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At least 667 records · Page 37Linked to original sources
PubMed1999

Nonspecific interactions alter lipopolysaccharide patterns and protein mobility on sodium dodecyl sulfate polyacrylamide gels.

In testing whether bacterial lipopolysaccharide (LPS) was a natural substrate for an esterase from the soil amebae Dictyostelium discoideum, we observed altered banding patterns of the LPS and changed protein mobility on sodium dodecyl sulfate (SDS) polyacrylamide gels after incubation of LPS with the enzyme. The initial interpretation of these results was that the enzyme had removed ester-linked acyl chains from the LPS, leading to a change in its migration on gels. However, esterase inactivated by treatment with either dithiothreitol (DTT), heat, or SDS generated the same mobility shifts. Bovine serum albumin (BSA) also induced the same change in the electrophoretic pattern. We conclude that the altered LPS patterns and protein mobility on SDS gels were caused by nonspecific interactions between LPS and protein.

Animals↗
PubMed1999

On the mechanism of background silver staining during sodium dodecyl sulphate-polyacrylamide gel electrophoresis.

A novel experimental procedure to study the factors that contribute to background silver staining in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) is described. A mechanism is proposed for background silver staining, which was found to result primarily from the redox initiator system using ammonium persulphate (APS) and an amine such as N,N,N',N'-tetramethylethylenediamine (TEMED).

Ammonium Sulfate↗
PubMed1999

Micellar electrokinetic chromatography as a complementary method to sodium dodecyl sulfate-polyacrylamide gel electrophoresis for studying limited proteolysis of proteins.

Micellar electrokinetic chromatography (MEKC) has been utilized as an analytical method to perform investigations on limited proteolysis of proteins. To this purpose partial proteolysis experiments with a series of proteinases were performed, utilizing as model protein pyruvate kinase (PK) from Escherichia coli, an enzyme that is regulated allosterically by fructose 1,6-bisphosphate (FBP). Data obtained with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and MEKC were compared; the profiles generated by submitting digests of PK treated with different proteinases in the presence and absence of FBP to electrophoretic analysis provided a useful adjunct for a better understanding of the effects of the allosteric activator on the conformation of the model enzyme. MEKC was also found to be a convenient technique for determining the kinetics of substrate proteolysis.

Animals↗
PubMed1999

Comparison between capillary and polyacrylamide gel electrophoresis for identification of Lolium species and cultivars.

In recent years variety discrimination has been achieved in a range of agricultural crops by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Previous works on genus Lolium have shown the effectiveness of this technique for the topic of cultivar identification. In the present research the potential of capillary gel electrophoresis (CGE) for identification of Italian (Lolium multiflorum Lam.), annual (Lolium rigidum Gaud.) and perennial (Lolium perenne L.) ryegrass cultivars in comparison with SDS-PAGE was investigated. Separation conditions of SDS-CGE were chosen in order to obtain electrophoretic data comparable with those of SDS-PAGE, at the expense of analysis speed. Both peak area and migration time of SDS-CGE electropherograms were reproducible. In the examined cultivars, a total of 27 (16.8-96.8 kDa) and 28 (13.2-111.5 kDa) protein subunits were detected by SDS-PAGE and SDS-CGE, respectively. The variability in seed storage protein composition was processed by numerical taxonomy. All cultivars were clearly identified by both electrophoresis systems. The orthogonality between SDS-PAGE and SDS-CGE was suggested by the lack of correlation among protein profiles obtained with the two separation systems. The nonredundant information from these analytical systems should provide a relevant benefit for identification of Lolium cultivars and wild biotypes that are extremely uniform phenotypically.

Electrophoresis, Capillary↗
PubMed2000

Two novel myogenic factors identified and isolated by sequential isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

A number of environmental factors were used experimentally to enhance myogenesis during muscle regeneration; however, many hormones and growth factors have been shown to have the ability to increase the rate of satellite cell division, but they only work on satellite cells that are already active in many animal experiments. Recently, the crushed muscle extract (CME) of rats was found to be able to trigger dormant adult rat satellite cells to re-enter the cell mytogenic cycle; however, the identity of the active factors present in rat CME remains unknown. In the present study, the CME was fractionated by the strategy of sequential isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) coupled with functional analysis by myoblast culture. Two satellite cell-specific myogenic factors were identified and purified from CME by this strategy. One of the factors has a molecular mass of around 7 kDa and another about 39 kDa. The factor of 39 kDa could be retained in heparin-Sepharose column and eluted with phosphate-buffered saline (PBS) containing 1 M NaCl, but the 7 kDa factor did not bind to the heparin column. These two purified myogenic factors could synergistically trigger the proliferation and differentiation of dormant satellite cells, whose progenies subsequently fuse in vitro, or fuse to pre-existing partially damaged muscle fibers to form full repair of the damaged muscle fibers or to form new myotubes to replace the completely damaged muscle fibers during the cascade of muscle healing and regeneration in vivo. The identities of these two myogenic factors are under study.

