Sodium Dodecyl Sulfate-polyacrylamide Slab Research Articles

Comparative Skeletal Muscle Proteomics Using Two-Dimensional Gel Electrophoresis.

The pioneering work by Patrick H. O’Farrell established two-dimensional gel electrophoresis as one of the most important high-resolution protein separation techniques of modern biochemistry (Journal of Biological Chemistry 1975, 250, 4007–4021). The application of two-dimensional gel electrophoresis has played a key role in the systematic identification and detailed characterization of the protein constituents of skeletal muscles. Protein changes during myogenesis, muscle maturation, fibre type specification, physiological muscle adaptations and natural muscle aging were studied in depth by the original O’Farrell method or slightly modified gel electrophoretic techniques. Over the last 40 years, the combined usage of isoelectric focusing in the first dimension and sodium dodecyl sulfate polyacrylamide slab gel electrophoresis in the second dimension has been successfully employed in several hundred published studies on gel-based skeletal muscle biochemistry. This review focuses on normal and physiologically challenged skeletal muscle tissues and outlines key findings from mass spectrometry-based muscle proteomics, which was instrumental in the identification of several thousand individual protein isoforms following gel electrophoretic separation. These muscle-associated protein species belong to the diverse group of regulatory and contractile proteins of the acto-myosin apparatus that forms the sarcomere, cytoskeletal proteins, metabolic enzymes and transporters, signaling proteins, ion-handling proteins, molecular chaperones and extracellular matrix proteins.

Sodium Dodecyl Sulfate Polyacrylamide Slab Gel Electrophoresis and Hydroxyethyl Cellurose Gel Capillary Electrophoresis of Luminescent Lanthanide Chelate-labeled Proteins with Time-Resolved Detection

Proteins labeled with a luminescent lanthanide chelate, {{2,2',2'',2'''-{4'-{[(4,6-dichloro-1,3,5-triazin-2-yl)amino]biphenyl-4-yl}-2,2':6',2''-terpyridine-6,6''-diyl}bis(methylenenitrilo)}tetrakis(acetato)}europium(III) (DTBTA-Eu(3+)),were analyzed with sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) slab gel electrophoresis and hydroxyethyl cellurose gel capillary electrophoresis with a time-resolved fluorometric detector. The metal ion of the luminescent lanthanide chelate did not dissociate from the ligand during electrophoresis, and the luminescence was retained. On a slab gel, the band of DTBTA-Eu(3+)-labeled streptavidin was apparently less broad than that of AlexaFluor 488-labeled streptavidin. DTBTA-Eu(3+) in SDS-PAGE slab gel is stable, and the gel after electrophoresis can be dried and stored for at least one year without luminescence fading. In capillary gel electrophoresis (CGE), five labeled proteins with different molecular weight were separated, and a good correlation was observed between the molecular weight and the migration time. Although the detection limits of these proteins determined in buffer solutions of the capillary electrophoresis were not better than those reported for CGE with laser-induced-detection, the detection limits of the same proteins in the present CGE system were not significantly deteriorated in serum solutions compared to those in buffer solutions, and the advantage of using time-resolved detection has been shown.

Changes in physicochemical properties and microstructure of dried squid during softening treatment

AbstractDried squid were prepared at 4 or 40 °C and softened first in water and then in alkaline solution. The physicochemical and structural changes in the dried squid during the softening treatment were examined. A significantly higher wet weight was observed for the 4 °C‐dried squid during the softening treatment compared with the 40 °C‐dried squid. The rupture stress and rupture energy of the 40 °C‐dried squid were significantly higher than those of the 4 °C‐dried squid during the softening treatment. The sodium dodecyl sulphate polyacrylamide slab gel electrophoresis (SDS‐PAGE) pattern of the 4 °C‐dried squid was almost the same as that of raw squid. The SDS‐PAGE pattern of the 40 °C‐dried squid showed many fragments of lower molecular weight. After soaking in distilled water the SDS‐PAGE pattern of the 40 °C‐dried squid did not change significantly; however, the SDS‐PAGE pattern of the 4 °C‐dried squid became the same as that of the 40 °C‐dried squid. Histological analysis by light microscopy showed the formation of muscle fibre bundles in the 40 °C‐dried squid. A higher water permeation was observed among the muscle fibres of the alkali‐softened 4 °C‐dried squid when compared with the alkali‐softened 40 °C‐dried squid. Copyright © 2003 Society of Chemical Industry

