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PubMed1992

Ultramicrodetection of proteins in polyacrylamide gels.

Here we report the development of a highly sensitive procedure to detect proteins within separation matrices which should facilitate the characterization of rare proteins. The procedure is based on photochemical reactions where very low amounts of silver are deposited around proteins and in a series of steps are converted to silver sulfide. When this conversion is carried out in the presence of [35S]thiourea the resulting radioactive silver sulfide allows detection down to femtogram quantities of protein. In this work we applied the above principle to proteins separated on sodium dodecyl sulfate-polyacrylamide gels, thus not influencing physical and chemical parameters which are important for separation. This procedure should find application in any technique where detection of very low or limited amounts of proteins are required.

Electrophoresis, Polyacrylamide Gel↗
PubMed1992

High-resolution one-dimensional polyacrylamide gel isoelectric focusing of various forms of elongation factor-2.

A system for analyzing covalent modifications of elongation factor-2 (eEF-2) by one-dimensional isoelectric focusing in slab polyacrylamide gels is described. Depending on the degree of phosphorylation, four species of eEF-2 could be resolved corresponding to the un-, mono-, bis-, and trisphosphorylated factor. Furthermore, the degree of ADP-ribosylation of the protein could also be assessed by this method. It was also shown that an acidic isoform of eEF-2 exists which appears not to be artifactual and that the relative level of this isoform appeared to vary between different cell types. By Western blotting the gels and using an antibody against eEF-2 it is possible to assess the state of phosphorylation of the factor in cells.

Animals↗
PubMed1992

Characterization of insulin-like growth factor binding proteins by two-dimensional polyacrylamide gel electrophoresis and ligand blot analysis.

Insulin-like growth factor binding proteins (IGFBPs) in pregnant baboon serum and tissue culture media obtained following explant culture of uteri from pregnant baboons were characterized by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (2D SDS-PAGE) followed by Western ligand blot analysis using 125I-labeled IGF-I. IGFBP-1 (Mr 30,000; pI 4-4.2), IGFBP-2 (Mr 34,000, pI 5.7-6.2), IGFBP-3 (doublet Mr 42-48,000; pI 6.2-6.8), and IGFBP-4 (Mr 24,000; pI 5.7-6.0) were clearly separated from one another. The authenticity of IGFBP-1, -2, and -3 was verified by immunoprecipitation using polyclonal antibodies followed by ligand blotting. Specificity of 125I-labeled IGF-I binding to IGFBPs was also determined by competitive binding studies using unlabeled IGF-I and -II. This technique allows for the identification of IGFBPs in complex biological fluids on the basis of their characteristic Mr and pI with or without the availability of specific antibodies and can be done rapidly using the mini 2D SDS-PAGE systems.

Animals↗
PubMed1992

Cytochrome c aided resolution of Lupinus albus isoperoxidases in a cathodal polyacrylamide gel electrophoresis system.

In a cathodal polyacrylamide gel electrophoresis system, three distinct groups of isoperoxidases from Lupinus albus were found to achieve retention factors (rf) dependent on the quantity of sample applied onto the gel. The possibility of extract-derived substances weakly associating with peroxidase samples was investigated. Association of the putative agents survived dialysis against electrophoresis buffer with and without 2 M CaCl2 and freeze-thaw treatments. The addition of polyvinylpolypyrrolidone and polyethylene glycol to the homogenization buffer also proved ineffective in eliminating the variation in isoperoxidase rf although differences in the zymogram profiles of these samples were evident. The addition of spermine and cytochrome c to samples was found to increase the rf of some peroxidase bands. Electrophoresis of samples with cytochrome c resulted in the resolution of peroxidase groups to distinct bands at rf independent of the quantity of peroxidase applied. Control experiments indicate that this treatment did not introduce any detectable artifacts.

Cell Wall↗
PubMed1992

Western blotting and immunochemical detection of histones electrophoretically resolved on acid-urea-triton- and sodium dodecyl sulfate-polyacrylamide gels.

We have developed a method for the efficient transfer of histones from acetic acid-urea-Triton X-100 (AUT)-polyacrylamide minislab gels to nitrocellulose. The AUT gel was equilibrated with 50 mM acetic acid and 0.5% sodium dodecyl sulfate and then with 62.5 mM Tris-HCl, pH 6.8, and 2.3% sodium dodecyl sulfate. An alkaline transfer buffer [25 mM 3-(cyclohexylamino)-1-propanesulfonic acid, pH 10, with 20% methanol] was used to electrophoretically transfer the strongly basic proteins from AUT or sodium dodecyl sulfate gels to nitrocellulose. The applicability of this approach in the immunochemical detection of ubiquitinated histone species is demonstrated.

