High Acrylamide Concentrations Research Articles

One‐Dimensional SDS Gel Electrophoresis of Proteins

AbstractElectrophoresis is used to separate complex mixtures of proteins, (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates) to investigate subunit compositions, and to verify homogeneity of protein samples. It can also serve to purify proteins for use in further applications. In polyacrylamide gel electrophoresis, proteins migrate in response to an electrical field through pores in the gel matrix; pore size decreases with higher acrylamide concentrations. The combination of gel pore size and protein charge, size, and shape determines the migration rate of the protein. This unit contains protocols for standard Laemmli gels, electrophoresis of peptides and small proteins, continuous SDS‐PAGE ultrathin gels, multiple single‐concentration gels, gradient gels, and multiple gradient gels.

Enzyme hydrolysis of grafted amylopectin

Previous methods of proof of grafting are based on separation of homopolymers from crude reaction products and further characterization of extracted component. This article reports the proof of grafting by a combined use of viscometry and enzyme hydrolysis that, to our knowledge, has not been reported so far. Two series of graft copolymers of amylopectin with polyacrylamide were synthesized using ceric ion-induced redox initiation technique. In the first series, a variation of ceric ion concentration at fixed acrylamide concentration and in the second series, a variation of acrylamide concentration at fixed ceric ion concentration were undertaken to effect a variation in the number and length of polyacrylamide chains. Qualitatively, it has been observed that there may be some homopolymers formed at a very high acrylamide concentration. The products may at best be a mixture of graft copolymer and homopolymer, but it contradicts the view that the products are purely physical mixtures of polysaccharide and polyacrylamide. © 1998 John Wiley & Sons, Inc. J Appl Polym Sci 70: 2627–2633, 1998

Comparison of DNA bending by Fos-Jun and phased A tracts by multifactorial phasing analysis.

Studies of DNA bending by Fos and Jun using different methods have yielded contradictory results. Whereas gel electrophoretic phasing analysis indicates that Fos and Jun bend DNA, results obtained through X-ray crystallography and ligase-catalyzed cyclization suggest that they do not. To test the assumptions underlying phasing analysis and to examine DNA bending by Fos and Jun, a multifactorial phasing analysis approach based on the distinct electrophoretic mobilities of DNA fragments of diverse shapes was developed. In this approach, the spacing between the bends, the length of sequences flanking the bends, and the acrylamide concentration in the gel are varied. Two closely spaced intrinsic bends with long flanking sequences had the same effect on electrophoretic mobility as a single bend corresponding to the sum of the bends when they were arranged in phase, and the difference between the bends when they were arranged out of phase. Based on the phase-dependent electrophoretic mobility variation of fragments containing intrinsic DNA bends of different magnitudes, three criteria for determination whether the phase-dependent mobility variation of protein-DNA complexes is caused by DNA bending were adopted. Complexes formed by the bZIP domains of Fos and Jun fulfilled each of these criteria. First, the electrophoretic mobility variation induced by Fos and Jun was proportional to that caused by an intrinsic bend over a broad range of acrylamide concentrations. Second, the mobility difference between fragments containing in phase and out of phase bends was reduced by an increase in the separation between the bends. The separation between the bends had the same effect on the electrophoretic mobility variation caused by Fos and Jun as well as intrinsic bends on long DNA fragments at low acrylamide concentrations. Third, on short DNA fragments analyzed at high acrylamide concentrations, two intrinsic bends separated by long spacers caused a larger decrease in electrophoretic mobility when they were out of phase than when they were in phase. This reversal of the phase dependence of the electrophoretic mobility variation was also observed for complexes formed by truncated Fos and Jun. Thus, the phase-dependent mobility variation of Fos and Jun complexes is due to DNA bending.

