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PubMed1984

Genetic polymorphism of apolipoprotein E: a variant form of apolipoprotein E2 distinguished by sodium dodecyl sulfate--polyacrylamide gel electrophoresis.

The apolipoprotein E2 ( apoE2 ) variant that possesses a cysteine substituted for an arginine at residue 158 in the amino acid sequence E2( Arg158 ----Cys) can be distinguished by sodium dodecyl sulfate-polyacrylamide gel electrophoresis from other forms of apoE, including E3 (the parent form), E4( Cys112 ----Arg), E2( Arg145 ----Cys), and E2( Lys146 ----Gln). The E2( Arg158 ----Cys) migrates as a distinctly separable band with a higher apparent molecular weight than the other forms. Chemical modification of apoE2 ( Arg158 ----Cys) with sulfhydryl reagents (2-bromoethyl)-trimethylammonium bromide or cysteamine, which convert cysteine to arginyl or lysyl analogues, respectively, abolishes the difference in apparent molecular weight and results in the co-electrophoresis of E2( Arg158 ----Cys) with other apoE forms. The mobilities of the other apoE variants are not affected by these modifications. These results suggest that the substitution site at residue 158 is a key location, important in modifying the behavior of apoE and in modulating its apparent molecular weight on sodium dodecyl sulfate-polyacrylamide gels. Furthermore, the technique used in this study may be very helpful in distinguishing specific mutant forms of apoE2 .

Amino Acid Sequence↗
PubMedDate not supplied

Derivatization of polyacrylamide gels with pallidin, a lectin of the cellular slime mold polysphondylium pallidum.

A procedure for covalent immobilization of (glyco) proteins in polyacrylamide gels is described. Pallidin, a cytosolic and membrane protein of Polysphondylium pallidum with the properties of a lectin, was derivatized into polyacrylamide gels. Activity of the derivatized gels was assessed in a cell adhesion assay, using rabbit erythrocytes. Immobilization resulted in stabilization of the binding activity in comparison with the agglutination activity of the soluble protein.

Acrylic Resins↗
PubMedDate not supplied

[Comparative study of the oncornavirus A and D proteins of human J-96 cells by the methods of isoelectric focusing and polyacrylamide gel electrophoresis].

Three peaks of 14C-radioactivity with buoyant densities of 1.23--1.24, 1.26 and 1.29 g/ml were detected in a cytoplasmic extract of J-96 cells upon equilibrium centrifugation in sucrose gradient. Electron microscopy of the 1.23--1.24 g/ml buoyant density fraction revealed particles 60--80 nm in diameter showing morphology characteristic of oncornavirus A. Isoelectric focusing in polyacrylamide gel showed polypeptides of extracellular D virus and oncornavirus A to differ in isofocusing points (pI). Proteins of extracellular D virus were localized in zones with pH 3.7, 4.0, 4.4, 4.7, 5.6, 6.5, 8.1, 9.45, and 10.0; polypeptide of intracytoplasmic oncornavirus A had the following isofocusing points: 4.0, 4.9, 6.7, 7.3, 9.0, 9.45 and over 10.0. Electrophoresis of polypeptides of D virus and intracellular oncornavirus A revealed differences in the molecular weights of the components. No proteins with molecular weights of 10,000, 12,000, 15,000, and 27,000 dalton characteristic of the extracellular D virus were found in oncornavirus A virions. The analysis of protein patterns obtained in parallel experiments of isoelectric focusing and polyacrylamide gel electrophoresis suggests that oncornaviruses A and D of J-96 cells differ in the characteristics (pI and molecular weight) of the structural polypeptide components.

Cell Line↗
PubMed1995

High-resolution SSCP analysis using polyacrylamide agarose composite gel and a background-free silver staining method.

