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Sodium Dodecyl Sulfate-polyacrylamide Gel Electrophoresis (sodium + dodecyl_sulfate-polyacrylamide_gel_electrophoresis)
Selected AbstractsPhysicochemical Changes in Alaska Pollock Surimi and Surimi Gel as Affected by Electron BeamJOURNAL OF FOOD SCIENCE, Issue 1 2004J. JACZYNSKI ABSTRACT: Alaska pollock surimi and surimi gels (cooked) were subjected to various doses of electron beam (e-beam). Shear stress of surimi gels increased as the dose increased up to 6 to 8 kGy and then decreased. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed gradual degradation of myosin heavy chain as the dose increased. The degradation was slower for frozen samples. The integrity of actin was slightly affected by high doses (25 kGy). The amount of sulfhydryl groups and the level of surface hydrophobicity of Alaska pollock surimi decreased as the dose increased, suggesting formation of disulfide bonds and hydrophobic interactions. The sulfhydryl groups and hydrophobicity of surimi gels increased as the dose increased up to 6 kGy and then decreased. [source] Characterization of bacteriocin produced by Streptococcus bovis J2 40-2 isolated from traditional fermented milk ,Dahi'ANIMAL SCIENCE JOURNAL, Issue 1 2009Md Harun-ur RASHID ABSTRACT A bacteriocin-producing strain Streptococcus bovis J2 40-2 was isolated from traditional fermented milk ,Dahi' in Bangladesh. Despite its narrow antimicrobial spectrum, it showed strong antimicrobial activity against extremely challenging and problematic organisms in foods, such as Listeria monocytogenes. Bacteriocin was sensitive to several proteolytic enzymes and showed antimicrobial activity over a wide pH range of 2.0,10.0. It was stable when heated to 110°C for 20 min, but lost 25% of its activity when heated to 121°C for 15 or 20 min. Optimum bacteriocin production (5600 AU/mL) was achieved when the strain was cultured at 37°C for 24 h in MRS medium rather than in TYLG, GM17, or skim milk medium. Bacteriocin was partially purified by ammonium sulfate precipitation (80% saturation), dialysis (cut-off MW: 3500) and gel filtration chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that bacteriocin had a molecular weight of approximately 4.5 kDa. [source] Physicochemical properties of the thermal gel of water-washed meat in the presence of the more soluble fraction of porcine sarcoplasmic proteinANIMAL SCIENCE JOURNAL, Issue 1 2007Yuji MIYAGUCHI ABSTRACT We investigated the physicochemical properties of the thermal gel of water-washed pork meat (WWM) in the presence of the soluble fraction of porcine sarcoplasmic protein (SP) obtained with ammonium sulfate at 75 percent saturation. Two precipitated fractions of SP were obtained at 0,50 percent and 50,75 percent saturation, named SP-f1 and SP-f2, respectively, and the soluble fraction obtained at 75 percent saturation, SP-f3, was used. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that SP-f3 contained mainly glyceraldehyde-3-phosphate dehydrogenase (GAPDH), while SP-f1 and SP-f2 had other SPs such as phosphorylase b, enolase, actin and phosphoglycerate mutase. The gel strength of WWM was greater when SP-f3 rather than one of various animal proteins such as bovine plasma (BP), egg white, or whey protein isolates (WPI), was added and SP-f3 had a gel-enhancing effect as good as that of polyphosphate (PP). The gel strength of WWM with added SP-f3 increased significantly with NaCl at 0.15 mol/L or more, but not in the absence of NaCl (0 mol/L). The effect of SP-f3 was evident at neutral pH and maximum gel strength was obtained at a pH above 6.0. Differential scanning calorimetric (DSC) analysis showed that an endothermic peak corresponding to myosin heads in WWM shifted to a lower temperature with the addition of SP-f3, as in the case of PP, though there was no such shift in the presence of other animal proteins (BP, egg white and WPI), suggesting that SP-f3 increases the gel strength of WWM through the dissociation of actomyosin similar to PP. Scanning electron microscopy (SEM) revealed wall-like structures among the protein strands in the WWM gel matrix in the presence of SP-f3. The results of DSC and SEM indicated that the formation of a gel network in meat products is reinforced with GAPDH in SP after the interaction between GAPDH and myofibrillar protein. [source] Enhanced sensitivity to cis -diamminedichloro-platinum(II) of a human carcinoma cell line with mutated p53 gene by cyclin-dependent kinase inhibitor p21WAF1 expressionCANCER SCIENCE, Issue 3 2003Mamoru Satou p53-mediated induction of p21WAF1, a cyclin-dependent protein kinase inhibitor, is known to protect cancer cells from the cytotoxic effects of anti-cancer drugs or ,-irradiation. Since the p53 gene is frequently inactivated in cancer cells, we examined whether p21WAF1 expression may alter the sensitivity of cancer cells with mutated p53 gene to anti-cancer drugs. Cells of a colon cancer cell line DLD-1 were transfected with p21WAF1 expression vector controlled by a tetracycline-repressable promoter and transfec-tants were cloned (Dp21,1). p21WAF1 expression induced by removal of tetracycline from culture media repressed cell proliferation and resulted in altered cell shape, suggesting induction of differentiation. Dp21,1 cells with p21WAF1 expression were more sensitive to cis -diamminedichloroplatinum(II) (CDDP) (IC50 value, 10 ,M) than those without p21WAF1 expression (IC50, 22 ,M). Sensitivity to doxorubicin was not different between Dp21,1 cells with and without p21WAF1 expression. DNA ladder formation was observed in Dp21,1 cells treated with CDDP, indicating that the enhanced sensitivity to CDDP involves apoptosis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cytosolic protein revealed that subunit protein bands with Mr 55 kDa and 44 kDa were markedly increased in cells with p21WAF1 expression. By