Sodium Dodecyl Sulfate Gel Electrophoresis Research Articles

Characterization of lipovitellin in eggs of the polychaete Neanthes arenaceodentata

The ooplasm of mature oocytes of the polychaete Neanthes arenaceodentata is characteristically filled with yolk platelets. A major component of these structures is lipovitellin, which provides energy and materials required by newly hatched larvae. The lipovitellin isolated and purified from the fertilized eggs of this polychaete was a high-density lipoprotein composed of protein (57%), lipid (42%) and carbohydrate (1%). The lipid component included phospholipids (92% of lipid), triacylglycerol (3% of lipid) and cholesterol (3% of lipid), while sodium dodecyl sulfate-gel electrophoresis showed the major protein component was a 120-kDa peptide. Microscopically, mature oocytes were present in the coelom along with phagocytic eleocytes. The presence of muscle fragments and oil droplets in eleocytes suggests that eleocytes play an important role in providing the protein and lipid needed for the assembly of lipovitellin in the oocytes.

Evaluation of the Allergenicity and Antigenicity of Bovine‐Milk αs1‐Casein Using Extensively Purified Synthetic Peptides

Alphas1-Casein (CAS1_BOVIN), the major allergen of cow's milk (CM), is widely used as hydrolysates in infant diet formulae and additive to other processed food items. To date, most of the reported B-cell epitope mapping were performed on polyethylene pins or cellulose-derivative membrane. We sought to locate the motifs critical for human-specific IgE and rabbit polyclonal IgG binding using extensively purified CAS1_BOVIN, synthetic peptides and derivatives. Thirteen overlapping peptides covering the whole CAS1_BOVIN encompassing 17 : 20 amino acid (AA) were synthesized by f-moc AA solid-phase polyamide peptide synthesis. In addition, six cyanogen bromide (CNBr) cleavage fragments were prepared. Limited hydrolysis, oxidized and reduced/alkylated derivatives were also produced. The preparations were purified by ion exchange, gel filtration chromatography, reversed phase and high-performance liquid chromatography. The homogeneity was visualized by sodium dodecyl sulfate (SDS) and poly acryl amide gel electrophoresis (PAGE) followed by IgE and IgG immunoblotting. IgE binding was measured by Biotin Streptavidin (Bio/strep) fluoro enzyme immuno assay (FEIA) or ELISA-inhibition. Eighteen CM allergy (CMA) sera from 45 clinically examined children (Melbourne) and five adults (Bergen) were selected. Individual sera and pools were used for mapping IgE-binding epitopes. Rabbit IgG sera and pools were used for locating the antigenic sites of the molecule. Results indicated that all the individual CMA sera and pools recognized the intact molecule and three of the CNBr fragments as major antibody-binding allergens. The N- and C-terminal peptides (CAS 16-35; CAS 136-155) showed high IgE-binding affinity. CAS 1-18 and CAS 181-199 showed high IgG bindings. Considering the diversity of the antibody specificities, a reasonable agreement between IgE and IgG epitopes were found at the N- and C-terminals of CAS1_BOVIN. Mapping IgE B-cell epitopes by direct Bio/strep FEIA allowed the development of a sensitive modified technique for detecting unlabelled, casein immune dominant peptides in food products.

Release of biotin from biotinylated proteins occurs enzymatically and nonenzymatically in human plasma

Studies in our laboratory and others indicate that biotin is released from biotinylated proteins in vivo and in vitro in human plasma. Using immunoglobulin G (IgG) as the model protein and four different biotinylating reagents, we investigated the mechanism of release. All of the biotin bonds shared an amide link to the carboxyl group of biotin but differed in the chemical links (amide, thioether, and hydrazone) between spacer arm and the various functional groups on IgG. Biotinylated IgG was incubated with phosphate-buffered saline, plasma, or plasma treated to either inactivate enzymes or remove all macromolecules. Released biotin was separated from bound biotin by ultrafiltration and quantitated by avidin-binding assay. As judged by high-performance liquid chromatography, greater than 95% of the released avidin-binding activity was biotin. We infer that the amide bond between the biotin and the spacer arm rather than the bond attaching the spacer to the protein was cleaved. Sodium dodecyl sulfate gel electrophoresis detected no proteolytic degradation of biotinylated IgG. Neither heat inactivation of plasma nor ultrafiltration of plasma to remove macromolecules completely eliminated biotin cleavage. We conclude that cleavage of biotin from protein occurs by both enzymatic and nonenzymatic mechanisms.

Prevention of artifactual protein oxidation generated during sodium dodecyl sulfate-gel electrophoresis.