We describe here an improved methodology for single-strand conformation polymorphism (SSCP) analysis of PCR products. The method utilizes a polyacrylamide agarose composite gel and background-free silver staining. In comparison with conventional pure polyacrylamide gel for PCR-SSCP analysis, the composite gels have a much greater mechanical strength and improved resolution. The background staining commonly seen in many silver staining protocols has been eliminated by incorporation of thiosulfate, which can prevent nonspecific deposits of silver salts. As shown in our titration tests, the composite gel and background-free silver staining together have allowed clear identification of point mutations in samples containing as little as 5% of the target sequences, the same sensitivity achieved by radioactive labeling methods.

DNA↗
PubMed1995

Alternative to polyacrylamide gels improves the electrophoretic mobility shift assay.

In this paper we outline a simplified protocol for the electrophoretic mobility shift assay utilizing TreviGel 500, a nontoxic alternative to polyacrylamide. The TreviGel 500 matrix combines the strength and resolution of polyacrylamide with the simplicity and flexibility of agarose in the casting of gels. Therefore, this method provides a simple, rapid and nontoxic alternative to current protocols for the investigation of protein: DNA interactions.

Base Sequence↗
PubMedDate not supplied

[Conjugates and carbohydrate chains of alpha 1-acid glycoprotein with polyacrylamide maintain the immunomodulating activity of native glycoprotein].

Translocation of carbohydrate glycoprotein N-chains onto soluble polyacrylamide was proposed as a method for studying the biological role of carbohydrate chains. N-linked carbohydrate chains of alpha 1-acid glycoprotein (AGP) were aminated at the reducing GlcNAc moiety and covalently attached to polyacrylamide (PAA). Thus "pseudo-AGP" was obtained where peptide core was replaced with PAA. The synthetic model mimics AGP by M(r) and carbohydrate content as well as the ratio of tetra-, tri- and diantennary and mono-, di-, tri- and tetrasialo chains. It was shown that the conjugate inhibits proliferation of lymphocytes like the parent AGP. Therefore, the property of AGP to inhibit the lymphocyte proliferation is attributed to its carbohydrate chains, whereas peptide core serves as carrier providing polyvalent interaction of multiple carbohydrate chains with cell.

Acrylic Resins↗
PubMed1994

Rapid silver staining and recovery of PCR products separated on polyacrylamide gels.

A rapid silver-staining procedure for DNA fragments in polyacrylamide gels is described. The time required for band detection is 15 min and the limit of sensitivity 3 pg/mm2. PCR products subjected to this rapid staining protocol are readily recovered from the gel by excision and elution by incubation at 95 degrees C for 20 min. Bands of up to 3 kb have been recovered and reamplified from either recently prepared or dried gels. The rapid staining protocol significantly decreases the processing time required for silver-stained polyacrylamide gels, which is of particular importance in diagnostic situations. The recovery protocol allows individual bands from complex mixtures to be easily recovered for sequencing or probe preparation.

Animals↗
PubMed1993

A simple apparatus for fast and inexpensive recovery of DNA from polyacrylamide gels.

A typical elution procedure for DNA should be easy, quick, inexpensive, highly efficient and must provide DNA of good purity and quality to carry out any further enzymatic reactions. Although a number of procedures and apparatus for the specific isolation of DNA from polyacrylamide gels have been described, none possess all these qualities. In this report, we describe the use of a simple apparatus with which DNA molecules can be eluted from polyacrylamide gels with a recovery efficiency of about 90% prior to ethanol precipitation. The quality of these elution products is good enough to allow their use in any further enzymatic reactions. In addition to the fact that more than a dozen individual samples can be eluted and isolated in less than two hours (less than one hour for a single sample), the estimated low cost per electroeluted sample makes it the most affordable and economical elution method available.

Base Sequence↗
PubMed1995

[Proteins from the outer membrane of Pasteurella--1. Polyacrylamide gel electrophoresis of outer membrane proteins--a possibility for differentiation of Pasteurella haemolytica strains].