im-munoblotting, these proteins were identified as c-Jun N-terminal kinase (JNK) 2 and p38 mitogen-activated protein kinase (MAPK) 8, respectively, both of which are believed to be involved in apoptosis induction by CDDP. These results suggest that p21WAF1 may enhance the sensitivity of colon cancer cells with mutated p53 gene to CDDP, possibly through the JNK and p38 MAPK pathways. (Cancer Sci 2003; 94: 286,291) [source] Two-dimensional electrophoresis with cationic detergents, a powerful tool for the proteomic analysis of myelin proteins.JOURNAL OF NEUROSCIENCE RESEARCH, Issue 4 2008Part 1: Technical aspects of electrophoresis Abstract The analysis of proteins in damaged myelin is crucial to clarify the mechanisms of dysmyelination and demyelination. In the present study, proteomic analysis of myelin using a modified two-dimensional electrophoresis (2-DE) method was carried out to obtain a better understanding of myelin biology. Although standard 2-DE (immobilized pH gradient isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis; IPG/SDS-PAGE) methods of analysis provide high resolutions of soluble proteins with isoelectric focusing points in the pH range of 4,8, major myelin components include highly basic proteins are compacted at the basic edge of the 2-DE gels and are not sufficiently separated for satisfactory analysis. In an attempt to improve the separation of these proteins, an alternative 2-DE method using the cationic detergents was applied. In part 1 of this study, we describe technical aspects of conditioning 2-DE using cationic detergent. In the accompanying paper (part 2), practical 2-DE analysis using cationic detergents is described to identify proteins in the purified CNS myelin fraction. We carried out benzyldimethyl- n -hexadecylammonium chloride (16-BAC)/SDS-PAGE 2-DE and tested 2-DE with four other cationic detergents. We found that 16-BAC was the most effective agent for separation of myelin proteins and that hexadecyltrimethylammonium bromide (cetyltrimethylammonium bromide; CTAB) was the most effective agent for solubilization of myelin proteins. The combination of 16-BAC/SDS-PAGE and CTAB/SDS-PAGE is a powerful tool for the analysis of myelin proteins, including highly basic, high-MW (MW > 100K), and integral membrane proteins. © 2007 Wiley-Liss, Inc. [source] Diversity of rice glutelin polypeptides in wild species assessed by the higher-temperature sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subunit-specific antibodiesELECTROPHORESIS, Issue 6 2008Nadar Khan Abstract In efforts to find genetic resources with high nutritional value of rice seed, we assessed the diversity of the major storage protein glutelin in 13 wild and 2 cultivated rice species by a unique SDS-PAGE method and subunit-specific antibodies. Maximum separation of microheterogeneous glutelin ,-polypeptides, which is a prerequisite for the diversity evaluation, could be attained by SDS-PAGE performed at higher temperature (45°C) than the generally employed temperatures (4,25°C). Seven antipeptide antibodies were raised against subunit-specific epitope sequences designed at five sites from four variable regions spanning the glutelin ,-polypeptides. High specificity of each antibody was confirmed using rice glutelin mutants, and demonstrated considerable variation in amino acid sequence and accumulation level of glutelin subunit in wild species, in combination with the higher-temperature SDS-PAGE. The degree of the variation was, however, changed according to the site of variable regions and the type of subunit. Some wild species accumulated nutritious GluB subunits more than cultivated rice. The wild species Oryza longiglumis and O. brachyantha had glutelin with low reactivity against most antibodies examined in this study, reflecting the significant divergence. Such wild species may hopefully serve as important genetic resources for nutritional improvement of cultivated rice. [source] Fast visible dye staining of proteins in one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gels compatible with matrix assisted laser desorption/ionization-mass spectrometryELECTROPHORESIS, Issue 7-8 2004Jung-Kap Choi Abstract A fast and matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) compatible protein staining method in one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (1- and 2-D SDS-PAGE) is described. It is based on the counterion dye staining method that employs oppositely charged two dyes, zincon (ZC) and ethyl violet (EV) to form an ion-pair complex. The protocol, including fixing, staining and quick washing steps, can be completed in 1,1.5 h depending upon gel thickness. It has a sensitivity of 4,8 ng, comparable to that of colloidal Coomassie Brilliant Blue G (CBBG) staining with phosphoric acid in the staining solution. The counterion dye stain does not induce protein modifications that complicate interpretation of peptide mapping data from MS. Considering the speed, sensitivity and compatibility with MS, the counterion dye stain may be more practical than any other dye-based protein stains for routine proteomic researches. [source] Analysis of proteins stained by Alexa dyesELECTROPHORESIS, Issue 6 2004Shijun Huang Abstract Alexa dye staining of proteins is used for the fluorescence microscopy of single particles that are sometimes multimolecular protein complexes. To characterize the staining, post-staining determination must be made of which protein(s) in a complex have been Alexa-stained. The present communication describes the use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for performing this determination. The Alexa-stained proteins are observed directly in gels by illumination with an ultraviolet transilluminator. The test multimolecular particle is bacteriophage T7. The protein capsid of T7 is a multimolecular complex that has both external and internal proteins. SDS-PAGE