Detection of giant myofibrillar proteins connectin and nebulin in fish meat by electrophoresis in 3-5 gradient sodium dodecyl sulfate polyacrylamide slab gels.

An improved method was investigated for sodium dodecyl sulfate polyacrylamide slab gel electrophoresis (SDS-PAGE) to facilitate the analysis of the giant myofibrillar proteins, connectin and nebulin, in fish meat by using jack mackerel (Trachurus japonicus) as the sample fish. It was established that separation of the alpha-connectin band from the beta-connectin band by SDS-PAGE could be achieved by using 3-5% gradient gels with glycerol to facilitate the formation of a gradient with polymerization at 35 degrees C. SDS-PAGE samples of white dorsal muscle from the jack mackerel were homogenized with a 2% SDS solution containing an inhibitor mixture (1 microg/mL of phenylmethanesulfonyl fluoride, 1 microg/mL of leupeptin, and 1 microg/mL of E-64) and heated at 50 degrees C for 20 min. Heating these samples at 100 degrees C for 2 min resulted in the disintegration of connectin but did not affect nebulin. A purified myofibril sample and a whole muscle sample showed similar changes in the overall rate of degradation of whole connectin and nebulin during the postmortem storage period, but it was clear that beta-connectin was cleaved from alpha-connectin during the preparation of myofibrils at the early stage postmortem. Storage of the SDS-PAGE samples at -85 degrees C was preferable to storage at -18 degrees C for a long period.

Purification of lactic acid dehydrogenase from crude bovine heart extract by pH-peak focusing counter-current chromatography

pH-peak focusing counter-current chromatography (CCC) was applied to the purification of lactic acid dehydrogenase (LDH) from a crude bovine heart extract using a cross-axis coil planet centrifuge (CPC). The experiment was performed with two sets of polymer phase systems composed of 16% (w/w) polyethylene glycol (PEG) 1000–12.5% (w/w) potassium phosphate buffer and 15% (w/w) PEG 1540–15% (w/w) ammonium sulfate each at various pH values. The best result was achieved from the PEG 1540–ammonium sulfate polymer phase system by adding a retainer (10 m M acetic acid) to the upper stationary phase and an eluter (100 m M sodium hydroxide) to the lower mobile phase. At a flow-rate of 0.5 ml/min, LDH was eluted as a sharp peak which was well resolved from other proteins. Collected fractions were analyzed by the LDH enzymatic activity and by sodium dodecyl sulfate–polyacrylamide slab gel electrophoresis to detect contaminated proteins. LDH was purified directly from crude bovine heart extract in a concentrated state.

Isolation of an aspartic proteinase precursor from the egg of a hard tick, Boophilus microplus.

An aspartic proteinase precursor, herein named BYC (Boophilus Yolk pro-Cathepsin) was isolated from eggs of the hard tick, Boophilus microplus. As judged by electrophoresis on sodium dodecyl sulfate polyacrylamide slab gel (SDS-PAGE), purified BYC presented 2 bands of 54 and 49 kDa, bearing the same NH2-terminal amino acid sequence. By Western blot analysis, BYC was also found in the haemolymph, indicating an extraovarian site of synthesis. Several organs were incubated in culture medium with [35S]methionine, and only the gut and fat body showed synthesis of BYC polypeptides. Protein sequencing of both the NH2-terminal and an internal sequence obtained after cyanogen bromide (CNBr) cleavage of BYC revealed homology with several aspartic proteinase precursors. Incubation at pH 3.5 resulted in autoproteolysis of BYC, which produced the mature form of the enzyme, that displayed pepstatin-sensitive hydrolytic activity against haemoglobin. Western blot analysis using anti-BYC monoclonal antibodies showed proteolytic processing of BYC during embryogenesis and suggested activation of the enzyme during development. A role of BYC in degradation of vitellin, the major yolk protein of tick eggs, is discussed.

