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PubMed1984

Use of cationic detergents for polyacrylamide gel electrophoresis in multiphasic buffer systems.

An improved system for polyacrylamide gel electrophoresis in the presence of cationic detergents, cetyltrimethylammonium bromide and cetylpyridinium chloride, respectively, is described. An acidic discontinuous buffer system generated according to the theory of multiphasic zone electrophoresis developed by T. M. Jovin (1973, Biochemistry 12, 871-904) was used. It was optimized with respect to the operational conditions and to the desirable range of relative mobility values for the proteins that have molecular weights from 16,500 to 90,300. Also presented is a procedure for the elimination of interference from cationic detergents frequently encountered during staining of gels. The electrophoretic system was suitable for fractionating a wide variety of proteins. The technique can also be used to provide an alternative estimate of molecular weight. To fully account for accurate estimations, the Ferguson relationship between mobility and gel concentration and the relation of molecular weight to mobility at a single gel concentration were both considered. Examples reported in this paper include the separation and/or molecular weight determination of several common proteins, histones, and microfibrillar and myofibrillar proteins. The results suggest that electrophoresis in the presence of cationic detergents offers the same degree of reliability in analysis of most proteins as is provided by the anionic detergent sodium dodecyl sulfate electrophoresis.

Buffers↗
PubMed1984

Quantitative assay of deoxyribonuclease activity after isoelectric focusing in polyacrylamide gels: pH control and effects of enzyme diffusion.

A method for the quantitative assay of nuclease activity in crude cell lysates after isoelectric focusing (IEF) in polyacrylamide slab gels is described. After IEF, an agarose overlay gel containing DNA is placed on the IEF gel and the nuclease activity quantified by the loss of ethidium bromide fluorescence of the DNA. With this method a linear response was obtained for 1 to 10 ng of DNase I. Various methods of pH equilibration after IEF were also evaluated. The use of a high buffer concentration in the overlay gel is recommended to control the pH during the enzyme reaction. An analytical solution for the diffusion of enzymes from the IEF gel to the overlay gel is also presented and an equation that may be used to choose optimum times for transfer of the enzyme from the IEF gel to the overlay gel is given.

Chemical Phenomena↗
PubMed1984

Evaluation of isoelectric focusing running conditions during two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis: variation of gel patterns with changing conditions and optimized isoelectric focusing conditions.

Five major isoelectric focusing (IEF) parameters--volt-hours; concentrations of acrylamide, NaOH, and H3PO4; and equilibration time--were systematically varied to determine the effect of each on two-dimensional IEF/sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel patterns and to optimize IEF conditions. Alterations in each parameter affected the gel pattern, frequently causing uncertainty in the identification of spots between conditions. The results emphasize the need for internal analytical consistency, and indicate that gel pattern comparisons between laboratories can be complicated if different IEF conditions are employed. The systematic evaluation indicated that optimized patterns were obtained when increased concentrations of NaOH and H3PO4 (to 50 and 25 mM, respectively) and run durations of 10,000 V-h or longer were used.

Buffers↗
PubMed1984

Rapid fluorescent staining of histones in sodium dodecyl sulfate-polyacrylamide gels.

The increase in the fluorescence intensity of 1-anilinonaphthalene-8-sulfonate (ANS) produced by core histones is higher than that produced by very lysine-rich histones (H1 and H5). In the presence of the anionic detergent sodium dodecyl sulfate (SDS) the enhancement of ANS fluorescence caused by these two groups of histones is roughly the same, but much lower than that observed for core histones in the absence of this detergent. However, the increase of ANS fluorescence produced by histone-SDS complexes is high enough to use it for the staining of these proteins separated in SDS-polyacrylamide gels. Histone bands are stained with ANS after electrophoresis and visualized by transillumination of the gel with a uv light source. The method described in this work allows the rapid detection of less than 0.5 microgram of histone per band.

Anilino Naphthalenesulfonates↗
PubMed1984

Peptide mapping by polyacrylamide gel electrophoresis after cleavage at aspartyl-prolyl peptide bonds in sodium dodecyl sulfate-containing buffers.

Protein samples prepared for sodium dodecyl sulfate-polyacrylamide gel electrophoresis are preferentially cleaved at aspartyl-prolyl peptide bonds upon heating at 110 degrees C. The presence of aspartyl-prolyl peptide bonds in a protein can therefore be detected by gel electrophoresis of heated samples and the resulting peptides mapped. The method of heat cleavage also works well with proteins in bands cut from electrophoresed gels using modified stacking conditions in the second electrophoresis. An immunoblotting procedure for peptide mapping of nanogram quantities of specific proteins in complex mixtures is demonstrated. Peptide maps produced by aspartyl-prolyl peptide bond cleavage of fructose-1,6-bisphosphatases from different sources show the effectiveness of the above techniques and suggest a conservation of aspartyl-prolyl peptide bonds in pig kidney and mouse and rat liver fructose-1,6-bisphosphatases.