Prevention of artifactual protein oxidation occurring during sodium dodecyl sulfate (SDS) acrylamide gel electrophoresis is critical for identifying physiological protein oxidation implicated in human diseases due to the routine use of gel electrophoresis to separate the multiple proteins in proteomic studies. To develop a methodology that completely prevents artifactual protein oxidation in SDS acrylamide gel electrophoresis, cytochrome c was electrophoresed on polyacrylamide gels and subjected to trypsin in-gel digestion followed by tryptic peptide analysis by mass spectrometry. It was found that degassing the acrylamide solution to remove molecular oxygen prior to gel polymerization is a crucial process to protect the electrophoresed protein from reactive oxygen species generated during electrophoresis. However, significant artifactual protein oxidation remains that can only be eliminated entirely, if proteins are electrophoresed on an SDS gel photopolymerized with flavin as the photoinitiator and thioglycolate included in the cathode buffer as a reactive oxygen species scavenger. Using this combination of methodologies, cytochrome c isolated from adult rat heart mitochondria was purified and digested followed by mass spectrometric analysis, demonstrating the requisite high resolution of the polyacrylamide gel and the entire elimination of artifactual oxidation.

Crystal structure of a phosphotransacetylase from Streptococcus pyogenes

Crystal structure of a phosphotransacetylase from <i>Streptococcus pyogenes</i>

Specific isomyosin proportions in hyperexcitable and physiologically denervated mouse muscle

We show here, by high resolution sodium dodecyl sulfate gel electrophoresis, that the proportions of myosin heavy chain (MyHC) isoforms of mouse muscles are specifically shifted by hereditary neuromuscular diseases. In wild-type and dystrophic MDX anterior tibial muscle (TA) about 60% of the MyHC is IIB, 30% IIX, at most 10% IIA and <2% type I (slow). In myotonic fast muscles, hyperexcitability leads to a drastic reduction of MyHC IIB which is compensated by IIA. Slow muscles, like soleus and diaphragm, were only marginally changed by myotonia. The MyHC pattern of TA of spinal muscular atrophy (SMA) ‘wobbler’ mice is shifted to a faster phenotype, with nearly 90% IIB. In the SMA mutant ‘muscle deficient’, all four adult isomyosins are expressed in the TA. These findings may be relevant for the future diagnosis of neurological disorders both in mouse disease models and in human patients.

Purification, characterization, and immunolocalization of paramyosin from the adult stage of Fasciola hepatica.

Paramyosin, a vaccine candidate in different helminthiases, was purified from the adult liver fluke Fasciola hepatica using two different procedures. The first started with a crude extraction of paramyosin in high-salt buffer followed by gel filtration chromatography and two precipitation-solubilization cycles; in the second, anion exchange chromatography replaced the gel filtration step. In both cases, the apparent molecular weight of the purified protein determined by sodium dodecyl sulfate gel electrophoresis under reducing and non-reducing conditions was 97 kDa and 200 kDa, respectively. The molecular weights were consistent with the presence of a dimeric protein linked by disulfide bridges. Western blot analysis showed that the dimeric and monomeric forms were both recognized by an antiserum raised against the F. hepatica 97 kDa band (alpha-FhPmy), and by an anti- Schistosoma mansoni paramyosin immune serum. Immunohistochemistry using alpha-FhPmy demonstrated the localization of paramyosin within the subtegumental muscle and in muscle cells surrounding the gut of adult parasites. We also observed labeling of extramuscular structures like testes, surface lamellae of the gut and the tegument of adult flukes.

Integration of isoelectric focusing with parallel sodium dodecyl sulfate gel electrophoresis for multidimensional protein separations in a plastic microfluidic [correction of microfludic] network.

An integrated protein concentration/separation system, combining non-native isoelectric focusing (IEF) with sodium dodecyl sulfate (SDS) gel electrophoresis on a polymer microfluidic chip, is reported. The system provides significant analyte concentration and extremely high resolving power for separated protein mixtures. The ability to introduce and isolate multiple separation media in a plastic microfluidic network is one of two key requirements for achieving multidimensional protein separations. The second requirement lies in the quantitative transfer of focused proteins from the first to second separation dimensions without significant loss in the resolution acquired from the first dimension. Rather than sequentially sampling protein analytes eluted from IEF, focused proteins are electrokinetically transferred into an array of orthogonal microchannels and further resolved by SDS gel electrophoresis in a parallel and high-throughput format. Resolved protein analytes are monitored using noncovalent, environment-sensitive, fluorescent probes such as Sypro Red. In comparison with covalently labeling proteins, the use of Sypro staining during electrophoretic separations not only presents a generic detection approach for the analysis of complex protein mixtures such as cell lysates but also avoids additional introduction of protein microheterogeneity as the result of labeling reaction. A comprehensive 2-D protein separation is completed in less than 10 min with an overall peak capacity of approximately 1700 using a chip with planar dimensions of as small as 2 cm x 3 cm. Significant enhancement in the peak capacity can be realized by simply raising the density of microchannels in the array, thereby increasing the number of IEF fractions further analyzed in the size-based separation dimension.