Peptide mapping of S-carboxymethylated hair and feather proteins using two-dimensional electrophoresis

The aim of this work was to develop a two-dimensional electrophoretic method for the multiple simultaneous peptide mapping of a broad spectrum of human and animal hair and avian feather proteins. The 14C labelled S-carboxymethylated proteins of hair and feather were separated in one dimension by sodium dodecyl sulphate-polyacrylamide slab gel electrophoresis and stained with Coomassie Blue. Each of the gel lanes containing the separated hair or feather proteins from one individual was cut and transferred at right angles on to a second slab gel. A solution of trypsin (5 μg/ml) in stacking gel buffer was poured on to the gel lane. Partial proteolysis of the hair or feather proteins proceeded in situ while the stacking gel buffer set. The second dimension of electrophoresis followed by staining and/or fluorography showed a characteristic pattern of peptides of proteins in the form of spots derived from each individual protein, leaving undigested proteins well separated on the diagonal. The protease digestion pattern was reproducible and characteristic of each sample. We were able to establish a finer peptide signature, for individual samples containing a mixture of many proteins, than was hitherto possible with previously published two-dimensional electrophoretic techniques. It was concluded that this technique may be of use in future evolutionary, ontogenetic and forensic studies using hair and feathers as a biological source material.

Quantification of apolipoproteins B-100, B-48, and E in human triglyceride-rich lipoproteins.

We have developed a procedure to quantify apolipoprotein (apo) B-100, apoB-48, and apoE in human triglyceride-rich lipoproteins. This procedure permits delipidation of small amounts of triglyceride-rich lipoproteins without appreciable losses, and quantification of these apolipoproteins in samples containing as little as 10 micrograms of protein. Delipidated triglyceride-rich lipoproteins are subjected to sodium dodecyl sulfate polyacrylamide slab gel electrophoresis, and the mass of apolipoproteins is estimated after densitometric scanning and volume integration of Coomassie blue-stained bands. The chromogenicities of apoB-100 and apoB-48 are virtually identical, and twofold lower than that of apoE. The standard curve for each apolipoprotein follows a power function over a wide protein range, permitting quantification of as little as 0.2 microgram of apoB-48 and as much as 30 micrograms of apoB-100 from a single application of triglyceride-rich lipoproteins to the gels. This method is suitable for routine use in studies of the intestinal and hepatic contributions to triglyceride-rich lipoproteins and their responses to postprandial lipemia.

Simple method of purification of Escherichia coli heat-labile enterotoxin and cholera toxin using immobilized galactose

A simple method for purification of heat-labile enterotoxin produced by enterotoxigenic Escherichia coli and for purification of cholera toxin using immobilized D-galactose column is described. A single run of column chromatography yielded homogeneous toxin as demonstrated by sodium dodecyl sulfate polyacrylamide slab gel electrophoresis.

Inhibition of protein synthesis and antiproliferative effect of the angiotensin converting enzyme inhibitor trandolaprilat in rat vascular smooth muscle cells