Animals↗
PubMed2000

Capillary zone electrophoresis of albumin-depleted human serum using a linear polyacrylamide-coated capillary: separation of serum alpha-and beta-globulins into individual components.

The separation of human serum globulins into individual components was investigated by capillary zone electrophoresis (CZE) using a linear polyacrylamide-coated capillary at pH 7.4. Prior to CZE analysis of globulin components present in serum, it was found that it was necessary to remove albumin. Preparation of albumin-depleted human serum with a HiTrap Blue column allowed the detection of alpha- and beta-globulin components as a series of peaks. Almost all the peaks, both narrow and broad, observed in CZE analysis could be assigned to six globulin components (alpha1-acid-glycoprotein, alpha1 -antitrypsin, haptoglobin, alpha2-macroglobulin, Gc-globulin, and transferrin) by using the technique of antibody-based indirect detection. The CZE results, obtained from serum preparations from three healthy adults and six patients, showed that the CZE system might be capable of detecting qualitative differences among individuals with regard to individual globulin components.

Acrylic Resins↗
PubMed2000

High-efficiency passive elution of bacterial lipopolysaccharides from polyacrylamide gels.

We recently described a method for recovering polyacrylamide-gel-separated bacterial lipopolysaccharides (LPS) based on the sensitive on-gel LPS detection (1-10 ng/band) with zinc-imidazole followed by passive elution from 32 microm average size gel microparticles into water. With this procedure, the recovery of rough- or semismooth-type LPS after 3 h elution is about 70-80%, while that of smooth LPS is only about 10%. Here we evaluated whether a simple replacement of water with other eluents would enhance the passive diffusion of LPS. We found that solutions of the detergents sodium dodecyl sulfate (SDS), sodium deoxycholate (DOC) and Triton X-100, or mixtures of the organic solvents acetonitrile and triethylamine and water, increased the recovery of a smooth LPS band from Vibrio cholerae O1 in a concentration-dependent manner. Furthermore, a quantitative recovery of rough or smooth LPS from V. cholerae O1, Escherichia coli O111:B4, E. coli K-235, or Serratia marcescens was feasible in 1% SDS or DOC after 3 h or in 5% triethylamine after only 2 min. A simple dilution of SDS or DOC or evaporation of triethylamine rendered the eluted LPS preparations compatible with biochemical activity determination, as tested by Limulus amebocyte lysate assay. Thus, this improved micropurification method may be a suitable interface between analytical gel electrophoresis and further characterization or use of LPS.

Detergents↗
PubMed1978

Two-dimensional polyacrylamide gel analysis on nuclear proteins from human tumor cell lines.

Two-dimensional polyacrylamide gel electrophoretic analysis was carried out on HeLa, KB, ALL and GW-39 cell "Chromatin Fraction II" proteins. Of the many proteins found, most were visualized in earlier studies on rodent tumors and normal tissues. Of these the greater density of protein CP and the presence of protein CG' differentiates tumors from the nontumor tissues. In samples of normal and mitogen stimulated human lymphocytes, protein CG' was absent and protein CP was present in small amounts. Two-dimensional patterns of 0.4N H2SO4 soluble nuclear proteins showed elevated amounts of proteins C16-18 in the GS-39 cell patterns. Proteins Hu1, G1, G2, G3 and G5 were detected only in human cell nuclei. The increased density of staining for protein CP and the presence of CG' are potential biological markers for neoplastic cells.

Animals↗
PubMed2000

Identification of proteins from one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis using electrospray quadrupole-time-of-flight tandem mass spectrometry.