Proteolytic peptide mapping.

AbstractAnalysis of intact proteins is hampered by the fact that even when using high-resolution techniques, like electrospray mass spectrometry, you will only be able to tell whether the protein has the expected mass. If you find a deviation from the expected you will, in most cases, be left wondering where (and perhaps what) the difference is. These observations are compounded when using low-resolution techniques like gel filtration or sodium dodecyl sulfate (SDS) gel electrophoresis. Although 2D gel electrophoresis is able to show a single charge difference, in the absence of additional information you are still left wondering where and what the differences are.KeywordsAmino Acid AnalysisDisulfide BridgeIntact ProteinPeptide MappingDigestion TimeThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Photoinhibition of photosystem I is accelerated by dimethyldithiocarbamate, an inhibitor of superoxide dismutase, during light-chilling of spinach leaves

In vivo photoinhibition of photosystem I (PS I) was investigated at chilling temperature using the leaves of the chilling-resistant spinach plant treated with an inhibitor of superoxide dismutase, diethyldithiocarbamate (DDC). When spinach leaves were treated with DDC during chilling at 4 °C for 12 h with a light intensity of 120 μmol m −2 s −1, the activity of PS I and the content of iron–sulfur centers declined to about 50% and 25% of the non-DDC-treated controls, respectively. A native green gel analysis of thylakoid membranes isolated from the DDC-treated leaves resolved a novel chlorophyll–protein complex, which was identified as the light-harvesting complex I (LHC I)-deficient PS I complex when examined by 77 K fluorescence spectroscopy and two-dimensional sodium dodecyl sulfate gel electrophoresis. The possible dissociation of LHC I as an early structural change in the PS I complex after DDC-induced photoinhibition of PS I is discussed.

The significance of carbohydrates on G-CSF: differential sensitivity of G-CSFs to human neutrophil elastase degradation.

It has been reported recently that granulocyte-colony stimulating factor (G-CSF) is degraded upon exposure to human neutrophil elastase (HNE), and this has a negative effect on the ability of the cytokine to promote the in vitro proliferation and maturation of CD34+ cells. This has important implications on the possible in vivo role of elastase in providing negative feedback to granulopoiesis by the direct antagonism of G-CSF. The cytokine used in that study was expressed in Escherichia coli [and was nonglycosylated (NG)], unlike the naturally occurring cytokine, which is an O-linked glycoprotein. As a Chinese hamster ovary-derived (glycosylated) cytokine is available, we compared the susceptibility of NG and glycosylated G-CSF to elastase degradation by incubating the cytokines with HNE and assessing its impact by sodium dodecyl sulfate gel electrophoresis and bioassay. We confirmed the ability of elastase to degrade NG G-CSF in a time- and concentration-dependent manner and found this was associated with a reduction in biological activity of the cytokine. Glycosylated G-CSF, however, was more resistant to elastase degradation, although prolonged exposure did lead to degradation and decreased biological activity. The significance of sugar residues on glycosylated G-CSF in providing protection against the effects of elastase was investigated using enzymatically deglycosylated G-CSF and a mutated form of the G-CSF molecule that was expressed in yeast but was NG. The possible role of HNE in serum-induced inactivation of NG G-CSF was also considered.

Method for cardiac myosin heavy chain separation by sodium dodecyl sulfate gel electrophoresis

Method for cardiac myosin heavy chain separation by sodium dodecyl sulfate gel electrophoresis

A calcium-dependent bacterial surface protein is involved in the attachment of rhizobia to peanut roots.

As part of a project to characterize molecules involved in the crack-entry infection process leading to nodule development, a microscopic assay was used to visualize the attachment of cells of Bradyrhizobium sp. strains SEMIA 6144 and TAL 1000 (labelled by introducing a plasmid expressing constitutively the green fluorescent protein GFP-S65T) to Arachis hypogaea L. (peanut). Qualitative and quantitative results revealed that attachment was strongly dependent on the growth phase of the bacteria. Optimal attachment occurred when bacteria were at the late log or early stationary phase. Cell surface proteins from the Bradyrhizobium sp. strains inhibited the attachment when supplied prior to the attachment assay. Root incubation with a 14-kDa protein (eluted from sodium dodecyl sulphate - gel electrophoresis of the cell surface fraction) prior to the attachment assay resulted in a strong decrease of attachment. The adhesin appeared to be a calcium-binding protein, since cells treated with EDTA were found to be able to bind to adhesin-treated peanut roots. Since this protein has properties identical to those reported for rhicadhesin, we propose that this adhesin is also involved in the attachment process of rhizobia to root legumes that are infected by the crack-entry process.