Using the sodium laurylsarcosinate method, outer membrane proteins (OMPs) of 63 Pasteurella haemolytica strains are isolated and their protein patterns obtained by SDS polyacrylamide gel electrophoresis (SDS-PAGE) are compared. A high degree of similarity became evident both within A and T biotypes in the molecular weight range of 25 to 50 KDa. Strain-to-strain variations are mainly limited to quantitative differences in individual protein bands. It is concluded that within both the A and T biotype groups equal sets of major proteins are present with differing amounts of individual constituents. Biotypes A and T can be clearly distinguished on the basis of marked variations in OMP profiles. OMPs of those AT strains included in comparative studies are exhibiting no general homogeneity, but a relative heterogeneity due to different protein compositions, which are, however, clearly distinct from the patterns of biotypes A and T. It is established unambiguously that isolates belonging to biotypes A or T, as well as AT strains, can be clearly distinguished from each other on the basis of SDS-PAGE analysis of OMP extracts. Thus, polyacrylamide gel electrophoresis of outer membrane proteins can be used for differentiation.

Bacterial Outer Membrane Proteins↗
PubMed1977

[Polyacrylamid-gel-electrophoresis of Haemophilus proteins (author's transl)].

After phenol-acetic-acid extraction the following Haemophilus strains resp. their proteins were subjected the polyacrylamid-gel-electrophoresis in presence of 8 M urea: Strains of the serovar A of H. paragallinarum: 0083, 1516, 1598, 2213, 1645, 1646, Löhren, 2671, 1385, 758, 17756; strains of serovar B of H. paragallinarum: 0222, 2600, 733, 2028, 1596, 2026, 1676, 245, the S and R-form of 2403 as well as the strains 782 and 1655, which were not serotyped; strains of H. paravium sp. nova (HINZ: Inst. J. Syst. Bacteriol. in press): 1762, 62 (Serovar 1), 2654, 2659 (Serovar 2), 780 (Serovar 3), 94 (Serovar 4) and 1254, 0002, 0003, which were not serotyped. H. parainfluenzae (NCTC 4101) and H. parasuis were examined in the same way. The Coomassie Blue-stained protein patterns show that each of the strains tested developed its characteristic protein pattern, with exception of the S- and R-form of the strain 2403, which developed identical pattern. Interrelations between electrophoretic pattern and biological properties such as biochemical activities or pathogenicity could not be proved. However, the procedure described seems to be suitable for strain- or clon-identification on the subspecies level. The electrophoresis apparatus, which was made according to our instructions was less expensive than corresponding available equipments and proved to be usable for the polyacrylamid-gel-electrophoresis.

Bacterial Proteins↗
PubMed1996

[Glycoconjugates on the basis of polyacrylamide--tools for studying lectins, antibodies, and glycosyltransferases in glycobiology, cytochemistry, and histochemistry].

The synthesis, physicochemical characteristics, and application for studying the carbohydrate-binding molecules of some analogues of human cell glycoconjugates, neoglycoconjugates, were reviewed. An approach to the synthesis of the polyacrylamide derivatives of carbohydrates based on the interaction of fully activated polyacrylic acid with omega-aminoalkyl glycosides was described. It provides highly reproducible results, is more simple than the previously known methods of the synthesis of such derivatives, and expands the range of synthetic possibilities because it can provide both the sugar-polymer type molecules and conjugates bearing various labels and effectors, sorbents, glycosurfaces, etc. In the first part of the review, the synthesis of polyacrylamide conjugates and their physicochemical properties were described. In its second part, the synthesis of some complex constructions, such as pseudoglycoproteins, pseudomucins, glycoparticles, and glycosurfaces, was outlined. Some examples of the application of the described conjugates in various fields of glycobiology were simultaneously discussed. Prospects of the further development of the presented approach in glycotechnology and medicine were also described.

Acrylic Resins↗
PubMed1998

Detection of the two short tandem repeat loci (HumTPO and HumLPL) in Japanese populations using discontinuous polyacrylamide gel electrophoresis.

Two short tandem repeat (STR) systems, HumTPO and HumLPL, were investigated in 218 unrelated Japanese living in Gunma and Fukui Prefectures by means of discontinuous, horizontal polyacrylamide gel electrophoresis and silver staining. Five and six alleles were identified at the HumTPO and HumLPL loci, respectively. No deviations from the Hardy-Weinberg equilibrium or differences in genotype frequency were found between the two Japanese populations in the two systems. Our detection methods offered several advantages including time-saving and safe electrophoretic procedures, and fast and easy silver-staining, in comparison with standard and conventional STR analysis using vertical polyacrylamide gel electrophoresis.

