Showing papers on "Gel electrophoresis published in 2023"

An efficient in‐gel digestion method on small amounts of protein sample from large intact gel pieces

Abstract: In-gel digestion is an essential method, but there has been concern that a large ratio of gel volume to protein volume will reduce digestion efficiency and make it difficult to recover peptides after digestion. Few methods for handling large gel pieces have been established. Therefore, we optimized the in-gel digestion method to handle large intact gel pieces containing small amounts of proteins. As a model for large intact gel pieces containing small amounts of protein, gel pieces of 7 × 10 mm in size obtained by short-range sodium dodecyl-sulfate polyacrylamide gel electrophoresis of 150 ng of total protein extracted from Arabidopsis cells were used. The use of a positive pressure manifold and 96-well filter enabled rapid and uniform liquid exchange and gel drying, even when handling multiple gel pieces, and reduced the labor required for a liquid exchange. This facilitated the study of conditions using multiple gels and enabled a detailed study of the enzyme solution permeation and elution methods. By using the optimized method, we were able to establish a method for recovering small amounts of protein from large intact gel pieces without compromising reproducibility or recovery rate.

Preparative Electrophoresis for HDL Particle Size Separation and Intact-Mass Apolipoprotein Proteoform Analysis

Abstract: The most abundant proteins on high-density lipoproteins (HDLs), apolipoproteins A-I (APOA1) and A-II (APOA2), are determinants of HDL function with 15 and 9 proteoforms (chemical-structure variants), respectively. The relative abundance of these proteoforms in human serum is associated with HDL cholesterol efflux capacity, and cholesterol content. However, the association between proteoform concentrations and HDL size is unknown. We employed a novel native-gel electrophoresis technique, clear native gel-eluted liquid fraction entrapment electrophoresis (CN-GELFrEE) paired with mass spectrometry of intact proteins to investigate this association. Pooled serum was fractionated using acrylamide gels of lengths 8 and 25 cm. Western blotting determined molecular diameter and intact-mass spectrometry determined proteoform profiles of each fraction. The 8- and 25 cm experiments generated 19 and 36 differently sized HDL fractions, respectively. The proteoform distribution varied across size. Fatty-acylated APOA1 proteoforms were associated with larger HDL sizes (Pearson's R = 0.94, p = 4 × 10-7) and were approximately four times more abundant in particles larger than 9.6 nm than in total serum; HDL-unbound APOA1 was acylation-free and contained the pro-peptide proAPOA1. APOA2 proteoform abundance was similar across HDL sizes. Our results establish CN-GELFrEE as an effective lipid-particle separation technique and suggest that acylated proteoforms of APOA1 are associated with larger HDL particles.

IgE cross-inhibition between Ara h 1 and Ara h 2 is explained by complex formation of both major peanut allergens.

Abstract: Surprisingly, IgE cross-reactivity between the major peanut allergens Ara h 1, 2, and 3 has been reported despite very low sequence identities.We investigated the unexpected cross-reactivity between peanut major allergens.Cross-contamination of purified natural Ara h 1, 2, 3, and 6 was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blot test, liquid chromatography-tandem mass spectrometry (LC-MS/MS), and sandwich enzyme-linked immunosorbent assay (ELISA). IgE cross-reactivity was studied with sera of peanut-allergic patients (n = 43) by ELISA and ImmunoCAP inhibition using both intact natural and recombinant allergens and synthetic peptides representing postulated Ara h 1 and Ara h 2 cross-reactive epitopes.Both purified nAra h 1 and nAra h 3 were demonstrated to contain small but significant amounts of Ara h 2 and Ara h 6 (<1%) by sandwich ELISA, SDS-PAGE/Western blot analysis, and LC-MS/MS. IgE cross-inhibition between both 2S albumins and Ara h 1 and Ara h 3 was only observed when using natural purified allergens, not recombinant allergens or synthetic peptides. Apparent cross-reactivity was lost when purified nAra h 1 was pretreated under reducing conditions, suggesting that Ara h 2 and Ara h 6 contaminations may be covalently bound to Ara h 1 via disulfide interactions.True cross-reactivity of both peanut 2S albumins with Ara h 1 and Ara h 3 could not be demonstrated. Instead, cross-contamination with small quantities was shown to be sufficient to cause significant cross-inhibition that can be misinterpreted as molecular cross-reactivity. Diagnostic tests using purified nAra h 1 and nAra h 3 can overestimate their importance as major allergens as a result of the presence of contaminating 2S albumins, making recombinant Ara h 1 and Ara h 3 a preferred alternative.