The potential of electrospray tandem mass spectrometry using a quadrupole-time-of-flight tandem mass spectrometer was evaluated for the identification of two unknown proteins from one-dimensional polyacrylamide gel electrophoresis (1-D-PAGE). Two proteins from cellular cultures of mammary epithelia were purified by 1D-PAGE. Their identification was achieved using peptide sequence tags generated by LC/Q-TOF-MS/MS, whereas MALDI-TOF mass mapping failed for these proteins obtained from simple 1D-PAGE separation.

Amino Acid Sequence↗
PubMed2001

Fluorescent labeling of proteins with nile red and 2-methoxy-2,4-diphenyl-3(2H)-furanone: physicochemical basis and application to the rapid staining of sodium dodecyl sulfate polyacrylamide gels and Western blots.

The fluorescent hydrophobic dye Nile red allows the rapid, sensitive, and general staining of proteins in sodium dodecyl sulfate (SDS)-polyacrylamide gels. Nile red staining does not preclude further electroblotting of protein bands onto polyvinylidene difluoride (PVDF) membranes. The resulting Western blot can be stained with the covalent fluorescent dye 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF) using a simple procedure. MDPF staining allows further N-terminal microsequencing and immunodetection of specific bands. This review considers the physicochemical, structural, and analytical studies that have led to the development of Nile red and MDPF staining methods. The usefulness of these procedures is discussed in comparison to other currently available fluorescent and nonfluorescent protein detection methods.

Blotting, Western↗
PubMed2001

Simultaneous, two-color fluorescence detection of total protein profiles and beta-glucuronidase activity in polyacrylamide gel.

A dichromatic method for measuring the specific activity of beta-glucuronidase from complex cell homogenates or partially purified protein fractions is presented. Dual fluorescence is achieved by using the green emitting fluorogenic substrate ELF 97 beta-D-glucuronide to detect beta-glucuronidase activity, followed by the red emitting SYPRO Ruby protein gel stain or SYPRO Ruby IEF gel stain to detect the remaining proteins in the electrophoretic profile. Both ELF 97 alcohol, the highly fluorescent hydrolytic product generated from the enzyme substrate, and the SYPRO Ruby total protein stains are maximally excited by ultraviolet illumination. ELF 97 alcohol emits maximally at 525 nm while the SYPRO Ruby dyes emit maximally at 610 nm. Since ELF 97 beta-glucuronide is a precipitating substrate, it allows precise localization of beta-glucuronidase activity with minimal band diffusion. The staining method is simple and direct, without the requirement for ancillary coupling reactions. Dichromatic protein detection is demonstrated after sodium dodecyl sulfate(SDS)-polyacrylamide gel electrophoresis, carrier ampholyte-mediated isoelectric focusing or two-dimensional gel electrophoresis.

Electrophoresis↗
PubMed2001

An improved sodium dodecyl sulfate-polyacrylamide gel electrophoresis system for the analysis of membrane protein complexes.

Membrane protein complexes such as the reaction center complexes of oxygenic photosynthesis or the complex I of mitochondira are composed of many subunit polypeptides. To analyze their polypeptide compositions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), a wide range of molecular sizes has to be resolved, especially in the low molecular mass range. We have improved the traditional Tris/HCI buffer systems adopting a Tris/2-(N-morpholino)ethanesulfonic acid (MES) buffer system containing 6 M urea. This gel system was used with an 18-24% acrylamide gradient for the separation of polypeptides with molecular masses from below 5 kDa to over 100 kDa. This buffer system can also be applied to the usual uniform concentration of acrylamide gel and also to minislab gels.

Acrylic Resins↗
PubMed2001

Glycerol-enhanced mini-polyacrylamide gel electrophoresis for the separation of differentially expressed DNA fragments in cDNA representational difference analysis.

Representational difference analysis (RDA) is a widely used technique in molecular biology. However, in practice, its efficiency depends on a rapid and reliable separation of the RDA fragments prior to cloning. To achieve this, we have compared and combined the separation efficiencies of conventional and MetaPhor agarose gel electrophoresis (MAGE) with a glycerol-enhanced mini-polyacrylamide gel electrophoresis (PAGE) system (Gem-PAGE). As anticipated, MetaPhor agarose provided significantly improved resolution over conventional agarose electrophoresis, but the latter remains useful to rapidly confirm the presence of RDA-enriched difference products and direct the concentration of MetaPhor agarose subsequently used for further fragment separation. Additional improvements in resolution were possible by using the Gem-PAGE system. The effect of glycerol on band definition of PAGE was most noticeable as the acrylamide to glycerol ratio (A:G) approached 1:2. Gels in which the A:G ratio was significantly above or below this resulted in both poor morphology and impaired resolution of the bands. By exploiting sequentially agarose-based and Gem-PAGE electrophoresis, the goal in RDA of "one band one product" is now realizable.

