native gel electrophoresis. By the simple procedure of preequilibrating both gel and buffer strips with appropriate ligands, this technique could be extended to investigate interactions between actin and other G-actin-binding proteins and other proteins whose stability is ligand dependent.

Actins↗

PubMed1990

Electrophoresis and densitometry of serum and urine in the investigation and significance of monoclonal immunoglobulins.

About 15% of monoclonal components or paraproteins are associated with malignancy when detected by simple electrophoresis on cellulose acetate membranes or agarose gel followed by protein staining. More sensitive methods for detecting monoclonal components result in a lower frequency of association with recognisable underlying disease. The sensitivity is dependent on the system used. Isoelectric focusing is 10 to 40 times more sensitive than simple electrophoresis at detecting monoclonal components. Electrophoretically separated bands may be quantitated densitometrically and at the present time this is the most satisfactory method for measuring monoclonal component concentration. Immunochemical methods for quantitating monoclonal components are limited by failure to react in a similar manner to polyclonal standards and giving problems of antigen excess resulting both in falsely elevated and low results. Electrophoretic methods must always be used in conjunction with these. The normal polyclonal ratio of immunoglobulin kappa:lambda light chain is disturbed by the presence of a monoclonal component. In 260 patients with myeloma we found that serum electrophoresis alone detected 90% while a disturbance in the kappa:lambda ratio detected 98%. However, 50% of monoclonal components less than 3 g/L were missed. Log kappa:lambda ratio correlates well with the densitometric scan of monoclonal components, both in vitro and in patients being monitored for treatment response in myeloma. This approach may complement or even replace densitometry for this purpose in the future.

Adult↗

PubMed1990

Two-dimensional electrophoresis of dog bronchoalveolar lavage fluid proteins.

Proteins of dog bronchoalveolar lavage fluid, obtained by washing the epithelial lining layer of lungs with phosphate-buffered saline, were separated by two-dimensional electrophoresis. Due to the low protein and high salt content of the bronchoalveolar lavage fluid, samples had to be concentrated and desalted. Following electrophoresis the protein spots were visualized by silver staining. Comparing the two-dimensional protein patterns of bronchoalveolar lavage fluid with that from serum, several lung-specific proteins were detected. The most prominent protein, most probably a surfactant-associated protein, showed isoforms with isoelectric points in the range of pH 4.2-4.8, and a molecular mass of 32 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis after reduction with dithiothreitol.

Animals↗

PubMed1990

Multiphasic electrophoresis with diversified spacers.

The principle and some applications of multiphasic electrophoresis with diversified spacers are described. The method combines previously published concepts of introducing highly diversified spacers into the stack of an isotachophoretic system with the theory of snow- and telescope-electrophoresis. The link between this electrophoretic method and isotachophoresis on the one hand and monophasic zone electrophoresis on the other hand is exemplified. The importance of the nature of the spacers as well as of the pH and ions in the leading and terminating zone is illustrated. Details are given concerning the experimental set-up. This electrophoretic technique could serve as an alternative to isoelectric focusing.

Blood Proteins↗

PubMed1990

Separation of enzymes from Candida boidinii crude extract by continuous flow zone electrophoresis.

Separation of the enzymes formate dehydrogenase, formaldehyde dehydrogenase and methanol oxidase from Candida boidinii crude extract has been explored using continuous flow zone electrophoresis in the VaP-22 and the scaled-up VaP-220 electrophoresis apparatus. Yields up to 95% and purification factors between 3 and 7 were obtained, together with separation of cell debris from the enzymes. Multiple injections of sample were used to obtain a protein throughput of 46.2 mg/h in the VaP-22. A tenfold higher throughput was achieved using the VaP-220. Correlation of the electrophoretic mobility in continuous flow zone electrophoresis with the elution behavior in ion-exchange chromatography confirmed the primary role of net surface charge in the separation of biological molecules. Proteins and enzymes with differences greater than 0.05 M elution molarities in ion-exchange chromatography can be separated. This corresponds to a preparative scale (mg/h or g/h) separation of proteins and enzymes whose difference in apparent electrophoretic mobility is greater than 0.70 x 10(-5) cm2/(V.s).

Alcohol Oxidoreductases↗

PubMed1991

Efficient, small scale electroelution of high molecular weight DNA from agarose gels by a miniature vertical electrophoresis cell.

A miniature, vertical electrophoretic system is described that allows quick elution and efficient harvesting of double-stranded, high molecular weight DNA fragments from agarose gels. A gel slice containing the band of interest is excised from the agarose gel after submarine electrophoresis and placed into an electrophoresis cell, assembled by inserting a truncated micropipette tip into a 2.0 mL polypropylene tube. The tip supports the agarose slice and is connected to dialysis tubing. Platinum wire electrodes are placed over the tip and at the bottom of the test tube. DNA molecules are quickly eluted into the dialysis bag (60 s to 10 min for bands ranging from 2 to 23 kb) and then easily recovered. The method is highly efficient and allows considerable time saving. Of special interest is its applicability to small starting amounts of DNA (less than 0.5 micrograms per band). Purity of the DNA is comparable, if higher, to that obtainable by conventional electroelution on dialysis bags and DEAE-cellulose membrane electrophoresis. The system is simple enough to be used as a routine method for eluting multiple bands in short times.

Bacteriophage lambda↗

PubMed1991

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.

Buffers↗

PubMed1991

Application of schlieren optics to real-time monitoring of protein electrophoresis in crosslinker-free linear polyacrylamide solution.