To investigate the effect of the angiotensin converting enzyme (ACE) inhibitor trandolaprilat on vascular smooth muscle cell growth, and to analyse its mechanism of action. Aortic vascular smooth muscle cells (VSMC) from Wistar-Kyoto rats were cultured, and cell proliferation was analysed using a cell synchrony technique. Proliferative activity was assessed by [3H]-thymidine uptake and doubling time. Protein synthesis was assessed by [3H]-leucine incorporation. Actin formation was measured using sodium dodecylsulphate-polyacrylamide slab gel electrophoresis and a densitometric assay. The effect of trandolaprilat on translational protein synthesis was also examined using the cell-free protein synthesis system of reticulocyte lysate and messenger RNA from VSMC. Trandolaprilat decreased [3H]-thymidine uptake and increased the doubling time of randomly cycling VSMC. The cell synchrony study revealed that this antiproliferative effect was due to increased transition time from S to G2-M. Decreased cell cycle progression during G2-M was reflected by inhibition of cellular protein synthesis during this period. Cellular protein in randomly cycling VSMC was also decreased by trandolaprilat. This decreased protein synthesis was probably produced by inhibition of RNA translation. The ACE inhibitor trandolaprilat reduces VSMC proliferation by lengthening the G2-M phase of the cell cycle, and produces a decrease in cellular protein content. This effect is probably mediated by inhibition of protein synthesis at the translational level.

Heat shock response in ovarian nurse cells ofAnopheles stephensi

Transcriptional and translational changes following temperature shock at 37, 39 or 41°C to ovarian cells of Anopheles stephensi were studied. Temperature shock at 39°C induced 6 puffs on polytene chromosomes in the nurse cells as revealed by ( 3 H) uridine incorporation studies. Only the 2R-19B puff was induced at 37°C and was found to be a major temperature shock locus remaining most active at all the 3 temperatures tested. Other temperature shock loci were activated only at 39°C. There was progressive inhibi tion of general chromosomal transcription with the rise of temperature. Transcription was drastically inhibited at 41°C but all the temperature shock loci still remained relatively active. Examination of ( 35 S)methionine labelled newly synthesized ovarian proteins using sodium dodecyl sulphate-polyacrylamide slab gels revealed that all the heat shock poly- peptides except the HSP 70 were synthesized in ovarian cells even at control temperature (29°C). Temperature shock induced the synthesis of HSP 70 and elevated the levels of other heat shock polypeptides (82, 30, 29, 23 and 17 KD). Present results suggest that the threshold level for induction of a complete heat shock response in mosquitos is higher (39°C) than the other dipteran insects studied and that a 41°C treatment is not lethal as in the case of Drosophila, Chironomus etc. These features reflect the adaptations of mosquitos to tropical climate and their dietary habit of warm blood meal.

A cross-sectional study of the effects of alcohol on the protein profile of rat lingual epithelium

Sodium dodecyl sulphate-polyacrylamide slab gel electrophoresis, and subsequent densitometric analysis of rat lingual epithelial proteins, showed that for expression of previously reported protein alterations in the lingual epithelium of alcoholic rats, chronic alcohol consumption is required. Alterations in the levels of a high molecular-weight glycoprotein and a 30 k dalton protein in the rat lingual epithelium became significant following 60 days of alcohol consumption. Alterations in the levels of a 28 k dalton protein required more time to become significant. The results provided no evidence of a precursor/product relationship between the high molecular-weight glycoprotein and the two lower molecular-weight proteins.

Synthesis and Assembly of a Novel Recombinant Ribulose Bisphosphate Carboxylase/Oxygenase

The Rhodopseudomonas sphaeroides form II ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBPC/O) expressed in Escherichia coli has been purified to electrophoretic homogeneity by dye-ligand column chromatography. The form II RuBPC/O made in E. coli exactly co-migrates with the R. sphaeroides enzyme in sodium dodecyl sulfate polyacrylamide slab gels. Immunological identity of the recombinant protein was verified by Western immunoblot analysis using antiserum directed against form II RuBPC/O. In addition, sequence determination of the amino terminus of the cloned gene product has established that the protein produced in E. coli under lac transcriptional control is identical to the R. sphaeroides enzyme. Based on comparisons of various catalytic properties, the RuBPC/O isolated from E. coli is functionally identical to the R. sphaeroides enzyme. The E. coli form II RuBPC/O should thus serve as an excellent model for subsequent molecular manipulation of this agriculturally important protein.