Animals↗
PubMed1985

Method for extraction of proteins from green plant tissues for two-dimensional polyacrylamide gel electrophoresis.

A method has been developed for the extraction of proteins from green plant tissues to be used in two-dimensional polyacrylamide gel electrophoresis. Three purification steps were necessary to overcome the problems due to streaking, charge heterogeneity, and other artifacts: After it was ground in liquid nitrogen, the powdered material was stirred in the presence of insoluble polyvinylpyrrolidone for binding phenols, and sodium ascorbate for binding quinones; proteins were precipitated with ammonium sulfate, and the sample was dialyzed. Hundreds of proteins could be detected after Coomassie blue staining using 200 micrograms of proteins from apical buds of Sinapis alba L.

Chemical Phenomena↗
PubMed1985

Rapid electrotransfer of proteins from polyacrylamide gel to nitrocellulose membrane using surface-conductive glass as anode.

A new type of inexpensive horizontal apparatus for the electrophoretic transfer of proteins from a gel to an immobilization membrane has been developed. In this system, gel and membrane were directly pressed between two flat electrodes. A surface-conductive glass was used as anode and a stainless-steel plate as cathode. Proteins could be transferred from polyacrylamide gel to nitrocellulose sheet, with a yield of at least 90% in 60-90 min, without overheating, using a voltage gradient of 30-40 V/cm. Moderate volumes of separate anodic (with 20% methanol) and cathodic (with 0.1% sodium dodecyl sulfate) buffers were suited for optimal transfer of proteins with relative molecular mass (Mr) in the 14,000-94,000 range.

Animals↗
PubMed1985

Assay of proteoglycan populations using agarose-polyacrylamide gel electrophoresis.

The agarose-polyacrylamide gel electrophoresis procedure for the analysis of proteoglycans originally described by C. A. McDevitt and H. Muir (1971, Anal. Biochem. 44, 612-622) has been modified to minimize trailing and to allow the analysis of crude samples, i.e., tissue extracts. A slab gel system was used, permitting reproducible analysis of many samples. Procedures are described that can be used to separate and quantify several subpopulations of proteoglycans and also to quantify the proportion of proteoglycans capable of aggregating with hyaluronic acid. Applications of the procedure include transfer to nitrocellulose paper followed by immunological detection of proteoglycans as well as fluorography of separated, radiolabeled proteoglycans.

Animals↗
PubMed1985

Identification of human alcohol dehydrogenase isozymes by disc polyacrylamide gel electrophoresis in 7 M urea.

Polyacrylamide gel electrophoresis in the presence of 7 M urea provides a simple, reproducible method for the identification of cathodic alcohol dehydrogenase (ADH) isozymes. Treatment of native ADH dimers with 7 M urea and 1 mM dithiothreitol results in a complete dissociation of the 40,000 Mr subunits. Electrophoresis of urea-dissociated ADH isozymes yields a single protein band for homodimers and two bands of equal intensity for heterodimers. The ADH subunits pi, alpha, gamma 2, gamma 1, and beta exhibit electrophoretic mobilities of 0.71, 0.79, 0.88, 0.95, and 1.0, respectively. Thus, the identity of any cathodic ADH isozyme can be determined from the electrophoretic mobilities of its component subunits.

Alcohol Dehydrogenase↗
PubMed1985

In situ detection of beta-lactamase activity in sodium dodecyl sulfate-polyacrylamide gels.

After beta-lactamase had been denatured by boiling in the presence of sodium dodecyl sulfate (SDS) and then electrophoresed in SDS-polyacrylamide gels, activity could be restored and could be detected in situ as specific molecular species. Renaturation was simple and facilitated by the presence of a carrier protein. The assay was sensitive, detecting 0.8 ng beta-lactamase activity in the gel.

Bacillus↗
PubMed1985

Reversible covalent immobilization of ligands and proteins on polyacrylamide gels.