of Alexa-stained bacteriophage T7 produces fluorescent capsid proteins each of which usually comigrates with an unstained protein. However, one Alexa-induced modification of protein migration was observed by SDS-PAGE. Mass spectrometry shows that the protein with modified migration is the major protein of the outer shell of the T7 capsid. The procedures used are generally applicable. The distribution of Alexa staining among T7 capsid proteins depends on the size of the dye molecule used. The larger the dye molecule is, the greater the preference for external proteins. [source] Highly sensitive and simple fluorescence staining of proteins in sodium dodecyl sulfate-polyacrylamide-based gels by using hydrophobic tail-mediated enhancement of fluorescein luminescenceELECTROPHORESIS, Issue 19-20 2003Chulhun Kang Abstract Fluorescein has an extremely low luminescence intensity in acidic aqueous media. However, when it was bound to proteins, subsequent increase of luminescence intensity took place. Furthermore, when a hydrophobic tail, such as aliphatic hydrocarbons, was introduced to fluorescein, more dramatic increase of luminescence intensity was observed upon binding to proteins. In the present study, by utilizing this luminescence enhancement, three hydrophobic fluorescein dyes (5-dodecanoyl amino fluorescein, 5-hexadecanoyl amino fluorescein, and 5-octadecanoyl amino fluorescein) were examined as noncovalent fluorescent stains of protein bands in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Effective incorporation of the dyes to proteins in gels was accomplished either simply by adding dyes at the protein fixation step, or by treating gels with a staining solution after the fixation. The sensitivity of this staining method using the fluorescein derivatives was approximately 1 ng/band for most proteins. For some cases, protein bands containing as low as 0.1 ng were successfully visualized. In addition, the detection sensitivity showed much less protein-to-protein variation than silver staining. This new staining method was also successfully applied to two-dimensional electrophoresis of rat brain proteins. Its overall sensitivity was comparable to that of silver staining. [source] Proteomics of snake venoms from Elapidae and Viperidae families by multidimensional chromatographic methodsELECTROPHORESIS, Issue 16 2003Jiraporn Nawarak Abstract Snake venoms contain a large number of biologically active substances and the venom components are very useful for pharmaceutical applications. Our goal is to separate and identify components of snake venoms in ten snake species from the Elapidae and Viperidae families using multidimensional chromatographic methods. The multidimensional chromatographic methods include reversed-phase high-performance liquid chromatography (RP-HPLC), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), lab-on-a-chip, two-dimensional electrophoresis (2-DE), and mass spectrometry. The venoms of eight snake species demonstrated major differences in hydrophobicity, molecular weight separations, and 2-DE protein distribution patterns. The 2-DE images showed major differences between families, within each family and even between the same species. Venoms of the Elapidae family showed many basic proteins with a wide range of molecular weights, while venoms of the Viperidae family showed wide ranges of pI and molecular weights, especially for Trimeresurus sp. The multidimensional chromatographic methods revealed specific differences in venom proteins intra-species as well as between species and families. We have isolated and identified proteins that may be unique for each species for further studies in the proteome of snake venoms and their potentially use in the pharmaceutical applications. [source] A versatile electrophoresis system for the analysis of high- and low-molecular-weight proteinsELECTROPHORESIS, Issue 11 2003Christophe Tastet Abstract A new, versatile, multiphasic buffer system for high-resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins in the relative molecular weight range of 300,000,3000 Da is described. The system, based on the theory of multiphasic zone electrophoresis, allows complete stacking and destacking of proteins in the above Mr range. The buffer system uses taurine and chloride as trailing and leading ion, respectively, and Tris, at a pH close to its pKa, as the buffering counterion. Coupled with limited variation in the acrylamide concentration, this electrophoresis system allows to tailor the resolution in the 6,200 kDa Mr range, with minimal difficulties in the post electrophoretic identification processes. [source] Delivery of bioactive, gel-isolated proteins into live cellsELECTROPHORESIS, Issue 9 2003Jennifer E. Taylor Abstract The delivery of proteins into live cells is a promising strategy for the targeted modulation of protein-protein interactions and the manipulation of specific cellular functions. Cellular delivery can be facilitated by complexing the protein of interest with carrier molecules. Recently, an amphipatic peptide was identified, Pep-1 (KETWWETWWTE WSQPKKKRKV), which crosses the plasma membrane of many cell types to carry and deliver proteins as large as antibodies. Pep-1 effectively delivers proteins in solution; but Pep-1 is not suitable for delivering sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) isolated proteins because Pep-1 complexes with cargo proteins are destroyed by SDS. Here, we report cellular delivery of SDS-PAGE-isolated proteins, without causing cellular damage, by using a nonionic detergent, Triton X-100, as carrier. To determine the specificity of our method, we separated antibodies against different intracellular targets by nonreducing SDS-PAGE. Following electrophoresis, the antibody bands were detected by zinc-imidazole reverse staining, excised, in-gel refolded with Triton X-100, and eluted in detergent-free