Usefulness of 8 kDa protein of Fasciola hepatica in diagnosis of fascioliasis.

This study was designed to detect and evaluate an antigenicity of low molecular weight proteins of Fasciola hepatica in fascioliasis. Low molecular weight protein of F. hepatica was purified by ammonium sulfate precipitation and Sephacryl S-100 HR gel filtration. The protein obtained was estimated to be 8 kDa on 7.5-15% gradient sodium dodecyl sulfate gel electrophoresis. Immunoblotting studies showed that the 8 kDa protein reacted with human fascioliasis sera, but not other trematodiasis sera. This result suggests that the 8 kDa protein of F. hepatica is one of diagnostic antigens in human fascioliasis without cross-reaction with other human trematodiasis.

Characterization of low molecular weight starch granule associated protein in common wheat by proteomic approaches

Starch granule proteins (SGPs) were extracted from wheat seeds and characterized using proteomic approaches: two dimensional electrophoresis (2-DE), 40cm long gradient sodium dodecyl sulfate (SDS) gel electrophoresis and electrospray ionization mass spectrometry. The majority of the low molecular weight (LMW) SGPs, especially in the range of 14-30kDa, was located on the surface of starch granule, and only a small number of LMW SGPs of molecular weight range from 30 to 55kDa was found within starch granules. The surface SGPs were mostly expressed in acidic, whereas the integral SGPs were expressed basic regions of 2-DE. Gene location of one surface SGP of approximately 14kDa was assigned on short arm of chromosome 4B. One of the LMW SGPs was identified by LC-ESI MS/MS endochitinase (30kDa). Intensity of this proteins was found to decrease during seed germination and seed maturation.

Allelic variation of the HMW glutenin subunits in Aegilops tauschii accessions detected by sodium dodecyl sulphate (SDS-PAGE), acid polyacrylamide gel (A-PAGE) and capillary electrophoresis

Variability of high molecular weight glutenin subunits (HMW-GS) was studied in198 accessions of Ae. Tauschii (2n=2x=14, DD) by sodium dodecyl sulphate(SDS-PAGE) and acid polyacrylamide gel electrophoresis (A-PAGE) and capillary electrophoresis (CE). A high allelic variation of HMW-GS, including some novel x- and y-type subunits and variable subunit combinations were observed. One accession(TD159) showed a x-type null form. The results by A-PAGE analysis revealed that the subunits Dx5 t and Dy10 t encoded by Glu-D t 1 locus in Ae. tauschii were different in relative mobilities in comparison with the subunits Dx5 and Dy10 found in bread wheats, whereas they had the same mobilities, respectively, when separated by SDS-PAGE. The higher resolution of Ae. tauschii HMW-GS separated by CE method showed two clear peaks in accordance with x- and y-type subunits, respectively,except the accession TD151 which possessed only subunit Dy12.1*t. The electro elution time of the x-type and y-type subunits were about 13–14 and 7–8minutes, respectively. Characterization of wheat HMW-GS was facilitated by using CE which provides high resolution and increases the speed of analysis in conjunction with the traditional gel electrophoretic methods. A total of 42HMW-GS alleles were identified, among which were several alleles not presently detected in bread wheats. Hence Ae. tauschii is potentially a valuable genetic resource for quality improvement of bread wheat.

Characteristics of oil–water emulsions stabilised by an industrial α-lactalbumin concentrate, cross-linked before and after emulsification, by a microbial transglutaminase

Industrial α-lactalbumin concentrate, cross-linked with a microbial transglutaminase, showed lower dilational surface viscosity at a planer oil–water interface than a non-cross-linked α-lactalbumin concentrate. Properties of emulsions containing cross-linked α-lactalbumin were influenced by the sequence of cross-linking and emulsification. Emulsions stabilised by α-lactalbumin concentrate (even without crosslinking) were generally unstable. While cross-linking before emulsification decreased the stability further, the emulsion stability was improved when cross-linking was carried out after emulsification. Results from the sodium dodecyl sulphate (SDS) gel electrophoresis of adsorbed protein suggested that, irrespective of the sequence of cross-linking and emulsification, the adsorbed protein was polymerised too extensively to be resolvable on the gel matrix. Results from the reverse-phase HPLC suggested that the amount of adsorbed protein in emulsions containing protein cross-linking before emulsification was lower than that containing cross-linking after emulsification.