One‐Dimensional SDS Gel Electrophoresis of Proteins

Electrophoresis is used to separate complex mixtures of proteins, to investigate subunit compositions, and to verify homogeneity of protein samples. It can also serve to purify proteins for use in further applications. In polyacrylamide gel electrophoresis, proteins migrate in response to an electrical field through pores in the gel matrix; pore size decreases with higher acrylamide concentrations. The combination of gel pore size and protein charge, size, and shape determines the migration rate of the protein. This unit contains protocols that gives the standard Laemmli method for discontinuous gel electrophoresis under denaturing conditions, and the standard method for full-size gels is adapted for the minigel format. Minigels provide rapid separation but give lower resolution. Several alternate protocols are provided for specific applications, including electrophoresis of peptides and small proteins, continuous SDS-PAGE, ultrathin gels, multiple single-concentration gels, gradient gels, multiple gradient gels, and multiple gradient minigels.

Polymerization of acrylamide in the conditions of fast atom bombardment mass spectrometry

The cause of the absence of acrylamide signals in the fast atom bombardment (FAB) mass spectra of its glycerol solutions has been studied. On the basis of analysis of certain features of FAB mass spectra, the disappearance of acrylamide signals in a matter of the first few seconds after sample exposure to the fast atom beam, degradation of the spectra, enhancement with the increase of acrylamide conentration and bombardment time, the change in the pattern of the glycerol matrix spectrum, the significant growth of viscosity of the irradiated sample (up to a glassy state at high acrylamide concentrations), the conclusion is made that the observed peculiarities arise due to beam-induced polymerization reactions. It is pointed out that the ions and radicals formed under the action of atom bombardment may play the role of initiators of polymerization reactions of acrylamide and other substances with analogous chemical properties, which should be taken into account properly as a possible source of errors in qualitative and quantitative FAB analysis.

Flexibility of the aldolase molecule measured using quenching-induced variations of the Forster distance for fluorescence energy transfer

The range of flexibility of the rabbit muscle aldolase molecule was studied using fluorescent labelled aldolase. The protein molecule was specifically labelled on the opposite sites of the enzyme subunit with the fluorescence energy donor and acceptor residues. Labelled aldolase with full enzymatic activity was used as a tool in the FRET studies between IAEDANS — donor or Cys-289 and IAF — acceptor on Cys-239. A range of Forster distance (R) was obtained by collisional quenching of the donor emission. The experiments of donor fluorescence quenching with wide range of acrylamide concentrations have shown the changes of donor-acceptor distances. In the absence of quencher the D-A distance distribution in characterized by an average value of 40.4 A, and a half-width of 0.13 A. A dramatic increase in half-width to 17.7A is observed after expositions of the enzyme to high acrylamide concentrations (0.13 M-0.68 M).

Microbial degradation of acrylamide monomer

Acrylamide, a neurotoxic monomer with extensive industrial applications was found to be degraded by the microorganisms present in a tropical garden soil. A bacterium capable of degrading acrylamide was isolated from this soil by enrichment. It was found to be aerobic, gram-negative, motile, short rod and identified as Pseudomonas sp. The bacterium degraded high concentrations of acrylamide (4 g/l) to acrylic acid and ammonia which were utilized as sole carbon and nitrogen source for growth. An amidase was involved in the hydrolysis of acrylamide, which could act on other short chain amides like formamide and acetamide but not on acrylamide analogues: methacrylamide and N,N-methylene bisacrylamide. The enzyme was sensitive to catabolite repression by succinate both in presence as well as absence of nitrogen source.

Inhibition of activity and quenching of intrinsic fluorescence of transglutaminase by acrylamide are independent events.

Addition of low concentrations of acrylamide to the assay mixture of erythrocyte transglutaminase leads to a strong inhibition of the enzyme with a mechanism consistent with a non-competitive inhibition against both substrates. For the quenching of the intrinsic fluorescence of the protein, much higher concentrations of acrylamide are required so that both phenomena appear independent of each other. In this particular case, therefore, acrylamide can be employed to obtain information on ligand-triggered conformational changes.