phosphate-buffered saline. When overlaid on cultured NIH 3T3 cells, the antibodies penetrated the cells localizing to their corresponding intracellular targets. These results are proof-of-principle for the delivery of gel-isolated bioactive proteins into cultured cells and suggest new ways for experimental protein therapy and for studying protein-protein interactions using gel-isolated protein. [source] Proteome analysis of human liver tumor tissue by two-dimensional gel electrophoresis and matrixassisted laser desorption/ionization-mass spectrometry for identification of disease-related proteinsELECTROPHORESIS, Issue 24 2002Jina Kim Abstract Hepatocellular carcinoma (HCC) is a common malignancy worldwide and is a leading cause of death. To contribute to the development and improvement of molecular markers for diagnostics and prognostics and of therapeutic targets for the disease, we have largely expanded the currently available human liver tissue maps and studied the differential expression of proteins in normal and cancer tissues. Reference two-dimensional electrophoresis (2-DE) maps of human liver tumor tissue include labeled 2-DE images for total homogenate and soluble fraction separated on pH 3,10 gels, and also images for soluble fraction separated on pH 4,7 and pH 6,9 gels for a more detailed map. Proteins were separated in the first dimension by isoelectric focusing on immobilized pH gradient (IPG) strips, and by 7.5,17.5% gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels in the second dimension. Protein identification was done by peptide mass fingerprinting with delayed extraction-matrix assisted laser desorption/ionization-time of flight-mass spectrometry (DE-MALDI-TOF-MS). In total, 212 protein spots (117 spots in pH 4,7 map and 95 spots in pH 6,9) corresponding to 127 different polypeptide chains were identified. In the next step, we analyzed the differential protein expression of liver tumor samples, to find out candidates for liver cancer-associated proteins. Matched pairs of tissues from 11 liver cancer patients were analyzed for their 2-DE profiles. Protein expression was comparatively analyzed by use of image analysis software. Proteins whose expression levels were different by more than three-fold in at least 30% (four) of the patients were further analyzed. Numbers of protein spots overexpressed or underexpressed in tumor tissues as compared with nontumorous regions were 9 and 28, respectively. Among these 37 spots, 1 overexpressed and 15 underexpressed spots, corresponding to 11 proteins, were identified. The physiological significance of the differential expressions is discussed. [source] Effects of different brush border membrane vesicle isolation protocols on proteomic analysis of Cry1Ac binding proteins from the midgut of Helicoverpa armigeraINSECT SCIENCE, Issue 6 2008Li-Zhen Chen Abstract Brush border membrane vesicles (BBMV) isolated from insect midguts have been widely used to study Cry1A binding proteins. Sample preparation is important in two-dimensional electrophoresis (2-DE), so to determine a suitable BBMV preparation method in Helicoverpa armigera for 2-DE, we compared three published BBMV preparation methods mostly used in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). All methods yielded similar types and numbers of binding proteins, but in different quantities. The Abdul-Rauf and Ellar protocol was the best of the three, but had limitations. Sufficient protein quantity is important for research involving limited numbers of insects, such as studies of insect resistance to Bacillus thuringiensis in the field. Consequently, we integrated the three BBMV isolation methods into a single protocol that yielded high quantities of BBMV proteins from H. armigera larval midguts, which proved suitable for 2-DE analysis. [source] Expression and characterization of ,-glucosidase III in the dwarf honeybee, Apis florea (Hymenoptera: Apoidea: Apidae)INSECT SCIENCE, Issue 4 2007CHANPEN CHANCHAO Abstract Alpha-glucosidase is synthesized in the hypopharyngeal glands located in the head of worker bees including Apis florea. To analyze the developmental stage-specific expression of the ,-glucosidase gene in A. florea, total RNA was isolated from eggs, and the heads of nurse and forager bees. By reverse transcription polymerase chain reaction (RT-PCR), it was shown that the highest expression levels of the ,-glucosidase III gene, in the three examined developmental stadia, were found in forager bees, with much lower expression levels in nurse bees and no detectable expression in eggs. A complete ,-glucosidase III cDNA was obtained by RT-PCR and sequenced. The 1 701 bp cDNA nucleotide sequence and the predicted 567 amino acids it encodes were assayed by BLASTn, BLASTp and BLASTx programs and revealed a 95% and 94% similarity to the A. mellifera,-glucosidase III gene at the DNA and amino acid sequence levels, respectively. For purification of the active encoded enzyme, forager bee heads were homogenized in sodium phosphate buffer solution and the crude extract (0.30 U/mg) sequentially precipitated with 95% saturated ammonium sulfate (0.18 U/mg), and purified by DEAE cellulose ion exchange chromatography (0.17 U/mg), and gel filtration on Superdex 200 (0.52 U/mg). After resolution through sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a single enzymically active band (73 kDa) was identified from renatured substrate gels. Excision of this band, elution of the protein and tryptic peptide digestives identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) revealed six matching masses to the A. mellifera (Q17958) and predicted A. florea,-glucosidase III protein with 12% coverage, supporting the probable purification of the same ,-glucosidase III protein as that encoded by the cloned cDNA. [source] Canine COL1A2 Mutation Resulting in C-Terminal Truncation of Pro-,2(I) and