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PubMed1986

Optimization of erythrocyte membrane glycoprotein fluorescent labeling with dansylhydrazine after polyacrylamide gel electrophoresis.

An improved procedure for the labeling of glycoproteins with dansylhydrazine subsequent to electrophoresis in polyacrylamide gels is reported. This procedure is derived from the work of Eckhardt et al. (1976, Anal. Biochem. 73, 192-197) and Weber and Hof (1975, Biochem. Biophys. Res. Commun. 65, 1298-1302) who showed that dansylhydrazine may be condensed with the aldehyde groups of oxidized glycoprotein carbohydrates and the resulting hydrazones reduced with dimethylamine borane and/or sodium borohydride. Using the known distribution of erythrocyte membrane glycoproteins as a benchmark the effect of variation of a number of process parameters was investigated and an optimal procedure identified. The procedure is shown to be relatively insensitive to moderate variations in reagent composition, pH, and time of incubation with dansylhydrazine solution or reducing agents. It is also shown that labeling patterns may be preserved in dried gels if dimethylsulfoxide is replaced or omitted from all of the process solutions and destaining is effected with 1 M sodium acetate, pH 5.6. While specifically developed for the labeling of erythrocyte membrane proteins, the procedure is demonstrated to be applicable to other glycoprotein containing preparations.

Dansyl Compounds↗
PubMed1986

Quantitation of proteins by elution of Coomassie brilliant blue R from stained bands after sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

A simple method for the extraction of Coomassie brilliant blue R from stained protein bands excised from polyacrylamide gels is described. Spectrophotometric measurement of the eluted dye forms the basis of a sensitive assay to quantitate proteins in gels in the range 0.5-10 micrograms. The method requires no unusual equipment and is suitable for measurement of multiple samples. The polypeptide is not extracted and remains available for further analysis. The technique has been applied to three proteins and gels of various acrylamide percentages.

Electrophoresis, Polyacrylamide Gel↗
PubMed1986

Detection of dextransucrase and levansucrase on polyacrylamide gels by the periodic acid-Schiff stain: staining artifacts and their prevention.

One use of the periodic acid-Schiff (PAS) stain is to detect dextransucrase and levansucrase activities on polyacrylamide gels by staining their polysaccharide products, dextran and levan. When gels with heavy dextran or levan bands were PAS stained, proteins other than dextransucrase and levansucrase also were stained, and a high background developed during storage. The staining of proteins other than dextransucrase and levansucrase is caused by the diffusion of the periodate-oxidized carbohydrate before and after staining. This diffusion could be greatly slowed, and the staining artifact decreased, by following the PAS stain by a crosslinking treatment of the carbohydrate-dye complex. Protein staining artifacts could be prevented by using chymotrypsin to remove the protein from the gel at the stage after polysaccharide synthesis but before the PAS stain.

Chymotrypsin↗
PubMed1986

Isoelectric focusing of proteins in rewetted ultrathin polyacrylamide gel layers.

Ultrathin layers of polyacrylamide gel bound to glass can be washed, air-dried, and stored for at least 1 year before rewetting in ampholyte solutions for isoelectric focusing. Short-term drying affects neither fluorescent banding of the ampholytes (not evident in conventional gels) nor resolution of complex protein mixtures while prolonged storage seems to have no deleterious effects. Layers are fully functional after soaking for 10 min in solutions that may contain 8 M urea and 10% sorbitol. Rewetting allows the rapid survey of different ampholytes, gradient stabilizers, separator compounds, or protein reagents and is adaptable to concentration modification of the pH gradient (alone or with a gel overlay), to focusing in a transverse urea gradient, and to electrophoresis across a preformed pH gradient. The procedure avoids protein modification by residual polymerizing reagents while adding to the convenience and economy of using ultrathin layers in relatively small formats.

Acrylic Resins↗
PubMed1986

Purification of small oligonucleotides by polyacrylamide gel electrophoresis and transfer to diethylaminoethyl paper.

The application of polyacrylamide gel electrophoresis with subsequent electroelution onto DEAE paper for the purification of small oligonucleotides is described. We demonstrate that synthetic DNAs and hydrolyzed RNAs as small as three nucleotides in length can be purified by this technique. The product is undegraded and homogeneous in length.

Electrophoresis, Polyacrylamide Gel↗
PubMed1986

Combined alcian blue and silver staining of glycosaminoglycans in polyacrylamide gels: application to electrophoretic analysis of molecular weight distribution.