Purification and properties of GM1 ganglioside beta-galactosidases from bovine brain.

Two GM1-beta-galactosidases, beta-galactosidases I, and II, have been highly purified from bovine brain by procedures including acetone and butanol treatments, and chromatographies on Con A-Sepharose, PATG-Sepharose, and Sephadex G-200. beta-Galactosidase I was purified 30,000-fold and beta-galactosidase II 19,000-fold. Both enzymes appeared to be homogeneous, as judged from the results of polyacrylamide disc gel electrophoresis. Enzyme I had a molecular weight of 600,000-700,000 and enzyme II one of 68,000, as determined on gel filtration. On sodium dodecyl sulfate polyacrylamide slab gel electrophoresis under denaturing conditions, enzyme II gave a single band with a molecular weight of 62,000, while enzyme I gave two minor bands with molecular weights of 32,000 and 20,000 in addition to the major band at 62,000. Both enzymes liberated the terminal galactose from GM1 ganglioside and lactosylceramide but not from galactosylceramide. Enzyme I showed a pH optimum of 4.0 and was heat stable, while enzyme II showed a pH optimum of 5.0 and lost 50% of its activity in 15 min at 45 degrees C. Enzyme I showed a pI of 4.2 and enzyme II one of 5.9.

A high molecular weight platelet aggregating factor in Crocus sativus

Bulbs of Crocus sativus, variety Cartwrightianus contain a protein factor with aggregating properties on human platelets. This factor was purified by different chromatographic techniques and shows a molecular weight of 42 000, as it was estimated by Sephadex G-75 column chromatography and sodium dodecyl sulfate (SDS) polyacrylamide slab gel electrophoresis.

Purification of a high-molecular-weight inhibitor of the calcium-activated proteinase

An inhibitor of the muscle calcium-activated proteinases has been purified from porcine skeletal muscle by using DEAE-cellulose column chromatography, thermal treatment, Sephacryl S-400 column chromatography in 6 M urea and Sephacryl S-300 column chromatography in 6 M urea. Sodium dodecyl sulfate polyacrylamide slab gel electrophoresis shows that the purified inhibitor is homogeneous and has a subunit molecular weight of 172000. The inhibitor inactivates both the low- and high-calcium-requiring forms of the calcium-activated proteinase but does not inhibit other proteinases against which it has been tried. It thus appears that the inhibitor is specific for the calcium-activated proteinase. Studies using homogeneous inhibitor and high-calcium-requiring proteinase show that one molecule of the inhibitor can inactive up to eight molecules of the calcium-activated proteinase. Inactivation of the calcium-activated proteinase by the inhibitor cannot be reversed by calcium concentrations as high as 25 mM, thus eliminating the possibility that the inhibitor functions by chelating calcium. The inhibitory peptide appears to be extremely susceptible to proteolysis during its isolation. Even in the presence of synthetic proteinase inhibitors different inhibitor preparations yield homogeneous inhibitory peptides ranging in molecular weight from 145000 to 172000. Preparative electrophoresis and column chromatography have been used to isolate putative proteolytic breakdown products of the 172 kDa peptide at 145, 114, 41 and 29 kDa.

High-performance liquid chromatography of proteins : Rapid chromatofocusing of plant enzymes

High-performance liquid chromatography was used to purify four different enzymes extracted from tobacco leaves. Phenylalanine ammonia-lyase (E.C. 4.3.1.5) and three O-methyltransferases (S-adenosyl- l-methionine: catechol O-methyltransferases, E.C. 2.1.1.6) were subjected to high-performance chomatofocusing. Parameters effecting the resolution of chromatography and the recovery of enzyme activity were investigated. The speed and high resolving power of chromatofocusing are major advantages for analytical or preparative purposes. The absorbance at 280 nm of chromatographic fractions was shown to arise mainly from small molecules and was not a measurement of protein concentration as indicated by subsequent high-performance size exclusion chromatography. Electrophoretic analysis of the active fractions on sodium dodecyl sulphate—polyacrylamide slab gels demonstrated the high degree of purification achieved by chromatofocusing.