Animals↗
PubMed1985

Subcellular distribution of protein kinase C of GH3 cells: quantitation and characterization by polyacrylamide gel electrophoresis.

Quantitative estimation of cytosolic Ca2+- and phospholipid-dependent protein kinase (PKC) activity was performed by polyacrylamide gel electrophoresis under nondenaturating conditions (PAGE). With this method less than 50 micrograms of cytosol protein can be accurately quantitated for PKC activity. The amount of cytosolic PKC activity recovered after PAGE was comparable to the amount obtained by DEAE-cellulose chromatography. Homogenization of GH3 cells in the presence of 2 mM EGTA/EDTA revealed that 80% of the total cellular PKC activity resided in the cytosol. However, omission of the ion chelator during cell disruption followed by subcellular fractionation and extraction of subcellular fractions by EDTA/EGTA showed that 80% of the total PKC was found in the lysosomal-mitochondrial and microsomal extracts. Detailed analysis of PKC activities demonstrated that cytosolic PKC was identical with the PKC solubilized by EDTA/EGTA from subcellular fractions. In conclusion, GH3 cells appear to contain one species of PKC with an apparent molecular weight of 90,000 which seems to be associated with membranes via a calcium-dependent mechanism (or mechanisms).

Animals↗
PubMed1987

Two-dimensional polyacrylamide gel electrophoresis analysis of phosphorylated, membrane-localized ras p21 proteins.

The ras p21 oncogene product migrates as a heterogeneous series of polypeptides as resolved by both one- and two-dimensional polyacrylamide gel electrophoresis (PAGE). We have prepared polyclonal rat serum antibody to ras p21 and used this as well as monoclonal antibodies to immunoprecipitate forms of p21 synthesized in vivo in transformed NIH3T3 cells. Two-dimensional PAGE of p21 resolved two distinct groups of polypeptides, one acidic (pI 4.8-5.3) which we call the "A" forms, and one less acidic or more basic (pI 6.5-7.0) which we call the "B" forms. It is the membrane-localized, B forms of v-ras-Ha p21 that are predominantly phosphorylated in vivo.

Animals↗
PubMed1988

Purification of galactose oxidase from Dactylium dendroides by affinity chromatography on melibiose-polyacrylamide.

Galactose oxidase is a fungal enzyme which is known to oxidize the C-6 hydroxymethyl of galactose and galactosamine to an aldehyde group. It has been widely used in glycoconjugate research, for example in the labeling of asialoglycoproteins. We have developed a simple affinity purification for galactose oxidase using melibiose-polyacrylamide. This affinity procedure was used to purify the enzyme from ammonium sulfate precipitates of culture filtrates of Dactylium dendroides. The material containing proteases and other contaminants is eluted in the buffer wash. The galactose oxidase is then specifically eluted from the column with buffer containing 0.1 M D-fucose or D-galactose. Using this procedure, the enzyme was also purified from commercial samples of galactose oxidase which contain high proteolytic activity.

Acrylic Resins↗
PubMed1992

Modification of acidic residues normalizes sodium dodecyl sulfate-polyacrylamide gel electrophoresis of caldesmon and other proteins that migrate anomalously.

Caldesmon migrates as a 140-kDa protein during polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS), although its true molecular mass is close to 90 kDa. Since caldesmon's high acidic residue content may be responsible for this anomaly, it was reasoned that modification of these residues, with a loss of negative charge, might restore normal electrophoretic migration. Therefore caldesmon was reacted with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in the presence of excess ethanolamine, which results in negatively charged carboxylates being converted to neutral amides without protein cross-linking. The absence of cross-linking was shown by rotary shadow electron microscopy. In accord with expectations, modified caldesmon migrated as a 94-kDa protein when compared to standards, which were much less affected by modification. The anomalous migration of caldesmon might be due to the repulsion of negatively charged SDS by caldesmon's acidic residues. Low binding of SDS to caldesmon is consistent with the fact that SDS, up to 1%, had little or no effect on the secondary structure of caldesmon, as monitored by circular dichroism. However, other mechanisms can also explain these observations. The abnormal migration of tropomyosin and calsequestrin, both of which have a high percentage of acidic amino acids, was also "normalized" by this treatment. Thus this method might have general application for the electrophoresis of proteins which have a high acidic residue content and migrate anomalously.

Calmodulin-Binding Proteins↗
PubMed1995

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of human whole saliva.

The purpose of this study was to characterize the electrophoretic patterns of human resting and stimulated whole saliva. Pooled and individual, uncentrifuged, unprocessed whole saliva from 20 healthy, unmedicated individuals was run on sodium dodecyl sulphate-polyacrylamide gel electrophoresis and stained with Coomassie blue R-250. Similar and characteristic patterns for whole saliva were observed for all samples, consisting of over 34 blue- and pink-violet-staining bands. Individual differences were usually in the size (or density) of the bands, not in the number. Repeat sampling of stimulated whole saliva over a period of weeks from the same individuals revealed a striking consistency of protein patterns, indicating a profound physiological stability of whole saliva. These results provide baseline data for whole saliva and suggest its use for future studies.