Ligands and proteins were covalently but reversibly immobilized on polyacrylamide gels using novel acrylic monomers whose syntheses are reported here. These reagents have an acrylyl group at one end for copolymerization into gels, an N-succinimidyl ester at the other allowing rapid immobilization of molecules having an available primary amino group, and a cleavable disulfide bond in the middle. Two immobilization methods were developed using these reagents. In the first method, a ligand with a primary amino group was treated with the immobilization reagent in anhydrous ethanol and the resulting amide derivative was purified and copolymerized with acrylamide and bisacrylamide resulting in the desired reversible immobilization. In the second method, the immobilization reagents (at densities up to 50 mumol/ml) were directly copolymerized with acrylamide and bisacrylamide to form activated gels of the desired shape and porosity. Proteins or other ligands in aqueous buffers were then added to the activated gels resulting in their covalent immobilization. Ligands or proteins immobilized using the methods reported here remained stably bound even when gels were subjected to boiling in detergents or high-ionic-strength buffers. Immobilized ligands were readily released (greater than 97%) from gels by treatment with quantitative amounts of aqueous dithiothreitol (DTT) under mild conditions. Immobilized proteins were also released (up to 87%) from the gels by DTT treatment. Small ligands (e.g., aminohexyl glycosides), active enzymes, and glycoproteins were immobilized, and then recovered, using these reagents.

Alkaline Phosphatase↗
PubMed1985

Silver staining of proteins in polyacrylamide gels: increased sensitivity through a combined Coomassie blue-silver stain procedure.

A combined Coomassie blue-silver stain method has been developed in sodium dodecyl sulfate-polyacrylamide gels for the detection of proteins using the model compounds bovine serum albumin, lysozyme, and recombinant DNA-derived human insulin. Sensitivity was enhanced 2.2 to 8.6 times by the new method relative to that of silver staining alone. The new method may also be useful in enhancing detection sensitivities of other proteins.

Electrophoresis, Polyacrylamide Gel↗
PubMed1985

A comparison of silver staining methods for detecting proteins in ultrathin polyacrylamide gels on support film after isoelectric focusing.

The application of silver staining methods to the detection of proteins on ultrathin isoelectric focusing gel systems requires the optimization of many steps in the procedure in order to obtain reproducible staining of proteins with acceptable levels of background. Three different methods which have been reported for detecting proteins by silver staining in sodium dodecyl sulfate-polyacrylamide gel systems were investigated. A major problem with staining ultrathin isoelectric focusing gels was found to be surface staining that was associated with gels cast on support films. A modification of the method of Poehling and Neuhoff (H.-M. Poehling and V. Neuhoff, 1981, Electrophoresis 2, 141-147) was found to give the best results.

Coloring Agents↗
PubMed1985

A specific iron stain for iron-binding proteins in polyacrylamide gels: application to transferrin and lactoferrin.

A new method for specifically staining the iron atoms present in transferrin and lactoferrin after polyacrylamide gel electrophoresis and isoelectric focusing is described. The stain, 3-(2-pyridyl)-5,6-bis(2-(5-furylsulfonic acid))-1,2,4-triazine, disodium salt, or Ferene S, will detect transferrin in 5 microliters of human serum, lactoferrin in 10 microliters of human whey, and 10 micrograms of purified primate (Macaca fascicularis) transferrin. This method of staining is very rapid as the serum transferrin bands can be seen within 5 to 10 min of staining.

Animals↗
PubMed1985

The determination of specific radioactivity of proteins eluted intact from polyacrylamide gels, utilizing a fluorescamine assay.

The methodology described permits the measurement of the specific radioactivity of diverse proteins resolvable by separatory techniques using cylindrical polyacrylamide gels. Following separation, the proteins are electroeluted; eluted protein is quantitated in the microgram range using a fluorescamine assay, while the major portion of the recovered sample is used for radioactivity measurement. These procedures have been adapted for use in tracer studies of protein metabolism. Their utility in kinetic investigations is demonstrated with data on the time course of changing specific radioactivities of human plasma albumin and apolipoprotein B labeled in vivo with a [3H]leucine tracer.

Apolipoproteins B↗
PubMed1985

The determination of molecular weights of biologically active proteins by cetyltrimethylammonium bromide-polyacrylamide gel electrophoresis.

A novel cetyltrimethylammonium bromide-polyacrylamide gel electrophoresis system which is useful for the separation of native forms of proteins consistent with their molecular weights is reported here. Many proteins examined in this system demonstrated the same association patterns which have been shown by other techniques to exist under nondenaturing conditions. In addition, biological activity could be assayed directly in the gel after electrophoresis. Based on the peculiar characteristics of cetyltrimethylammonium bromide, a possible explanation which may account for the behavior of proteins in this system is presented.

Cetrimonium↗
PubMed1985

Determination of collagen band positions on polyacrylamide gel without staining.

Protein band positions on a slab polyacrylamide gel were determined without staining, by blotting the bands onto a nitrocellulose membrane which was then stained to visualize the electrophoretic pattern. The unfixed, unstained bands were cut from the gel according to the copy pattern placed underneath the slab gel. Collagen alpha 1 and alpha 2 chains were separately extracted from the gel using this procedure.

Amino Acids↗