Physicochemical and nitrogen solubility properties of Bacillus proteinase hydrolysates of sodium caseinate incubated with transglutaminase pre- and post-hydrolysis.

Sodium caseinate (NaCN), hydrolyzed with Protamex, a Bacillus proteinase preparation, to 0.5, 1.3, and 17.5% degrees of hydrolysis, was incubated with transglutaminase (TGase) for 3, 42, and 290 min at enzyme/substrate ratios of 1, 1, and 10% (w/w), respectively, pre- and post-hydrolysis. The electrophoretic, reversed-phase high-performance liquid chromatography (RP-HPLC) and nitrogen solubility profiles of the modified products were investigated. Combinations of hydrolysis and incubation with TGase generated products displaying novel physicochemical and nitrogen solubility properties. Significant changes in sodium dodecyl sulfate (SDS) and urea polyacrylamide gel electrophoresis profiles were apparent in the modified caseinate samples. Extensive TGase cross-linking resulted in polymers that were unable to enter the resolving gel during SDS polyacrylamide gradient gel electrophoresis. Extensive combined enzymatic modification resulted in peptides eluting earlier on RP-HPLC than limited combined enzymatic modification or limited hydrolysis. Combination of enzymatic treatments resulted in significantly (P < 0.005) improved solubility around pH 4.6, compared to incubation with TGase or hydrolysis of NaCN alone.

Identification of Staphylococcus aureus exotoxins by combined sodium dodecyl sulfate gel electrophoresis and matrix-assisted laser desorption/ ionization-time of flight mass spectrometry.

Staphylococcus aureus is an important human pathogen whose pathogenesis involves the synthesis of cell wall associated virulence factors and secreted toxins with damaging effects on the host cells. Most of these pathogenic factors are synthesized in a growth-phase dependent manner as a response to environmental stress like heat, lack of nutrients or other deleterious conditions. Conventional identification of these pathogenic factors is based on Western blot analysis or enzyme-linked immunosorbent assay (ELISA) and is limited by the commercial availability of antibodies against these toxins. We report here the use of matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry for monitoring the pathogenic factors of S. aureus. For the identification of pathogenic factors, a methicillin sensitive strain of S. aureus, ATCC-29213, was grown at 37 degrees C or 42 degrees C in brain-heart infusion broth and harvested during the early stationary phase of growth. Secreted proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, enzymatically digested with trypsin and analyzed by MALDI-TOF mass spectrometry. When grown at 42 degrees C, alpha- and beta-hemolysins were found to accumulate in S. aureus supernatants while the concentration of protein A was slightly decreased. The identity of some of these toxins was confirmed by Western-blot analysis. MALDI-TOF mass spectrometry combined with sodium dodecyl sulfate gel electrophoresis represents a rapid and simple approach to characterize the virulence of S. aureus strains which seems to be particularly valuable for the identification of S. aureus exotoxins for which ELISA is not established.

Intersexual variations in the venom of the Brazilian ‘armed’ spider Phoneutria nigriventer (Keyserling, 1891)

Venom from male and female specimens of the medically most important Brazilian Ctenidae spider Phoneutria nigriventer (Keyserling, 1891) has been compared. Males showed a slightly higher venom yield (386 μg) than equal sized females (296 μg), while adult females showed nearly a three times higher venom yield (1079 μg). High-pressure liquid-chromatography analyses revealed differences in the venom composition between males and females. A single peak in HPLC (peak 11) was only detected in venom from females carrying egg-sacs, and sodium dodecyl sulfate–gel electrophoresis showed a series of high molecular weight proteins only in the male venom pool. The median lethal dose (LD 50) in mice for female venom was 0.63 μg kg −1 (95% confidence interval [0.54; 0.71] and 0.61 [0.56; 0.73] μg kg −1) for females with egg-sacs, when compared to the male venom which showed a LD 50 of 1.57 [1.46; 1.88] μg kg −1. The venom of both sexes was also tested in insects using a termite bioassay with doses of 2, 3, 4, and 5 μg per termite. No effect was detected for the lowest dose of female venom, whereas all the other venom doses from both sexes caused a decreased paralysis time and death of the termites. Comparing the venom of both sexes, it was observed that female venom provoked a faster reaction than male venom. The results indicate that males and females of P. nigriventer have differing venom composition which lead to different effects in biological assays.