Study of chemical gelation dynamics of acrylamide in water by real-time pulsed nuclear magnetic resonance measurement

Chemical gelation process of acrylamide monomers in water has been studied by a real-time pulsed nuclear magnetic resonance (NMR) measurement. The temporal change of molecular mobility has been detected by a real-time observation of the spin–spin relaxation time T2. The influence of the concentration of crosslinker and acrylamide on gelation dynamics has been studied, and it becomes clear that these factors have strong effects on the gelation dynamics. The effects of linear polymerization and network formation on the motional state of water have been studied. The formation of three-dimensional cages drastically reduces the mobility of water. In low acrylamide concentration, the results of NMR measurement mainly reflect the state of water. In high acrylamide concentration, on the other hand, they reflect both polymer network and water, and furthermore, in high crosslinker concentration the spatial heterogeneity of gels is observed. In this paper, we demonstrate that a real-time NMR measurement is a useful tool for a direct observation of the temporal change of molecular mobility during polymerization and gelation process.

Human parainfluenza virus 3: Purification and characterization of subviral components, viral proteins and viral RNA

A simple method was established that allowed large quantities of human parainfluenza 3 (PF3) virions to be isolated from tissue culture cells. The purity of the virus was sufficient for biochemical analysis of virion proteins. The density of PF3 virions was 1.18–1.20. Purified virions contained seven viral proteins with estimated molecular weights of: L, 180000; P, 83000; HN, 69000; NP, 66000; f 0, 60000; F 1, 51000; and M, 38000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. There were three phosphoproteins, P, NP and M, and two glycoproteins, HN and F (includes f 0 and f 1). F 1,2, the activated, cleaved, fusion glycoprotein (60000 Da), consisting of two disulfide-linked subunits, f 1 and F 2, was seen only under nonreducing conditions. Because of its small size (approximately 9000 Da) F 2 could be seen only on gels with high acrylamide concentrations. As in other enveloped viruses, cellular actin (43 000 Da) was present in purified virions. Several minor bands migrating between NP and M represented breakdown products of NP. Solubilization of the virion membrane in low salt buffer with non-ionic detergent resulted in the loss of HN and F. In high salt buffer, the M protein was also removed. Nucleocapsids isolated by CsCl centrifugation contained L, P, NP and small amounts of M. Nucleocapsids isolated in the presence of the ionic detergent, sarcosyl, contained only the NP protein. The density of nucleocapsids was 1.29–1.30. Genomic 50S RNA isolated from nucleocapsids had an estimated molecular weight of 5 × 10 6.

Protein structural changes accompanying formation of enzymatic transition states: tryptophan environment in ground-state and transition-state analogue complexes of adenosine deaminase.

The accessibility of protein tryptophan fluorescence to the quenching agent acrylamide has been studied in adenosine deaminase and in binary complexes of the enzyme with ground-state or transition-state analogues. Although the enzyme contains three tryptophan residues, Stern-Volmer plots are linear with all the fluorescence quenchable at high acrylamide concentrations. Tryptophan fluorescence is less easily quenched in the binary complexes than in the free enzyme, indicating a decrease in the accessibility of these residues. The greatest decrease in accessibility is found for the transition-state analogue complexes. Although the affinities of the transition-state analogues studied span a range of 10(6), the Stern-Volmer constants of the complexes are the same within experimental error. Thus, as measured by this technique, changes in enzyme conformation accompanying formation of these complexes are similar for all transition-state analogues. Resonance energy transfer from tryptophan as donor to ligand as acceptor successfully explains the differing abilities of ligands to quench the enzyme's intrinsic fluorescence upon formation of complexes in the absence of acrylamide. On the basis of Forster distance calculations, it is likely that the residues partially quenched upon formation of transition-state analogue complexes are distant from the active site.