Severe Osteogenesis ImperfectaJOURNAL OF BONE AND MINERAL RESEARCH, Issue 6 2001Bonnie G. Campbell Abstract RNA and type I collagen were analyzed from cultured skin fibroblasts of a Beagle puppy with fractures consistent with type III osteogenesis imperfecta (OI). In a nonisotopic RNAse cleavage assay (NIRCA), the proband's RNA had a unique cleavage pattern in the region of COL1A2 encoding the C-propeptide. DNA sequence analyses identified a mutation in which nucleotides 3991-3994 ("CTAG") were replaced with "TGTCATTGG." The first seven bases of the inserted sequence were identical to nucleotides 4002-4008 of the normal canine COL1A2 sequence. The resulting frameshift changed 30 amino acids and introduced a premature stop codon. Reverse-transcription polymerase chain reaction (RT-PCR) with primers flanking the mutation site amplified two complementary DNA (cDNA) fragments for the proband and a single product for the control. Restriction enzyme digestions also were consistent with a heterozygous mutation in the proband. Type I procollagen labeled with [3H]proline was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Increased density of pC-,2(I) suggested comigration with the similarly sized pro-,2(I) derived from the mutant allele. Furthermore, ,-chains were overhydroxylated and the ratio of ,1(I):,2(I) was 3.2:1, consistent with the presence of ,1(I) homotrimers. Analyses of COL1A2 and type I collagen were both consistent with the described heterozygous mutation affecting the pro-,2(I) C-propeptide and confirmed a diagnosis of OI. [source] FILM FORMING MECHANISM AND MECHANICAL AND THERMAL PROPERTIES OF WHEY PROTEIN ISOLATE-BASED EDIBLE FILMS AS AFFECTED BY PROTEIN CONCENTRATION, GLYCEROL RATIO AND PULLULAN CONTENTJOURNAL OF FOOD BIOCHEMISTRY, Issue 3 2010MAHAMADOU ELHADJI GOUNGA ABSTRACT Tensile strength (TS), elongation at break (EAB) and elastic modulus (EM) of edible films prepared from 5, 7 and 9% whey protein isolate (WPI) plasticized with different levels of glycerol (Gly) (WPI : Gly = 3.6:1, 3:1 and 2:1) were investigated in order to completely characterize WPI-Gly films. On increasing protein concentration an increase in TS and EAB was observed. On the other hand, increasing Gly led to a decrease in TS and EM, while EAB increased. The addition of pullulan (Pul) into the film forming solution (FFS) increased EAB while TS, EM and thermal properties were reduced. This suggested that Pul had a similar effect as plasticizers. Films with higher Pul content showed lighter protein bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Fourier transform infrared spectroscopy showed that hydrogen bonding was high in WPI : Pul films as compared with the control. This is attributed to the protein-polysaccharide interactions brought about by the dominance of Pul in the FFS. PRACTICAL APPLICATIONS This work describes some physical properties of films based on blends of whey protein isolate (WPI) and pullulan (Pul), made after a previous study on some characteristics of films based on pure WPI plasticized by glycerol. The most studied proteins in the edible films technology being gluten and WPI, the use of Pul in mixture with WPI is considered as a new investigation to explore the utilization of WPI-Pul in edible film and coating materials applied to food products. Furthermore, the use of WPI-Pul films and coatings could potentially extend the shelf life and improve the stability of the coated products as shown by the resultant properties in this investigation and previous works. [source] PROTEOLYTIC ACTIVITY OF LACTOBACILLUS SAKEI, LACTOBACILLUS FARCIMINIS AND LACTOBACILLUS PLANTARUM ON SARCOPLASMIC PROTEINS OF PORK LEANJOURNAL OF FOOD BIOCHEMISTRY, Issue 3 2004ANNA LISA BASSO The aim of this study was to assess the proteolytic activity of Lactobacillus sakei (DSM 6333), L. plantarum (B21), and to a lesser extent, L. farciminis (DSM 20184) on meat sarcoplasmic proteins. The protein composition was assayed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and capillary electrophoresis after incubation of meat extract inoculated with bacteria. All strains showed proteolytic activity: a band about 94 kDa disappeared in samples inoculated with L. farciminis and L. plantarum and strongly decreased in those inoculated with L. sakei. The intensity of the bands with a molecular weight between 94 and 38 kDa decreased in all samples. Capillary electrophoresis analysis ascertained the disappearance of the fractions corresponding to 8.64 and 8.66 min retention time in all samples. The bands corresponding to 94 kDa and 38 kDa were, respectively, identified as glycogen phosphorylase muscle isoform and glyceraldehyde 3-phosphate dehydrogenase, by in situ digestion of protein gel bands and peptide map analysis using Matrix Assisted Laser Desorption/Ionization - Time of Flight Mass Spectrometry (MALDI-TOF MS). [source] PURIFICATION OF AMYLASE FROM TILAPIA BY MAGNETIC PARTICLEJOURNAL OF FOOD PROCESSING AND PRESERVATION, Issue 1 2010MING CHANG WU ABSTRACT Recent development in magnetic carrier technology involves the use of nonmagnetic substrates attached to superparamagnetic particles forming functionally modified magnetic support to isolate a particular enzyme in a controllable magnetic field. In this study, the superparamagnetic particles were modified by epichlorohydrin and other agents to cross-link with starch to form the purification support. This affinity support was able to bind the amylase and was used in the purification of amylase from Taiwan tilapia. After ammonium sulfate precipitation of amylase from Taiwan tilapia, the modified superparamagnetic particles were able to purify the crude amylase by 20.78-fold with recovery of activity of 75.6%. The molecular weight of the amylase was estimated to be 66.1 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both crude and purified amylase reached an optimum