Electrophoresis, Polyacrylamide Gel↗
PubMed1999

Separation of proteins from stressed rice (Oryza sativa L.) leaf tissues by two-dimensional polyacrylamide gel electrophoresis: induction of pathogenesis-related and cellular protectant proteins by jasmonic acid, UV irradiation and copper chloride.

We have used three kinds of stresses, including the signaling compound jasmonic acid, an environmental stressor, UV irradiation, and a heavy metal salt copper chloride, to study changes in the protein patterns in rice (Oryza sativa L.) leaf tissues using two-dimensional polyacrylamide gel electrophoresis. However, instead of using lysis buffer containing urea (O'Farrell, J. Biol. Chem. 1975, 250, 4007-4021) for extraction of proteins from rice seedling tissues, we used Tris-HCl buffer (commonly used for extraction of proteins for separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) for extraction of proteins and resolved these extracted proteins by the usual method of O'Farrell. Furthermore, the induction of a large number of proteins was clearly observed over controls. No spots corresponding to these induced proteins were found in the control experiment, indicating qualitative changes in protein patterns after various stress treatments. A total of 12 out of 13 proteins could be N-terminally sequenced from jasmonic acid-treated rice leaf tissues, and one protein was sequenced from UV-irradiated leaf tissues. These proteins showed high homology to pathogenesis-related (thaumatin-like protein, a PR5 class protein; a beta-1,3-glucanase precursor; an intracellular PR protein encoded by PBZ1 gene, and an antifungal protein) and cellular protectant (glutathione transferase, EC 2.5.1.18; and ascorbate peroxidase) proteins, from plants, including rice. Results presented here suggest a role for jasmonic acid in the self-defense mechanisms of rice plants.

Amino Acid Sequence↗
PubMed2000

Deamidation as a widespread phenomenon in two-dimensional polyacrylamide gel electrophoresis of human blood plasma proteins.

The human plasma protein patterns obtained by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) is a good model system for post-translational modifications because of the existence of several "ladders" of protein spots [Anderson, N. L., Anderson, N. G., Electrophoresis 1991, 12, 883-906], so-called "trains" of spots. Our investigation of several proteins, amongst others beta2-microglobulin and the haptoglobin chains, found the differences in isoelectric points (p/) to be due to deamidation of asparagines. After enzymatic cleavage with endopeptidases in the 2-D polyacrylamide gel, the asparagine and deamidated asparagine containing peptides were separated and quantified by reversed-phase HPLC. In order to separate these peptides, a neutral pH system was established and, as a result, the differences in hydrophobicity of asparagine-containing and deamidated asparagine-containing peptides increased. But how do deamidated asparagines contribute to the observed spot pattern? One spot in the 2-D gel consists of a mixture of protein species with the same number of deamidated asparagines but on different sequence position sites. The difference between the spots in the "ladder" is a growing number of negative charges introduced in the protein by an increasing number of deamidated asparagines. As a consequence, the mass difference between two spots is exactly 1 Da, which is shown in this paper for intact protein masses and the corresponding deamidated peptides.

Amides↗
PubMed2002

Quantitative evaluation of proteins in one- and two-dimensional polyacrylamide gels using a fluorescent stain.