Functional and structural organization of chlorophyll in the developing photosynthetic membranes of Euglena gracilis Z. V-separation and characterization of pigment-protein complexes of the differentiated thylakoids

Low temperature sodium dodecyl sulfate polyacrylamide gel electrophoresis following mild solubilization of Euglena thylakoid components allowed to resolve, in addition to the main CP1, CPa and LHCP chlorophyll-protein complexes, the additional CP1a and LHCP' green bands. A carotenoid enriched band CPc can be separated from CPa using high acrylamide concentration. Pigment and polypeptide composition of these complexes were analyzed by absorption and fluorescence measurements and two dimensional gel electrophoresis. Spectral properties of CP1 and CP1a indicate an heterogenous organization of chlorophyll and the presence of significant amount of chlorophyll b in these complexes. They both contain a major 68 kilodalton polypeptide associated with three minor low molecular weight polypeptides in CP1a. CPa and CPc exhibit a characteristic fluorescence emission at 687 nm and they each contain one polypeptide of 54 and 41 Kda respectively. LHCP and LHCP' are less abundant than in higher plant thylakoids and they contain a lower proportion of chl b (chl a: chl b=3). They include two polypeptides of 26 and 29 Kda.

Purification and properties of the activating enzyme for iron protein of nitrogenase from the photosynthetic bacterium Rhodospirillum rubrum.

The oxygen-labile, activating enzyme for iron protein from the photosynthetic bacterium, Rhodospirillum rubrum, was purified 11,800-fold using a combination of chromatophore washing, DE52-cellulose chromatography, hydroxylapatite chromatography, reactive red-120 cross-linked agarose chromatography, reactive red-120 cross-linked agarose chromatography, and Sephadex G-75 gel filtration. Activating enzyme appeared homogeneous on silver-stained sodium dodecyl sulfate-polyacrylamide gels, and the staining intensity of the activating-enzyme band was correlated with the activating-enzyme activity observed in in vitro assays. Either formaldehyde fixation or higher acrylamide concentration was required to accurately assess the purity of activating enzyme on silver-stained gels. Activating enzyme was stable for 30 days at 4 degrees C. Dithiothreitol was a necessary component for the stability of partially purified activating enzyme. NaCl inhibited the coupled assay for activating enzyme. The pI of activating enzyme was determined to be 6.5. Activating enzyme is composed of a minimum of 336 amino acids and a minimum calculated Mr is 32,032. The Mr of activating enzyme was estimated to be 21,700 by analytical gel filtration and 32,800 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An absorption maximum at 280 nm was observed for the activating enzyme.

Cytotoxicity of cyclophosphamide and acrylamide in glioma and neuroblastoma cell lines cocultured with liver cells

A method for the cocultivation of nervous system cells with liver cells has been developed. When acrylamide was tested using the cocultivation procedure no significant liver metabolism-mediated changes in the cytotoxicity in C6 culture were detected. In neuroblastoma NIE115 cultures the toxicity was increased by induced chick hepatocytes at high acrylamide concentrations.

Carcinogen-mediated induction of SV40 DNA amplification is enhanced by acrylamide in Chinese hamster CO60 cells.

The exposure of SV40-transformed Chinese hamster cells (line CO60) to 50-150 micrograms/ml of monomeric acrylamide for 24 h resulted in a very weak induction of the amplification of the SV40 DNA inserts as measured by in situ hybridization with radioactive SV40 DNA as probe. The weak SV40 DNA amplification observed might result from a weak DNA-damaging activity as biologically illustrated by the capacity of high concentrations of acrylamide to irreversibly inhibit the DNA synthesis rate of CO60 cells. Moreover, acrylamide synergistically enhanced both the cytotoxicity and the induction of SV40 DNA synthesis by the carcinogens ethylmethane sulfonate, benzo[a]pyrene, mitomycin C, 4-nitroquinoline-1-oxide, 8-azaguanine and 5-fluorodeoxyuridine. Although a weak inducer of SV40 DNA amplification by itself, acrylamide potentiated the genotoxicity of a series of chemical carcinogens. This finding should be taken into consideration when assessing the risk of this widely used chemical.