at a pH of 8.0 and temperature of 50C, and the enzyme was stable between 20 and 50C. PRACTICAL APPLICATIONS Because of the rapid development of high technology such as carrier supports for enzyme purification, the development, research and application of magnetic carriers are timely needed. The present study demonstrated that the affinity superparamagnetic particles could be used as a carrier support to absorb and purify the amylase and that technology of affinity purification can be widely used in protein purification. Compared with the traditional chromatography used in the purification of proteins, this novel affinity superparamagnetic particle technology is rapid, has low operation cost, requires simple facilities, and involves easy separation and recovery of the enzymes. [source] Polyphenol Oxidase from Apple (Malus domestica Borkh. cv Bramley's Seedling): Purification Strategies and CharacterizationJOURNAL OF FOOD SCIENCE, Issue 1 2006Deirdre M. Ni Eidhin ABSTRACT Polyphenol oxidase (PPO) was isolated from Bramley's Seedling apples with 75.7-fold purification and 26.5% recovery by ammonium sulfate precipitation, phenyl sepharose chromatography, ion exchange chromatography, and hydroxyapatite chromatography. Molecular weight was estimated to be about 45 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS PAGE). Optimum PPO activity was at pH 6.5 and greater than 50% activity was retained during storage for 72 h at pH 5.5 to 6.5. Optimum temperature for activity was 30 °C and the enzyme had residual activity of greater than 50% during storage for 72 h at 20 °C to 30 °C and for 24 h at 40 °C to 50 °C. Of the substrates tested, activity was greatest with 4-methylcatechol followed by catechol, pyrogallol, and (,)epicatechin. The most effective inhibitors tested were sodium metabisulfite and ascorbic acid. [source] Partially Purified Collagen from Refiner Discharge of Pacific Whiting Surimi ProcessingJOURNAL OF FOOD SCIENCE, Issue 8 2005Jin Soo Kim ABSTRACT The physicochemical properties of acid-soluble collagen (ASC) from refiner discharge and the partially purified collagen (PPC) from both the refiner discharge and the fish skin were evaluated. Yield of collagen from refiner discharge was 34% higher in PPC than ASC. Mercury, lead, cadmium, and chromium contents of PPC from refiner discharge were not detected. There was no difference in the pattern of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) between ASC and PPC from refiner discharge. PPC from refiner discharge showed better functional properties than that from skin and was similar to ASC: whiteness, solubility, emulsifying activity, and cooking stability. Therefore, PPC from refiner discharge could be used as a new resource. [source] Effect of Chemically Modified Soy Proteins and Ficin-tenderized Meat on the Quality Attributes of SausageJOURNAL OF FOOD SCIENCE, Issue 1 2003R. Ramezani ABSTRACT: The purpose of this investigation was to use ficin-tenderized meat and cysteine-modified soy proteins in the production of bologna and to evaluate the effect of these modifications on water-holding capacity (WHC), emulsion stability (ES), texture, and protein solubility. The effect of ficin on meat protein was also evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Results indicated that both ficin-tenderized meat and modified soy proteins substantially improved WHC, ES, and other quality factors. SDS-PAGE results showed the disappearance of several protein bands in ficin-treated meat. Solubility of meat proteins increased when ficin was used for meat tenderization. The results of this study indicated that some quality attributes of meat products can be improved by enzymatic and chemical modification of protein sources in the manufacture of meat products. [source] Effects of Transglutaminase on SDS-PAGE Patterns of Wheat, Soy, and Barley Proteins and their BlendsJOURNAL OF FOOD SCIENCE, Issue 7 2002A. Basman ABSTRACT: Transglutaminase (TG) catalyzes the formation of nondisulfide covalent crosslinks between pep-tide-bound glutaminyl residues and ,-amino groups of lysine residues in proteins. TG can be used for polymerizing proteins from 1 or more sources through formation of intermolecular crosslinks. This study investigated, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, polymers created by the action of TG on proteins of wheat, soy, barley, wheat-soy, and wheat-barley blends. Electrophoretic results showed that with increasing incubation time, the crosslinking reaction is substantially increased, with progressive decrease or disappearance of some protein monomers. Densitometric results showed that soy proteins were the best substrates of TG while barley and wheat proteins were similar in reactivity. [source] Purification and Characterization of an ,-L-Rhamnosidase from Aspergillus terreus of Interest in WinemakingJOURNAL OF FOOD SCIENCE, Issue 2 2001M.V. Gallego ABSTRACT: An enzyme with ,-L-rhamnosidase activity was purified to homogeneity from a culture filtrate of Aspergillus terreus after growth in a medium containing L-rhamnose as the sole carbon source. The biosynthesis of this enzyme was repressed by glucose. The enzyme had a molecular mass of 96 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an isoelectric point of 4.6 as determined by analytical isoelectric focusing. The pH and temperature optima for the enzyme were found to be 4.0 and 44 °C, respectively. Using p-nitrophenyl-,-L-rhamnopyranoside as a substrate, the enzyme exhibited Michaelis-Menten kinetics with KM and Vmax values of 0.17 mM and 84 U/mg, respectively. The enzyme was inhibited competitively by L-rhamnose (K1 2.5 mM). Divalent cations such as Ca2+ Mg2+ Zn2+ and Co2+ stimulated the a-L-rhamnosidase activity, whereas this was inhibited by Hg2+ and Cd2+. Ethanol (12% v/v) and glucose (21% w/v) decreased enzyme activity by approximately 20%, while this was not