Disc Electrophoresis Gel Research Articles

Electrophoretic artifacts arising from the use of thiol-containing reagents.

Thiol reagents migrate as a curtain behind the salt front when loaded with the sample solution onto disc-electrophoresis gels. In immobilized pH gradients (IPG) the same compounds are driven by electrophoresis and electroosmosis from the alkaline to the neutral and acidic regions of the gradients. In either case, a dose-dependent sideways spreading results in spurious reduction between adjacent lanes if samples with and without reducing agent are loaded side by side.

Isolation, characterization and function of laccase from Trichoderma

Of fourteen natural isolates of Trichoderma, no correlation was found between substrate weight loss and phenol oxidase (PO) activity in rice straw cultures. The highest PO producer from these laccase-positive strains was subjected to UV mutagenesis in order to select high and low PO activity mutants. There was no significant difference in substrate weight loss for mutant strains with six times higher and six times lower PO activity than the parent strain. Nor did the enzyme activity result in decreased growth inhibition by inhibitory phenolic compounds. PO enzyme from the parent Trichoderma and one of its high-PO-activity mutants was subsequently purified by ethanol precipitation from liquid cultures optimized by supplementation with copper sulphate and cycloheximide. Protein staining and activity staining of disc electrophoresis gels showed that only one PO enzyme of approximately 71 000 Da was produced. The enzyme could be defined as a laccase (benzenediol: oxygen oxidoreductase E.C. 1.10.3.2) because it catalysed the oxidation of syringaldazine and p-phenylenediamine in the absence of hydrogen peroxide, and because it was inhibited by cetyltrimethylammonium because but not by cinnamic acid. No specific in-vivo function could be assigned to this enzyme.

Characterization of Lipase of Pseudomonas fluorescens 27 Based on Fatty Acid Profiles

The objectives of this research were to isolate lipase from Pseudomonas fluorescens 27, to compare the purity of the partially purified lipase preparation with crude extract, and to determine if bands of lipase activity revealed by disc gel electrophoresis liberated different free fatty acids from milk fat. Lipases were isolated from a shaken skim milk culture of P. fluorescens 27 by using ion-exchange chromatography on DEAE cellulose (Whatman DE 32) and gel filtration on Sephadex G-150. The principal lipase-rich fractions from gel filtration represented 6.2% of total lipolytic activity. Disc gel electrophoresis of partially purified enzyme revealed two protein bands. These protein bands were cut from disc electrophoresis gels and used as an enzyme source for reaction with butter oil. Free fatty acids were isolated from the assay mixture, separated, and quantified by gas chromatography. Data from gas chromatographic analysis indicated that P. fluorescens 27 produces at least two different lipases.

On the heterogeneity of catalase from goat liver. Purification and characterization.

Catalase [EC 1.11.1.6] from goat liver was purified by acetone and ammonium sulfate fractionations followed by DEAE-cellulose and Bio-Gel A-1.5 m column chromatography. The purified enzyme was found to be homogeneous ultracentrifugally and the molecular weight of the native goat liver catalase (GLC) was estimated by gel filtration to be approximately 220,000. SDS-gel electrophoresis results indicated that catalase from goat liver consists of four apparently identical subunits, with a molecular weight of about 60,000; this is similar to the subunit structure of bovine liver catalase (BLC). The enzyme activity of GLC was slightly less than that of BLC, which apparently correlated with the lower R.Z. value (A405nm/A280nm) of GLC than of BLC. The optimal pH of the enzyme activity of GLC was found to be around 7, the same as that of BLC. The CD spectrum of native GLC generally resembled that of BLC, although the band intensities and band positions differed slightly. However, the α-helix content of GLC was found to be about 30% by CD measurement, slightly less than that of BLC. The effect of pH on the stability of GLC was studied in terms of α-helical conformation and the fluorescence of tryptophanyl residues and 8-anilinonaphthalene-1-sulfonate (ANS) bound to the enzyme. These results indicated that GLC may dissociate into subunits at extreme pH's. On the other hand, precise densito-metric tracing of disc electrophoresis gels indicated heterogeneity of the purified GLC; there were four components, with isoelectric points of 5.80, 5.97, 6.01, and 6.10. It was found that three out of the four components varied electrophoretically on treatment with thiol reagents such as 2-mercaptoethanol (ME) and dithiothreitol (DTT).