Appearance of covalently bound antigen in immune complexes formed during the activation of complement

The effects of complement activation on the antigenic component of immune complexes have been studied, using 125I-BSA-rabbit anti-BSA-IgG complexes as models. Polyethylene glycol precipitates of 4 types of IC (those formed in native normal human serum, or NHS-containing EDTA, NHS-EDTA, and preformed soluble IC incubated in NHS or NHS-EDTA immediately after preparation) were analysed by sodium dodecyl sulphate-polyacrylamide slab gel electrophoresis. A characteristic difference in the distribution of 125I-BSA in the gel was observed between the NHS- and NHS-EDTA-treated samples. With the former type of IC, a significant part (16–23%) of the label was found in gel fractions of mol. wt. exceeding that of the antigen (80–300 kDa vs. 69 kDa), whereas with NHS-EDTA-treated IC only a minimal amount (4–5%) of the radioactivity was detected in these fractions. These findings indicate that complement activation results in the covalent binding of complement to the antigenic component of IC. The practical importance of these observations is discussed. In addition, a marked difference in precipitability was observed between IC formed in NHS and preformed IC incubated in NHS.

Cytolytic T cell granules. Isolation, structural, biochemical, and functional characterization.

The cytoplasmic, dense granules of cloned T cell lines were isolated and analyzed for their functional and biochemical properties. Isolated granules of approximately 90% homogeneity, in the presence of Ca, effect strong tumoricidal and hemolytic activity. Tumor cell lysis is complete in less than 30 min, with less than 2 micrograms granule protein corresponding to a killer/target ratio of 3-6:1 by assuming 50% yield for granule isolation. The granules contain a set of unique proteins, responsible for cytolytic activity and designated K1 to K6, in the molecular weight range of 14,000 to 75,000, as defined by sodium dodecyl sulfate (SDS) polyacrylamide slab gel analysis under reducing and nonreducing conditions. Cytolysis mediated by isolated granules is accompanied by the assembly of tubular complexes of 160 A (poly P1) and of approximately 70 A width (poly P2) that are inserted into membranes and form ultrastructural membrane lesions. As shown by immunofluorescence and by Percoll gradient fractionation, cytolytic granules are detected in cells of cytolytic T cell lineage and not in the T cell lymphomas E14 and S194. Poly perforin 1 assembled by CTLL-2 upon stimulation with concanavalin A (Con A) and phorbol myristate acetate (PMA) was isolated by detergent extraction and gel filtration. Poly P1 is composed of disulfide-linked subunits that, after reduction, co-migrate with certain granule proteins. The results are compatible with the hypothesis that the dense granules of cytolytic T cells contain cytolytic proteins that polymerize to disulfide-linked tubular poly perforins in a Ca-dependent reaction and may cause cytolysis by membrane insertion and transmembrane channel formation.

Photoaffinity labelling of Ca2+ channels with [3H]azidopine

A 1,4-dihydroypyridine arylazide photoaffinity ligand, [3H]azidopine (50.6 Ci/mmol), has been synthesized. [3H]Azidopine binds reversibly with a Kd of 350 pM to guinea-pig skeletal muscle membranes in the absence of ultraviolet light. The reversible [3H]azidopine binding is inhibited stereoselectively by 1,4-dihydropyridines, phenylalkylamine Ca2+ channel blockers and La3+. Covalent incorporation into membrane proteins after photolysis was investigated by sodium dodecyl sulfate polyacrylamide slab gel electrophoresis. [3H]Azidopine is photoincorporated specifically into a protein of Mr∼145 000. The covalent labelling of the Mr ∼145 ooo band is inhibited stereoselectively by drugs and cations which block the reversible [3H]azidopine binding. It is suggested that [3H]azidopine is photoincorporated into a subunit of the putative Ca2+ channel.