The characteristics of protein detection and quantitation with SYPRO Ruby protein gel stain in one- and two-dimensional polyacrylamide gels were evaluated. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses of three different purified recombinant proteins showed that the limits of detection were comparable to the limits of detection with ammoniacal silver staining and were protein-specific, ranging from 0.5 to 5 ng. The linearity of the relationship between protein level and SYPRO Ruby staining intensity also depended on the individual protein, with observed linear dynamic ranges of 200-, 500-, and, 1000-fold for proteins analyzed by SDS-PAGE. SYPRO Ruby protein gel stain was also evaluated in two-dimensional electrophoretic (2-DE) analysis of Escherichia coli proteins. The experiment involved analysis of replicates of the same sample as well as dilution of the sample from 0.5 to 50 nug total protein across gels. In addition to validating the 2-DE system itself, the experiment was used to evaluate three different image analysis programs: Z3 (Compugen), Progenesis (Nonlinear Dynamics), and PDQuest (Bio-Rad). In each program, we analyzed the 2-DE images with respect to sensitivity and reproducibility of overall protein spot detection, as well as linearity of response for 20 representative proteins of different molecular weights and pI. Across all three programs, coefficients of variation (CV) in total number of spots detected among replicate gels ranged from 4 to 11%. For the 20 representative proteins, spot quantitation was also comparable with CVs for gel-to-gel reproducibility ranging from 3 to 33%. Using Progenesis and PDQuest, a 1000-fold linear dynamic range of SYPRO Ruby was demonstrated with a single known protein. These two programs were more suitable than Z3 for examining individual protein spot quantity across a series of gels and gave comparable results.

Bacterial Proteins↗
PubMed2002

Mass spectrometric identification of mitochondrial oxidative phosphorylation subunits separated by two-dimensional blue-native polyacrylamide gel electrophoresis.

Blue-native polyacrylamide gel electrophoresis is a powerful tool for the separation of intact membrane protein complexes mainly applied to the analysis of the enzymes of the mitochondrial oxidative phosphorylation system (OXPHOS). Combined with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), it reveals a two-dimensional pattern showing the individual subunits of the five OXPHOS multi-enzyme complexes. This pattern is useful in the diagnostic analysis of several diseases related to disorders in the oxidative phosphorylation system. However, in order to use this method for systematic diagnostic purposes and to be able to link disease with absence or reduced expression of specific subunits, an unambiguous identification of the individual subunits is necessary. In this study, we completed this task, implementing peptide mass fingerprinting and mass spectrometric sequence analysis. In the course of these analyses, we discovered a novel variant of a cytochrome c oxidase subunit VIc.

Electron Transport Complex IV↗
PubMed1976

Preparation and properties of bacterial chromatophores entrapped in polyacrylamide.

The immobilization of Rhodospirillum rubrum chromatophores was successfully performed by entrapping them in polyacrylamide. Their photophosphorylating activity was about 40% of native chromatophores. The temperature and pH optima for immobilized chromatophores were similar to the native photosynthetic apparatus and kinetic parameters showed that the rate of photophosphorylation in polyacrylamide particles was diffusion controlled. Light penetration of the gel particles was not a limiting parameter. Immobilization considerably increased the stability of the chromatophores towards denaturation.

Acrylamides↗
PubMed1989

Direct N-terminal sequence analysis of rat liver plasma membrane glycoproteins separated by two-dimensional polyacrylamide gel electrophoresis.

Nine previously uncharacterized membrane glycoproteins from normal rat liver have been analyzed by amino acid sequencing from two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) after transblotting to Immobilon-P membranes. Three of these components show altered levels of expression in liver tumors. A single electroblotted polyacrylamide gel yielded sufficient quantities of these glycoproteins for amino acid sequencing and the N-terminal structure could be determined for four of them. The remaining five glycoproteins of interest were not sequenceable in this manner, presumably because they had blocked N-termini. Prior to electrophoresis, two enrichment methods were applied to the crude liver membrane preparations: affinity chromatography with concanavalin A to isolate the plasma membrane glycoproteins and then fast protein liquid chromatography on Superose 12 to obtain components having a specific range of molecular weights. These materials were next subjected to 2-D PAGE using pH 4-6 carrier ampholytes in the first dimension and 7.5% sodium dodecyl sulfate gels in the second. The proteins were then electroblotted to Immobilon-P membranes and located by staining with Coomassie Brilliant Blue R-250. Our results demonstrate that N-terminal sequencing (gas-phase) can be achieved on polypeptides obtained from approximately 250 micrograms of total glycoproteins applied to a single 2-D gel.

Amino Acid Sequence↗