Visualization of Yeast Chromosomes Using Clamped Homogeneous Electric Field (CHEF) Electrophoresis v1

Abstract: I inherited this protocol while working as a postdoctoral researcher in the laboratory of Dr. Stewart Scherer in the 1990s. Literature that influenced protocol development is listed in the References section. This protocol describes how to isolate chromosome-sized DNA from yeast cells. The yeast cells are encased in agarose, then the cell is digested away. The result is unsheared chromosomes in an agarose plug that can be loaded onto electrophoresis gels or subjected to additional analysis such as digestion with restriction enzymes. The protocol details two sets of electrophoresis conditions for separating the chromosomes using a BioRad CHEF-DR III variable angle electrophoresis system. Because the protocol was developed for work with Candida albicans, the conditions are most useful for chromosomes in the size range of approximately 0.5 to 5 Mb. The "short protocol" (Condition 1) separates the smaller C. albicans chromosomes (approximately 1 Mb) without running them off the bottom of the gel. The "long protocol" (Condition 2) separates the larger chromosomes (approximately 3 Mb) with approximately 0.1 Mb resolution but risks running smaller DNA molecules off the gel. We typically run samples using both electrophoresis protocols and combine the resulting information to understand the karyotype.

Effect of Some Herbicides on the Total Seed Proteins Patterns of Maize and Sorghum Using Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS–PAGE)

Abstract: Although herbicides are important in controlling weeds in corn and sorghum and increasing their yields, some of these chemicals have severe side effects on the environmental components. These negative effects may be extended to crop seed quality. This investigation was carried out with

Grain Characteristics, Moisture, and Specific Peptides Produced by Ustilaginoidea virens Contribute to False Smut Disease in Rice (Oryza sativa L.)

Abstract: The fungus Ustilaginoidea virens, the causative agent of false smut in rice (Oryza sativa L.), is responsible for one of the severe grain diseases that lead to significant losses worldwide. In this research, microscopic and proteomic analyses were performed by comparing U. virens infected and non-infected grains of the susceptible and resistant rice varieties to provide insights into the molecular and ultrastructural factors involved in false smut formation. Prominent differentially expressed peptide bands and spots were detected due to false smut formation as revealed by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional gel electrophoresis (2-DE) SDS-PAGE profiles and were identified using liquid chromatography-mass spectrometry (LC-MS/MS). The proteins identified from the resistant grains were involved in diverse biological processes such as cell redox homeostasis, energy, stress tolerance, enzymatic activities, and metabolic pathways. It was found that U. virens produces diverse degrading enzymes such as β-1, 3-endoglucanase, subtilisin-like protease, putative nuclease S1, transaldolase, putative palmitoyl-protein thioesterase, adenosine kinase, and DNase 1 that could discretely alter the host morphophysiology resulting in false smut. The fungus also produced superoxide dismutase, small secreted proteins, and peroxidases during the smut formation. This study revealed that the dimension of rice grain spikes, their elemental composition, moisture content, and the specific peptides produced by the grains and the fungi U. virens play a vital role in the formation of false smut.

Methylsensitive comet assay: analysis of dna methylation level in glioblastoma t98g cell line

Abstract: Methylsensitive comet electrophoresis is based on the assessment of the level of DNA migration from individual lysed cells after treatment with methylsensitive restriction enzymes. Using model human lymphocytes, the optimal combination of restriction intensity and electrophoresis time was selected and a new approach for evaluating the relative level of DNA methylation was proposed. It was established that in the cells of the T98G culture, which are actively proliferating, the level of methylation is higher than in cells arrested at the G1 phase of the cell cycle. At the same time, the level of DNA methylation in G1 cells of the T98G line is significantly lower compared to lymphocytes.