Preparative electrophoresis of histones in solubilizable polyacrylamide gels

Histones from the mouse testis have been fractionated on 17% acrylamide gels containing 0.19% Bis-acrylylcystamine (BAC), 2.5 M urea and 0.4% Lubrol-WX at pH 2.7. Polyacrylamide gels which contain BAC, a cross-linking agent with disulfide bonds, can be solubilized in the presence of 2-mercaptoethanol, at pH 8.3. Polymerization is carried out at 40°C and in the presence of 6.5–7.5% tetramethylethylenediamine (TEMED) at pH 8.3. Gels containing high acrylamide concentration ( > 15%) are not soluble if polymerized at lower pH or TEMED concentrations. Following electrophoresis, the gels are stained with Coomassie brilliant blue R for the visualization of protein bands. The stained protein bands are excised and adjusted to pH 8.3 prior to their solubilization. The histones are recovered by ion-exchange chromatography and are free of soluble gel components and dye. Two variants of histone H2B (H2B·1 and H2B·2) have been isolated at approx. 95% purity using this single purification step.

Analysis of skin fibroblast proteins in Duchenne muscular dystrophy: 1. Sodium dodecyl sulphate polyacrylamide gel electrophoresis

AbstractProtein profiles of skin fibroblasts from patients with Duchenne muscular dystrophy (DMD) and normal individuals have been compared using polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (SDS) combined with various detection systems. Protein patterns of normal and DMD fibroblasts obtained usin 10 % polyacrylamide gels showed no qualitative or quantitative differences. The use of acrylamide gels results in increased resolution. No qualitative or quantitative differences were observed using Coomassie Brilliant Blue R‐250 staining. However, Coomassie Brilliant Blue lacks the sensitivity to detect many minor components. Therefore a silver staining procedure was used, which resulted in greatly increased detection sensitivity. Background staining did not increase with gel concentration, but stain development proceeded more slowly at higher acrylamide concentrations. No qualitative differences were observed between normal and DMD profiles. Fibroblasts radiolabelled with [35S]‐methionine were analysed using gradient gels. No qualitative differences between DMD and normal patterns were observed by autoradiography of dried gels. However, quantitative analysis of slicing of gel rods revealed differences between normal and DMD fibroblasts. Two methods of statistics were used to analyse this data, firstly a method that accounts for inter‐experimental variation (termed “batch statistics”) and secondly the standard t‐test. Regions of 73 000 to 68 000 and 48 000 molecular weight were decreased in DMD samples and this finding was significant by both methods of statistical analysis. Regions of 175 000 and 53 000 molecular weight were significantly elevated in DMD preparations only by t‐test, whereas a regions of 32 × 103 molecular weight was only significantly elevated when tested by batch statistics.

Fluorographic detection of nucleic acids labelled with weak β-emitters in gels containing high acrylamide concentrations

Fluorographic detection of nucleic acids labelled with weak β-emitters in gels containing high acrylamide concentrations

Polyacrylamide gels with modified cross-linkages

The retention of low molecular weight proteins during electrophoresis through gradient polyacrylamide gels was improved when a gradient of N,N′,N″-triallyl citric triamide (TACT) was superimposed on the gradient of acrylamide and N,N′-methylenebisacrylamide (MBA). Gels cross-linked only with N,N′-(1,2-dihydroxyethylene)bisacrylamide (DHEBA) are soluble in dilute periodic acid or dilute aqueous solutions of bases. DHEBA cross-linked gradient gels have a smaller pore structure at high acrylamide concentrations and a more open structure at low acrylamide concentrations than gels cross-linked with MBA. Proteins labeled with tritium and carbon-14 were fractionated through DHEBA cross-linked gradient gels and the isotopes measured after solution of the gel with periodic acid. The mild solubilizing conditions enhanced isotope resolution. The characteristics of several cross-linking molecules are discussed and reasons advanced for the superiority of those with acrylamido end groups.

Improvement of pore gradient electrophoresis by increasing the degree of cross-linking at high acrylamide concentrations

Improvement of pore gradient electrophoresis by increasing the degree of cross-linking at high acrylamide concentrations