Adult↗
PubMed1984

Separation of rat liver lysosome membrane adenosine triphosphatase activities by polyacrylamide gel electrophoresis.

Solubilization of rat liver lysosome membranes with octyl glucoside or lauryl sarcosinate and analysis of ATPase activities in sections of polyacrylamide gels after electrophoresis revealed one major peak at pH 8 and two peaks at pH 5. The pH 8 ATPase peak was not localized in the same peak with pH 5 ATPase activity, suggesting that these were catalyzed by different proteins. Ca2+- and Mg2+-ATPase activities at pH 8 were present in the same major peak, with Ca2+ activity predominating. The pH 8 Ca2+-ATPase was also not present in the same area of the gels as Ca2+-ADPase.

Adenosine Triphosphatases↗
PubMed1975

Rapid preparative isolation of major erythrocyte membrane proteins using polyacrylamide gel electrophoresis in sodium dodecylsulfate.

1. We describe a simple method for preparative, sodium dodecylsulfate/polyacrylamide gel electrophoresis of the major proteins in human erythrocyte membranes. 2. The method is based on extraction of prestained proteins from gel slabs. Three different fluorescent dyes (o-phthalaldehyde, fluorescamine and 1-dimethyl-aminonaphthalene-5-sulfonylchloride) have been used for pretaining. The method allows separation of up to 75 mg membrane protein and isolation of mg quantities of all major erythrocyte ghost proteins, while preserving the high resolution of analytical polyacrylamide gel electrophoresis. 3. Yield depends on extraction conditions and the molecular weight of the proteins being eluted. It ranges from 43-48% for protein 1 (apparent mol. wt approx. 310000) and 72-78% for protein 3(apparent mol. wt 87 000-93 000) to 87-93% for protein 6 (apparent mol. wt 35 000). 4. The labile behaviour of the high molecular "spectrin" bands (bands 1 and 2) is described. Extraction at room temperature tends to split these proteins into products of lower molecular weight. In contrast, the minor protein components 2.1 and 2.2 tend to aggregate yielding components 1 and 2. 5. N-terminal amino acid analyses have been performed on proteins 1, 2, 3, 4A, 4B, 5 and 6. Each of these bands contains several N-terminals, most of which appear constant. Some additional N-terminal amino acids vary from one donor to the next.

Amino Acid Sequence↗
PubMed1976

Determination of Triton X-100 binding to membrane proteins by polyacrylamide gel electrophoresis.

The molecular weight of proteins in protein-detergent complexes can be determined from ultracentrifugation experiments if the amount of bound detergent is known. A new sensitive method to measure the binding of the nonionic detergent Triton X-100 to proteins has been developed. For the membrane proteins studied, less than 50 mug of protein was required to achieve an accuracy of 10% in the determination of the detergent-protein weight ratio. The proteins were equilibrated with the detergent by electrophoresis into polyacrylamide gels containing radioactively labelled Triton X-100. The gels were then sliced and the amount of bound detergent calculated from the increase in radio-activity in the slices containing the protein zone. The amounts of protein were determined by amino acid analysis of identical protein zones cut from gels running parallel.

Electrophoresis, Polyacrylamide Gel↗
PubMed1980

Identification of surface proteins of a bacterial membrane using thiolactone-activated polyacrylamide beads.

A new method for the determination of protein accessibility in membranes and membrane fractions using a resin, 'Enzacryl' polythiolactone, is described. Enzacryl polythiolactone is a hydrophilic polymer of acrylamide and acrylamide derivatives with thiolactone ring substituents. The binding of enzymes and proteins to this resin is accomplished very simply by mixing them together in a simple aqueous buffer. Groups which react with the polymer in the pH range 5--9 include aliphatic and phenolic hydroxyls and aliphatic amino groups. Surface proteins of Bacillus licheniformis membrane and solubilised membrane fractions are bound irreversibly to this resin. Inaccessible proteins remaining in the fractions are solubilised with sodium dodecyl sulphate and examined by sodium dodecyl sulphate polyacrylamide gel electrophoresis.

Acrylic Resins↗
PubMed1981

Detection of the associated state of membrane proteins by polyacrylamide gradient gel electrophoresis with non-denaturing detergents. Application to band 3 protein from erythrocyte membranes.