Acrylic Resins↗
PubMed2000

Improved detection of amylase activity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with copolymerized starch.

An improved method, based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for detection of amylase activity is described. This method will allow better characterization of certain amylases than that obtained by the Davis technique. The main features of the technique are: (i) identification of amylase bands and molecular mass determination are possible in the same gel; (ii) the hydrolysis of copolymerized substrate during electrophoretic separation is prevented using very low temperatures instead of inactivating agents such as chelating agents; and (iii) the technique is applicable to reveal amylase activity in a wide range of biological samples. The method is not useful for enzymes sensitive to SDS and for high molecular mass amylases.

Amylases↗
PubMed2001

Electric light scattering from single-stranded DNA in linear polyacrylamide solutions.

The electric light scattering (ELS) of ssDNA (calf thymus, 10 kbp, 55 micrograms/mL) in denaturing polyacrylamide (PAA) solutions was studied as a function of applied sinusoidal electric field and polymer concentration. Electric fields of strengths up to 300 V/cm and of frequencies between 100 and 5000 Hz were applied. It was found that the ELS effect increases with the field strength and decreases at high frequencies. The dependence of the ELS effect of ssDNA on polymer concentration passes through a maximum at 1% PAA. The relaxation times of decay of the ELS effect increase with increasing polymer concentrations. It was demonstrated that ELS is a useful method for investigation of ssDNA behavior in the course of pulse-field electrophoresis in polymer solutions.

Acrylic Resins↗
PubMed2001

Heparan sulfate, heparin, and heparinase activity detection on polyacrylamide gel electrophoresis using the fluorochrome tris(2,2'-bipyridine) ruthenium (II).

The paper shows the ability of the fluorochrome tris(2,2'-bipyridine) ruthenium (II) (Rubipy) to detect heparan sulfate, heparin, and heparinase activity of M3 murine mammary adenocarcinoma cells as well as bacterial heparinases I, II, and III in native polyacrylamide gel electrophoresis (PAGE). The technique is based on the electrophoretic mobility of high molecular weight heparins and subsequent staining with Rubipy (50 micrograms/mL). The minimum content of heparin detected by fluorescence in a UV transilluminator was 25-50 ng. The number of Rubipy molecules bound to heparin, determined in relationship to the number of disaccharide units (DU), showed that two to six heparin disaccharide units are bound by each fluorochrome molecule. Scatchard plot analysis showed one Rubipy-binding site (Kd = (8.56 +/- 2.97) x 10(-5) M). Heparinase activity was determined by densitometric analysis of the fluorescence intensity of the heparin-containing band of the gel. While heparinase I (EC 4.2.2.7.) degraded heparin and, to a lower degree, partially N-desulfated N-acetylated heparin (N-des N-Ac), heparinase II (no EC number) could efficiently degrade heparan sulfate (HS) and partially N-des N-Ac heparin. Finally, heparinase III (EC 4.2.2.8.) degraded HS almost exclusively. Only heparin and N-des N-Ac heparin were substrates for M3 tumor cell heparinases. We describe a qualitative, sensitive and simple method to detect heparinase activity and determine its substrate specificity using Rubipy fluorescence with heparin and heparan sulfate in multiple biological samples tested in parallel.

2,2'-Dipyridyl↗
PubMed2001

The use of polyacrylamide gel electrophoresis in animal systematics, phylogeny, and ecophysiology.

In the 1980s, the use of polyacrylamide gel electrophoresis (PAGE) was popular. But more recent developments in other electrophoretic techniques have resulted in this method being less widely used. However, it is adequate for the needs of naturalists for issues which do not require high-performance methods, such as in systematics, phylogeny, studies of intraspecific or clinal variability, ecology and ecophysiology. We illustrate the application of PAGE by the results of a research program on marine and terrestrial invertebrates which was conducted from 1979 to 1992, but which is still used to initiate graduate students to the research. Thus, issues faced by the zoologist can be clarified by this method, not yet obsolete and still useful in natural history despite considerable advances in other electrophoretic methods.

Animals↗