affected by SO2. [source] Vegetative Storage Protein with Trypsin Inhibitor Activity Occurs in Sapindus mukorassi, a Sapindaceae Deciduous TreeJOURNAL OF INTEGRATIVE PLANT BIOLOGY, Issue 4 2009Shi-Biao Liu Abstract A vegetative storage protein (VSP) with trypsin inhibitor activity in a deciduous tree, Sapindus mukorassi, was characterized by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western-blot, immuno-histochemical localization, light- and electro-microscopy, together with analysis of proteinase inhibitor activity of the purified VSP in vitro. There were two proteins with molecular masses of about 23 and 27 kDa in a relatively high content in the bark tissues of terminal branches of S. mukorassi in leafless periods. The proteins decreased markedly during young shoot development, indicating their role in seasonal nitrogen storage. Immuno-histochemical localization with the polyclonal antibodies raised against the 23 kDa protein demonstrated that the 23 kDa protein was the major component of protein inclusions in protein-storing cells. The protein inclusions were identified by protein-specific staining and should correspond to the electron-dense materials in different forms in the vacuoles of phloem parenchyma cells and phloem ray parenchyma cells under an electron microscope. So, the 23 kDa protein was a typical VSP in S. mukorassi. The 23 and 27 kDa proteins shared no immuno-relatedness, whereas the 23 kDa protein was immuno-related with the 22 kDa VSP in lychee and possessed trypsin inhibitor activity. The 23 kDa protein may confer dual functions: nitrogen storage and defense. [source] Verticase: a Fibrinolytic Enzyme Produced by Verticillium sp.JOURNAL OF INTEGRATIVE PLANT BIOLOGY, Issue 11 2007an Endophyte of Trachelospermum jasminoides Abstract Plant endophytes are among the most important resources of biologically active metabolites. Twenty-three endophyte strains residing in Trachelospermum jasminoides were cultivated in vitro with the cultures assayed for the fibrinolytic substance production. As a result, the culture of Verticillium sp. Tj33 was shown to be the most active. A fibrinolytic enzyme designated as verticase was subsequently purified from the supernatant of Verticillium sp. culture broth by a combination of DEAE-52, Sephadex G-75 and hydrophobic column chromatographies. Verticase, with its molecular mass of 31 kDa and pI of 8.5, was demonstrated to be homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing electrophoresis. Verticase is an enzyme that hydrolyzes fibrin directly without activation of plaminogen. It was stable in a broad pH range from 4 through to 11 with the optimal reaction pH value and temperature shown to be around 9,10 and 50,60 °C, respectively. The fibrinolytic activity of verticase was severely inhibited by phenylmethylsulfony fluoride, indicating that verticase was a serine protease. [source] Fluctuation of Vegetative Storage Proteins in the Seedlings of Swietenia macrophylla, Analogous to the Seasonal Changes of Those in the Shoot of the Adult TreeJOURNAL OF INTEGRATIVE PLANT BIOLOGY, Issue 3 2007Ya-Qin Han Abstract In order to identify appropriate plant materials for studying the gene expression and biological function of vegetative storage proteins (VSPs) in woody plants, the VSPs in the seedlings of Swietenia macrophylla King were investigated by using light microscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western-blotting. The seed of S. macrophylla was rich in storage proteins that accumulated in the vacuoles of cotyledon parenchyma cells in appearance of compact spherical grains. The growth and development of S. macrophylla seedlings were characterized by an obvious growth rhythm. The storage proteins in seeds disappeared during seedling growth while VSPs appeared in the stem 2 weeks after seedling leaves matured. Thereafter, the VSPs in the seedling stem almost exhausted during new shoot growth, and when the leaves of new shoot just matured, both the stem beneath the new shoot of seedlings and the stem of new shoot started to accumulate VSPs. Nitrogen application dramatically increased the level of VSPs, but had little influence on the dynamics of VSP consumption and accumulation in seedling stem. Together with these data, the fluctuation of VSPs in seedlings was very similar to that in the branches of the adult trees. In addition, seedlings are easy to be treated due to their small size. Our results suggested that S. macrophylla seedlings were suitable for investigating the biological roles of VSPs and the mechanism of nitrogen storage in trees. [source] Cloning and Characterization of a Novel Purple Acid Phosphatase Gene (MtPAP1) from Medicago truncatula Barrel MedicJOURNAL OF INTEGRATIVE PLANT BIOLOGY, Issue 2 2006Kai Xiao Abstract A novel purple acid phosphatase gene (MtPAP1) was isolated from the model legume Medicago truncatula Barrel Medic. The cDNA was 1 698 bp in length with an open reading frame (ORF) of 1 398 bp capable of encoding an N-terminal signal peptide of 23 amino acids. The transcripts of MtPAP1 were mainly detected in leaves under high-phosphate conditions, whereas under low-phosphate conditions the transcript level was reduced in leaves and increased in roots, with the strongest hybridization signal detected in roots. A chimeric gene construct fusing MtPAP1 and GFP was made in which the fusion was driven by the CaMV35S promoter. Transgenic Arabidopsis plants carrying the chimeric gene constructs showed that the fusion protein was mainly located at the apoplast based on confocal microscopic analysis, showing that MtPAP1 could be secreted to the outside of the cell directed by the signal peptide at the N-terminal. The coding region of MtPAP1 without signal peptide was inserted into the prokaryotic expression vector pET-30a (+) and overexpressed in Escherichia coli BL21 (DE3). The acid phosphatase (APase) proteins