Oligomeric and polymeric fragments of glycosaminoglycans may be separated for rapid analysis by electrophoresis through a 10% polyacrylamide matrix. A ladder-like series of bands is observed, in which adjacent major bands correspond to species differing in chain length by one disaccharide unit. The component species are detected by a combined alcian blue and silver staining protocol. Detection limits are less than 50 ng per band, or approximately 2-5 micrograms total load for polydisperse samples. Densitometry of the stained gel may be used to determine molecular weight averages and distribution. The applicable molecular weight ranges are approximately 4000 to 100,000 for hyaluronate, or 1500 to 40,000 for chondroitin and dermatan sulfate samples of moderate charge density heterogeneity.

Alcian Blue↗
PubMed1986

Enzymatic detection of native and derivatized horseradish peroxidase in sodium dodecyl sulfate polyacrylamide gels.

We developed procedures for the restoration of peroxidatic activity in native horseradish peroxidase (HRP) and HRP conjugated to wheat germ agglutinin (WGA-HRP) following electrophoresis in SDS-polyacrylamide gels (SDS-PAGE). After extraction of SDS with isopropanol from gels containing HRP and WGA-HRP, the peroxidatic activity in these probes could be demonstrated by tetramethylbenzidine (TMB) chemistry. This procedure also showed HRP enzyme activity in electrophoresed tissue homogenates containing HRP. Both free HRP as well as WGA-HRP preparations contain several molecular weight species that display peroxidatic activity. These findings are important for cell biological studies utilizing these substances as molecular probes. The procedures described here should be useful for the analysis of the enzymatically active molecular forms of these frequently used markers in vitro and in vivo.

Animals↗
PubMed1986

Applications of chemical cleavage procedures to the peptide mapping of neurofilament triplet protein bands in sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

A procedure for examining possible sequence homology in the triplet neurofilament proteins using a sodium dodecyl sulfate-polyacrylamide gel electrophoresis system is described. Five different chemical reagents (cyanogen bromide, BNPS-skatole, hydroxylamine, formic acid, and nitrothiocyanobenzoic acid) have been used for peptide mapping studies. Potential applications of this technique are discussed.

Amino Acid Sequence↗
PubMed1986

Renaturation of dextranase activity from culture supernatant fluids of Streptococcus sobrinus after sodium dodecylsulfate polyacrylamide gel electrophoresis.

A rapid and reproducible method for the assay of individual fractions of the multicomponent dextranase activity of Streptococcus sobrinus after reduction/denaturation and electrophoresis in acrylamide gels is described. Multiple forms of dextranase, possible virulence factors in the formation of dental caries by oral Streptococci (S. mutans, S. sobrinus, S. sanguis, S. cricetus, and S. rattus), have been separated in sodium dodecylsulfate-polyacrylamide gels into which an indicator substrate, blue dextran, has been incorporated, and identified after renaturation to remove the reducing/denaturing agents of the Laemmli buffer system.

Animals↗
PubMed1987

In situ staining procedure for the detection of ornithine transcarbamylase activity in polyacrylamide gels.

Ornithine transcarbamylase deficiency is a human genetic disease potentially susceptible to gene therapy. A murine model system exists for the disease in the sparse-fur (spf) mouse. Before gene therapy studies can be performed it is necessary to have practical methods which could detect successful gene transfer. Therefore we have developed an in situ staining procedure for the detection of ornithine transcarbamylase activity in polyacrylamide gels. Following electrophoretic separation under nondenaturing conditions inorganic phosphate cleaved from carbamyl phosphate in gels as a result of enzymatic activity was precipitated as phosphomolybdic acid and visualized by reduction with ascorbic acid. Results from the procedure correlated with ornithine transcarbamylase activity as measured by solution assay for citrulline, the other product of the reaction. This procedure readily distinguished mutant forms of ornithine transcarbamylase as exemplified by the murine spf mutation and resolved ornithine transcarbamylases of all animals tested into multiple forms. The procedure further distinguished ornithine transcarbamylases of animals of several different genera while yielding virtually identical patterns of the enzyme from species within the same genus. This procedure also suggested that the human enzyme was more labile than murine ornithine transcarbamylase; direct thermolability studies confirmed this finding.

Animals↗
PubMed1987

Rapid isoelectric focusing in a vertical polyacrylamide minigel system.

A rapid method is described for the resolution of proteins employing isoelectric focusing in a vertical polyacrylamide minigel system. Isoelectric focusing can be performed in only 3 h, utilizing low voltage, under either native conditions or denaturing conditions in the presence of 8 M urea. The procedure permits the application of larger sample volumes containing more protein than other isoelectric focusing procedures, and provides the additional advantages of slab gels over tube gels for analytical purposes. The procedure is also well adapted for use in two-dimensional electrophoretic techniques, making it possible to complete a two-dimensional gel in 1 day.