Purification and some properties of violet-colored acid phosphatase from spinach leaves.

The violet-colored acid phosphatase was purified from leaves of spinach (Spinacia oleracea). The purified preparation was found to be homogeneous by electrophoresis and ultracentrifugation. Concentrated solution of the enzyme had an intense violet color with a broad absorption band between 410 nm and 700 nm. The peak was at around 530 nm. The molecular weight of the enzyme was approximately 92000. The enzyme was composed of two subunits of equal size and contained manganese. The result of staining of the enzyme in disc electrophoresis gel by periodic acid-Schiff reagent indicated that the enzyme was glycoprotein.

Isolation, chemical and electron microscopical characterization of neutral-salt-soluble type III collagen and procollagen from fetal bovine skin.

Extraction of fetal bovine skin at neutral pH and in the presence of protease inhibitors solubilized substantial amounts of type I and type type III collagen and Type III procollagen. Type I and Type III collagen were separated from each other by salt precipitation, and DEAE-cellulose chromatography was used to separate collagen from procollagen. The main chain constituents in type III collagen and procollagen were disulfide-bonded gamma and pgamma components, respectively. Amino acid composition, cross striation banding as observed using electron microscopy, cyanogen bromide peptide patterns in disc electrophoresis gels and resistance of the disulfide regions to pepsin digestion indicated a close similarity to previously described insoluble type III collagen, which was solubilized by limited pepsin digestion. Electron microscopy of long-spacing-segment crystallites and evidence for an extended form of the disulfide-bonded cyanogen bromide peptide suggested that neutral-salt-soluble type III collagon is longer at its C-terminal end by about 10 to 20 amino acid residues than pepsin-treated material. A small elongation was also indicated in the N-terminal portion of the molecule. Procollagen has an additional N-terminal extension with a length of about 160 A, but no difference was observed between collagen and procollagen at their C-terminal ends.

Studies on dextranase. VI. Some physicochemical properties and amino acid compositions of dextranases from Brevibacterium fuscum var. dextranlyticum and Peniclllium funiculosum IAM 7013.

Some physicochemical properties and amino acid compositions of dextranases from Brevibacterium fuscum and Penicillium funiculosum IAM 7013 were investigated. The values of 4.35S and 4.37S, respectively, were obtained for the sedimentation constant (S20, w) of dextranases from B. fuscum and P. funiculosum, and their isoelectric points (pI) were also determined 4.17 and 4.19, respectively. Gel filtration on Bio Gel P-100 indicated the molecular weight 5.5×104 for B. fuscum and 4.4×104 for P. funiculosum, and their intrinsic viscosities were 0.038 dl/g and 0.027 dl/g, respectively. Amino acid analysis suggested that dextranase of B. fuscum was found to be composed of more than 429 amino acid residues of 17 amino acids and the enzyme of P. funiculosum contained more than 349 of the residues of 18 amino acids. The composition of the enzymes was very similar except for two residues of cystine which were contained in only P. funiculosum dextranase. Dextranase of B. fuscum contained 11 moles of neutral sugars as glucose and 3 moles of amino sugars as glucosamine per one mole of the enzyme, and in P. funiculosum, the former was 10 moles and the later was found to be 1 mole. The results of staining of the enzymes in disc electrophoresis gels by periodic acid and fuchsin-sulfite, and the observations above mentioned indicated that both dextranases were glycoprotein.