Polyacrylamide gradient gel electrophoresis was carried out in micellar solutions of various detergents which differ in degree of potency to denature proteins. From the application of this method to band 3 protein from erythrocyte membranes, it was suggested that the procedure was useful in studying the molecular state of membrane proteins. The electrophoretic behaviors of human and bovine band 3 protein did not show any species specificity in either a denature state and a state resembling the native state. As well as in nonionic detergent solutions, the dimeric and tetrameric structures of bovine band 3 protein were preserved in sodium deoxycholate solution, in which protein complexes maintained in nonionic detergent solutions are frequently dissociated. Even in dodecyltrimethylammonium bromide solution, which is a denaturant for water-soluble proteins, part of the band 3 protein was still present as the oligomer. The results suggest that the oligomeric form of band 3 protein is the stable structure and that the dimer and tetramer possibly coexist in membranes.

Animals↗
PubMed1982

Tryptic peptide map analysis of the major human blood platelet membrane glycoproteins separated by two-dimensional polyacrylamide gel electrophoresis.

Washed platelets were surface-labelled by lactoperoxidase catalyzed iodination and either the platelets or membranes were solubilized in detergent and applied to a wheat germ agglutinin-Sepharose column and a Lens culinaris lectin Sepharose column coupled sequentially. The glycoproteins eluted from the lectin columns were separated by two-dimensional gel electrophoresis. Alternatively, labelled whole platelets or membranes were solubilized and then directly separated by two-dimensional polyacrylamide gel electrophoresis. Spots corresponding to specific glycoproteins identified by apparent isoelectric point (pI), apparent molecular weight (Mr), staining and labelling characteristics were cut from the gels and analyzed by tryptic peptide mapping. The maps of the individual glycoproteins(GP) Ia, Ib, IIa, IIb, GP4-4.5 132-135, IIIa, IIIb and IIIc were all different. Glycoproteins with the same Mr but different pI were distinct with the exception of regions of GP Ib. There were minor differences in the maps of glycoproteins separated in the reduced or non-reduced state. Tryptic peptide maps provide a valuable additional parameter for the identification and characterization of platelet glycoproteins.

Blood Platelets↗
PubMed1977

Analysis by two-dimensional polyacrylamide gel electrophoresis of liver ribosomal subunnit proteins obtained from free and membrane-bound polysomes of unfasted animals.

Ribosomal proteins were analyzed by means of two-dimensional gel electrophoresis. To insure that the analysis included only that fraction of the ribosome actively participating in protein synthesis, only polysomal-bound ribosomes were used. This differs from previously reported analyses of liver ribosomal proteins. The ribosomal proteins were prepared from ribosomes of polysomal origin from membrane-bound and free polysomes. Membrane-bound and free liver polysomes were isolated from unfasted mice. The polysomes were purified on hydroxyapatite under conditions known to result in polysomes and ribosomes that are active in both endogenous and synthetic mRNA translation. Moreover, this is the first time that liver ribosomal protein was obtained and analyzed from animals that have not been starved prior to sacrifice. The puromycin-released ribosomes were dissociated into subunits and ribosomal proteins were analyzed by means of two-dimensional polyacrylamide gel electrophoresis. When 100-200 mug samples of the ribosomal subunit proteins were analyzed by two-dimensional electrophoresis, approximately 32 major proteins were detected for the 60 S ribosomal subunit and 25 major proteins for the 40 S ribosomal subunit. A total of 13 "fractional" ribosomal proteins was also detected in the ribosomal subunit profiles. No differences in number or mobility of the ribosomal proteins were found between the membrane-bound and free ribosome populations. We describe a system in which all ribosomal proteins are completely solubilized and quantitatively move from the first to the second dimension gel. Thus the total sample is separated and fractionated. This procedure elimates artifacts due to incomplete solubilization of ribosomal proteins, which is common for the transfer from the first- to second-dimension gel. Therefore, a more detailed and accurate analysis is achieved.

Animals↗
PubMed1976

Esterification of J chain and its effect on electrophoretic mobility in sodium dodecyl sulfate polyacrylamide gels.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis yields mobilities indicative of molecular weights of approximately 27 000 for human J chain and approximately 14 000 for nurse shark J chain, in contrast to values of approximately 15 500 and approximately 12 200, respectively, obtained by other methods. The relatively high content of acidic amino acids of human J chain as compared to nurse shark J chain suggested that the greater error in the sodium dodecyl sulfate determined molecular weight for human J chain may be due to a charge anomaly. The overall net negative charge on human J chain was decreased by forming methyl esters of the carboxyl groups, resulting in a sodium dodecyl sulfate determined molecular weight of approximately 17 700. Methylated nurse shark J chain did not show a significant difference in sodium dodecyl sulfate electrophoretic mobility from the non-methylated form. These results form the basis for a possible explanation of an occasional artifact in a widely used analytical method.

Animals↗