Electrophoresis, Polyacrylamide Gel↗
PubMed1987

Quantitative molecular hybridization with unfractionated, solubilized cells using RNA probes and polyacrylamide gel electrophoresis.

Molecular hybridization with RNA probes was performed on unfractionated cells solubilized in guanidine thiocyanate solutions. Unhybridized probe was digested with ribonuclease, and protected probe fragments were resolved by polyacrylamide gel electrophoresis (PAGE). Since the same medium was used both for solubilization of the cells and as the hybridization buffer, RNA purification was not required and the analysis of large numbers of samples was facilitated. Using this method, specificity is superior to dot blot analysis because the size of hybridized fragments is determined and the signal of the probe hybridized to target RNA is separated from the background by PAGE.

Animals↗
PubMed1987

Tryptic peptide analysis of nanogram quantities of proteins: radioiodination of proteins detected by silver staining in polyacrylamide gels.

The silver-staining procedure for detecting proteins in polyacrylamide gels has been modified so that the polypeptides remain suitable for subsequent radioiodination and tryptic peptide analysis. The procedure, which involves a silver-staining/destaining protocol that minimizes crosslinking, is more rapid than many other methods, and can detect as little as 1 ng of protein. Following elimination of silver, the proteins can be radioiodinated and digested with trypsin by a modification of the method described by J. H. Elder, R. A. Pickett, J. Hampton, and R. A. Lerner (1977, J. Biol. Chem. 252, 6510-6515). Together, these improvements have increased the sensitivity of the tryptic peptide mapping technique by three orders of magnitude and thereby enabled us to perform reproducible structural analysis of femtomolar quantities of proteins.

Electrophoresis, Polyacrylamide Gel↗
PubMed1987

Ketimine rings: useful detectors of enzymatic activities in solution and on polyacrylamide gel. Detection of L-amino acid oxidase, glutamine transaminase, pantetheinase, and acylase I.

A simple procedure has been developed for the detection of L-amino acid oxidase, glutamine transaminase, pantetheinase, and acylase I in solution and on polyacrylamide gels. The method is based on the strong absorbance at 296 nm of some ketimine rings which can be directly produced by the enzymatic reaction or formed by the reaction of the enzymatic product with 3-bromopyruvate. The procedure allows one to visualize up to about 1-10 mU of enzyme.

Amidohydrolases↗
PubMed1987

Bacterial adherence on replicas of sodium dodecyl sulfate-polyacrylamide gels.

A method for determining which molecules in a complex mixture of proteins can function as bacterial receptors was devised. Salivary proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose. Bacteria that were metabolically labeled with 3H or externally labeled with 125I were incubated on the nitrocellulose replicas. After 18 h at 4 degrees C, the unbound cells were removed by repeated washing of the replicas, and the bands to which the radiolabeled bacteria bound were visualized by autoradiography. By this technique, Fusobacterium nucleatum, which adheres via carbohydrate residues on receptor molecules, and Staphylococcus aureus, which recognizes the peptide portion of fibronectin, were shown to bind specifically to their respective receptors. These results suggest that this method can be useful for profiling bacterial binding to either the carbohydrate or the protein portions of molecules present in complex mixtures, such as those composing biological fluids or tissue substrates. Structural specificities, such as recognition sequences formed by certain oligosaccharides, could be further investigated by adding the appropriate simple sugars, as well as oligosaccharide inhibitors, to the incubation medium. The latter approach is particularly important since most glycoproteins carry multiple N- and O-linked carbohydrate substituents that could serve as bacterial receptors.

Bacterial Adhesion↗
PubMed1987

Location and purification of enzymes on polyacrylamide gels using dialyzable fluorescent markers.

A method for the location of proteins/enzymes by polyacrylamide gel electrophoresis using dialyzable low-molecular-weight fluorescent peptide markers is described. The markers prepared by treating the peptic digest of casein with fluorescamine showed several bands on gel electrophoresis which helped in locating the proteins. The located desired protein could subsequently be purified by extraction.

Aldose-Ketose Isomerases↗
PubMed1987

Detection of a few picograms of DNA on polyacrylamide gels by silver staining.

DNA fragments separated on polyacrylamide gels are silver stained in ethanolamine solution. The staining procedure can be completed in 3 1/2 h. Illumination of the gels on a black background increases the sensitivity of detection compared with the usual transillumination. The limit level of detection is 3-5 pg per band with a cross-sectional area of 5 mm2. Five to fifty picograms of DNA may be detected quantitatively by scanning the gels. The method will detect 0.1 to 1 ng per band of low-molecular-weight RNA components.

Animals↗