Species Distribution of J Chain

Abstract A polypeptide chain, other than the heavy and light chains and the secretory component, has been observed in secretory immunoglobulin A (IgA) of both rabbit (1) and human (2) origin. This J chain, as it is called, is readily detectable as a fast-moving band on alkaline-urea disc electrophoresis gels of L-chain fractions from reduced and alkylated or S-sulfonated polymeric immunoglobulins (1, 2). It has been suggested that this component functions to join together the two 7S subunits of both 11S secretory and serum IgA (1). The presence of an analogous chain was recently described in studies involving human macroglobulinemic IgM (2) and in ovine (3) and porcine (Zikan, personal communication) secretory immunoglobulins. In the present investigation several other animal species representing key positions on the phylogenetic scale were examined in an attempt to establish whether or not this J chain is a common component of phylogenetically distinct polymeric immunoglobulin molecules.

Pyruvate Decarboxylase

Pyruvate decarboxylase, isolated from active dry bakers' yeast, dissociates into subunits of one-half the molecular weight at alkaline pH. This has been determined by chromatography on Sephadex G-200 over the pH range 6.5 to 8.0. The conditions at which protein dissociation is observed are the same as the conditions at which the cofactors thiamine pyrophosphate and Mg2+ are released and can be separated from the protein. When the holoenzyme is reconstituted in the presence of thiamine-PP and Mg2+ the active enzyme is a dimer of the same molecular weight as the original native enzyme. When the apoenzyme, subjected to the reconstitution procedure in the absence of thiamine-PP and Mg2+, is chromatographed on Sephadex G-200 at pH 6.5 the protein is eluted at an intermediate position, suggesting a monomer-dimer equilibrium which favors the monomer. It appears that thiamine-PP in addition to its catalytic role also functions in the formation of a stable dimer which is the active holoenzyme. The presence of a subunit association step in the reconstitution process which requires thiamine-PP and Mg2+ identifies a site which may be related to the unusual requirement for excess thiamine-PP in the reconstitution process. A method for selectively staining pyruvate decarboxylase on disc electrophoresis gels with a fuchsin staining reagent is described.

The demonstration of tryptophan, tyrosine and carbohydrate-containing proteins in disc electrophoresis gels

The demonstration of tryptophan, tyrosine and carbohydrate-containing proteins in disc electrophoresis gels

A method of staining aldolase activity in the protein fractions separated by Disc electrophoresis

The aldolase activity was detected in the disc electrophoresis gels by the formation of formazane dye from nitroblue tetrazorium reduced by NADH which was formed by the action of glyceraldehyde 3-phosphate dehydrogenase.

Studies on aldolases of fishes and other marine animals by disc electrophoresis

Using the method of staining aldolase activity in disc electrophoresis gels, 5) it was demonstrated that the muscle and liver extracts of various fishes and other marine animals contained isozymes of aldolase.Young yellowtail Seriola quinqueradiata TEMMINCK et SCHLEGEL, flatfish Microstoma kitaharai JORDAN et STARKS, swellfish Fugu pardale TEMMINCK et SCHLEGEL, and sea bream Sebastes matsubarai HILGENDORF, contained in their muscle and liver extracts 2, 3, 5, and 5 aldolase isozymes, respectively.In muscle extracts of prawn Penaeus japonicus BATE, 3 aldolase isozymes were separated. In muscle extracts of Squilla oratoria de HAAN and of sphincter muscle extract of Atrina pectinata LINNÉ, aldolase activity was contained in a single fraction. In the mantle extracts of cuttlefish Sthenoteuthis bartrami LESCUEUR and Octopus vulgaris CUVIER, 3 aldolase isozymes were separated.

Analysis of the proteins in human seminal plasma

The proteins in human seminal plasma were analyzed by cellulose acetate electrophoresis, immunoelectrophoresis, and disc electrophoresis gel diffusion. Cellulose acetate electrophoresis resulted in four bands, while disc electrophoresis produced fifteen which were compared to those formed with serum. Specific identification was made by immunoelectrophoresis and disc electrophoresis gel diffusion with antisera against human serum and twelve individual proteins. Human seminal plasma was shown to contain six proteins, three of which were identified by reactions with specific antisera as albumin, transferrin and immunoglobulin G, and three by their location as a prealbumin an alpha 1 globulin and an alpha 2 globulin.

Destaining apparatus for disc electrophoresis gels by current at right angles to gel axis

Destaining apparatus for disc electrophoresis gels by current at right angles to gel axis