extracted from bacterial culture were found largely based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An enzyme activity assay demonstrated that the APase activity in the transformed bacteria was 3.16-fold higher than that of control. The results imply that MtPAP1 functions to improve phosphorus acquisition in plants under conditions of phosphorus (P) stress. (Managing editor: Li-Hui Zhao) [source] Alteration of argyrophilic nucleolar organizer region associated (Ag-NOR) proteins in apoptosis-induced human salivary gland cells and human oral squamous carcinoma cellsJOURNAL OF ORAL PATHOLOGY & MEDICINE, Issue 4 2001Yasuhiro Morimoto Abstract: The level of argyrophilic nucleolar organizer regions (AgNORs) and AgNOR-associated proteins (Ag-NOR proteins) varies with cell activity, including ribosomal biogenesis occurring in proliferating cells. Proteins associated with some AgNORs are detected by a specific silver staining. To investigate a possible relationship between apoptosis and the AgNORs or Ag-NOR proteins, we examined the changes of AgNORs and Ag-NOR proteins during apoptosis in a human salivary gland cell line, HSG cells, and a human oral squamous carcinoma cell line, SCC-25 cells. Apoptosis was induced by treatment of HSG and SCC-25 cells with okadaic acid. Proteins prepared from HSG and SCC-25 cells treated with varying concentrations of okadaic acid (OA) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by transferring to transfer membranes and staining for Ag-NOR proteins by modified Western blot analysis. Four major bands (110 kDa, 43 kDa, 39kDa, and 37 kDa) were detected in the proteins obtained from the control cells. The level of the 110-kDa protein decreased in the proteins prepared from OA-induced apoptotic cells; however, the reaction intensity of the other three bands was changed in apoptotic cells. An additional band of an 80-kDa Ag-NOR protein appeared and increased in the apoptotic cells. Cellular fractionation of HSG cells and SCC-25 cells was done with or without apoptotic induction. An 80-kDa Ag-NOR protein was detected in the nuclear fraction prepared from the apoptotic cells, while the 110-kDa protein decreased in the nuclear fraction of these cells. The 110-kDa Ag-NOR protein may be nucleolin (C23) as deduced from its AgNOR staining features, including molecular weight. The 80-kDa protein may be the cleavage product of the 110-kDa protein. In the cell-free apoptotic system, in which intact nuclei of HSG cells were incubated with the cytosol fraction of apoptotic HSG and SCC-25 cells, the 80-kDa Ag-NOR protein was detected in nuclei incubated with the cytosol fraction of apoptotic cells, while the level of the 110-kDa protein decreased. The changes of Ag-NOR proteins in nuclei prepared from SCC-25 cells incubated with cytosol fractions prepared from HSG and SCC-25 cells were identical to those of the HSG cells. The alternation of AgNORs in apoptosis-induced HSG cells was also examined using double staining with Hoechst 33342 and silver nitrate. Hoechst staining revealed typical apoptotic nuclei, which exhibited highly fluorescent condensed chromatin in OA-treated HSG cells. Silver grains representing AgNORs were not detected in the cells undergoing apoptosis. The dual-imposition view confirmed that AgNORs, which are visible as dots in nucleoli in the control cells, disappeared from the apoptotic nuclei of HSG cells. Our results indicate that the 110-kDa nucleolar Ag-NOR protein is associated with apoptosis and is cleaved during apoptosis. [source] Lipid,protein modifications during ascorbate-Fe2+ peroxidation of photoreceptor membranes: protective effect of melatoninJOURNAL OF PINEAL RESEARCH, Issue 3 2006Margarita H. Guajardo Abstract:, The rod outer segment (ROSg) membranes are essentially lipoprotein complexes. Rhodopsin, the major integral protein of ROSg, is surrounded by phospholipids highly enriched in docosahexaenoic acid (22:6 n3). This fluid environment plays an important role for conformational changes after photo-activation. Thus, ROSg membranes are highly susceptible to oxidative damage. Melatonin synthesized in the pineal gland, retina and other tissues is a free radical scavenger. The principal aim of this work was to study the changes in the ROSg membranes isolated from bovine retina submitted to nonenzymatic lipid peroxidation (ascorbate-Fe2+ induced), during different time intervals (0,180 min). Oxidative stress was monitored by increase in the chemiluminescence and fatty acid alterations. In addition we studied the in vitro protective effect of 5 mm melatonin. The total cpm originated from light emission (chemiluminescence) was found to be lower in those membranes incubated in the presence of melatonin. The docosahexaenoic acid content decreased considerably when the membranes were exposed to oxidative damage. This reduction was from 35.5 ± 2.9% in the native membranes to 12.65 ± 1.86% in those peroxidized during 180 min. In the presence of 5 mm melatonin we observed a content preservation of 22:6 n3 (23.85 ± 2.77%) at the same time of peroxidation. Simultaneously the alterations of membrane proteins under oxidative stress were studied using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Loss of protein sulfhydryl groups and increased incorporation of carbonyl groups were utilized as biomarkers of protein oxidation. In membranes exposed to Fe2+ -ascorbate, we observed a decrease of protein thiols from 50.9 ± 3.38 in native membranes to 1.72 ± 2.81 nmol/mg of protein after 180 min of lipid peroxidation associated with increased incorporation of carbonyl groups into proteins from 7.20 ± 2.50 to 12.50 ± 1.12 nmol/mg of protein. In the SDS-PAGE we observed a decrease in the content of all the proteins, mainly rhodopsin, as a consequence of peroxidation. Melatonin, prevent both lipid peroxidation and protein oxidation. [source] | ||||||||||||||||