Molecular sieving effect of crosslinker-free linear polyacrylamide solutions could be visualized in a real-time mode using schlieren optics for electrophoresis of proteins. Since linear polymer solutions are going to find application in capillary electrophoresis (Zhu, M., et al., J. Chromatogr. 1989, 480, 311-319), the above approach may be useful for obtaining basic data of electrophoresis of proteins and nucleic acids in such media under conditions free from the predominant wall effect pertinent to the use of a capillary.

Cross-Linking Reagents↗

PubMedDate not supplied

A practicable two-dimensional electrophoresis of urinary proteins as a useful tool in medical diagnosis.

Practical experience with a rapid two-dimensional electrophoresis technique for routine analysis of urinary proteins is discussed. The method consists of cellulose acetate electrophoresis in combination with sodium dodecyl sulfate (SDS) electrophoresis, performed together with Coomassie Blue gel staining on "PhastSystem". Over 400 analyses were performed within the time of two years. Most patients were from nephrological, urological and kidney-transplant departments. Some of them were from obstetric, pediatric or oncological departments. A systematic discussion and evaluation of the two-dimensional protein pattern with typical examples is outlined.

Blood Proteins↗

PubMed1991

Capillary zone electrophoresis for the quantitation of oligosaccharides formed through the action of chitinase.

Capillary zone electrophoresis with fluorescence detection was used to analyze the products formed by chitinase acting on N-acetylchitooligosaccharide-fluorescent conjugates. Six oligosaccharides of the structure [N-acetylglucosamine(1----4)]n (where n = 1-6) were conjugated to 7-amino-1,3-naphthalene disulfonic acid by reductive amination. Each oligosaccharide-fluorescent conjugate was purified by preparative gradient polyacrylamide gel electrophoresis, semi-dry electrotransfer to a positively-charged nylon membrane and recovered by washing the membrane with salt solution. The products formed by treating each oligosaccharide-fluorescent conjugate with chitinase were analyzed by capillary zone electrophoresis. The chitinase treatment hexasaccharide-fluorescent conjugate was also examined kinetically to study the action pattern of this enzyme.

Amination↗

PubMed1992

High resolution-separation of DNA bands by electrophoresis with a long gel in a fluorescence-detection DNA sequencer.

A high-resolution separation of DNA bands is achieved by electrophoresis with a long gel in DNA base sequencing using fluorescence detection. We separate 760 and 761 base DNA fragments using the 93 cm migration electrophoresis optimized for the separation of DNA bands. A T7 DNA polymerase and an Mn++ buffer are used in sequencing reactions to obtain fluorescence peaks of uniform strength, and the peak areas in the spectrum are used for recognizing the peak number in a cluster of successive peaks. This method is successfully applied to the DNA fragment spectrum obtained by 93 cm migration electrophoresis, which results in a single-band differentiation of bands of 1040 base DNA.

Base Sequence↗

PubMedDate not supplied

Preparative continuous separation of biological particles by means of free-flow magnetophoresis in a free-flow electrophoresis chamber.

For sorting, cells or cellular components can specifically be labeled by antibody-coated magnetic beads. We have developed a device for continuous magnetic sorting based on the flow-chamber of a free-flow electrophoresis system. Magnetically labeled particles are injected into a given continuously flowing chamber buffer and pass an inhomogeneous magnetic field, configurated perpendicular to the flow direction. According to its magnetic moment, the magnetic material is deviated into the direction of the magnetic forces, while nonmagnetic material passes the field without interaction. The magnetic forces can be changed with the electrical current of the solenoids producing the magnetic field. As in the free-flow electrophoresis system, the particle fractions are collected in different vials. On-line control of the experiments can be performed by an optical scanning system. Experiments with model particles achieved a sorting purity of more than 99% at a rate of up to 5 X 10(8) particles per hour. In experiments with blood cells, a high enrichment of either B-or-T-lymphocytes was obtained. In contrast to free-flow electrophoresis, there is no limitation, in principle, regarding the type of chamber buffer to be used. This allows an optimal adaptation of the buffer conditions to the requirements of vital sorting. The preliminary results so far confirm this conclusion.

B-Lymphocytes↗

PubMedDate not supplied

Two-dimensional electrophoresis of the total polypeptides in ripe red grape berries.

The total polypeptide composition of mature grape berries was analyzed by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional electrophoresis (isoelectric focusing in the first dimension followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension), followed by Coomassie Blue and nitrate silver staining, respectively. Adapted methods for total protein preparation of grapes and for two-dimensional gel electrophoretic separation of polypeptides are presented. The grape patterns presented up to 52 fractions with Mrs ranging from 15,000 to 110,000. The polypeptides displayed pIs from 4.6 to 7.3. A group of spots from Mr 28,000 to 83,000 and with a pI from 4.6 to 5.4 was strongly silver stained. The Mr 28,000 spot, pI 4.6, was revealed to be a complex of four fractions. Reproducible separations were obtained with the different carrier ampholyte mixtures tested.

Electrophoresis, Gel, Two-Dimensional↗

PubMed1992

A new concept for sequencing DNA by capillary electrophoresis.

It is proposed that the scaling symmetry of constant charge density with increasing molecular weight, which prevents the separation by electrophoresis of DNA molecules in solution (with respect to molecular weight) be broken by the attachment of a perturbing entity (protein, virus or charged sphere) to one end of the molecule. An application of this idea to a concept for sequencing DNA by capillary electrophoresis is discussed, and the possibility of using the reattachment of the RecA protein to separate large segments of DNA in solution by electrophoresis following sequence-specific cleavage is mentioned.

Base Sequence↗