SDS-PAGE-Based Quantitative Assay of Hemolymph Proteins in Honeybees: Progress and Prospects for Field Application

Abstract: In human and veterinary medicine, serum proteins are considered to be useful biomarkers for assessing the health and nutritional status of the organism. Honeybee hemolymph has a unique proteome that could represent a source of valuable biomarkers. Therefore, the aims of this study were to separate and identify the most abundant proteins in the hemolymph of worker honeybees to suggest a panel of these proteins that could represent useful biomarkers for assessing the nutritional and health status of the colonies and, finally, to analyze them in different periods of the year. Four apiaries were selected in the province of Bologna, and the bees were analyzed in April, May, July, and November. Thirty specimens from three hives of each apiary were sampled and their hemolymph was collected. The most represented bands obtained after 1D sodium-dodecyl-sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were cut from the gel, and the proteins were identified using an LC-ESI-Q-MS/MS System. A total of twelve proteins were unmistakably identified; the two most abundant proteins were apolipophorin and vitellogenin, which are known biomarkers of bee trophic and health status. The two other proteins identified were transferrin and hexamerin 70a, the first being involved in iron homeostasis and the second being a storage protein. Most of these proteins showed an increase from April to November, mirroring the physiological changes of honeybees during the productive season. The current study suggests a panel of biomarkers from honeybee hemolymph worth testing under different physiological and pathological field conditions.

Characterization of acrosin and acrosin binding protein as novel CRISP2 interacting proteins in boar spermatozoa.

Abstract: BACKGROUND Previously we reported that cysteine rich secretory protein 2 (CRISP2) is involved in high molecular weight complexes in boar spermatozoa. These CRISP2 protein complexes are formed at the last phase of sperm formation in the testis and play a role in sperm shaping and functioning. OBJECTIVES To identify CRISP2 interacting partners whereby their interactions were investigated under different conditions, namely non-capacitating conditions, after the induction of in vitro sperm capacitation and subsequently during an ionophore A23187 induced acrosome reaction. MATERIALS AND METHODS The incubated pig sperm samples were subjected to protein extraction. Extracted proteins were subjected to blue native gel electrophoresis and native immunoblots. Immunoreactive gel bands from were excised and subjected to liquid chromatography mass spectrometry (LC-MS) analysis for protein identification. Protein extracts were also subjected to CRISP2 immunoprecipitation and analyzed by LC-MS for protein identification. The most prominent CRISP2 interacting proteins that appeared in both independent LC-MS analyses were studied with a functional in situ proximity interaction assay to validate their property to interact with CRISP2 in pig sperm. RESULTS Blue native gel electrophoresis and native immunoblots revealed that CRISP2 was present within a ∼150 kDa protein complex under all three conditions. Interrogation of CRISP2-immunoreactive bands from blue native gels as well as CRISP2 immunoprecipitated products using mass spectrometry consistently revealed that, beyond CRISP2, acrosin and acrosin binding protein (ACRBP) were among the most abundant interacting proteins and did interact under all three conditions. Co-immunoprecipitation and immunoblotting indicated that CRISP2 interacted with pro-acrosin (∼53 kDa) and ACRBP under all three conditions and additionally to acrosin (∼35 kDa) after capacitation and the acrosome reaction. The colocalization of these interacting proteins with CRISP2 was assessed via in situ proximity ligation assays. The colocalization signal of CRISP2 and acrosin in the acrosome seemed dispersed after capacitation but was consistently present in the sperm tail under all conditions. The fluorescent foci of CRISP2 and ACRBP colocalization appeared to be redistributed within the sperm head from the anterior acrosome to the post acrosomal sheath region upon capacitation. DISCUSSION AND CONCLUSION These results suggest that CRISP2 may act as a scaffold for protein complex formation and dissociation to ensure the correct positioning of proteins required for the acrosome reaction and zona pellucida penetration. This article is protected by copyright. All rights reserved.

Bacterial Leakage Evaluation Through DNA-DNA Checkerboard Hybridization Technique in Morse Taper Implant-Abutment Connections: An In Vitro Study.

Abstract: Purpose: The objective of this in vitro study was to evaluate the activity of local gel containing metronidazole (MN) in the leakage area, which was analyzed by the DNA-DNA checkerboard hybridization method. Materials and Methods: Thirty-six sets of Morse taper/mini-pillar implants were used in this study. These implants were equally divided into the following three groups: MN gel (test group), no MN gel (negative test group), and no gel (control). The gel was prepared with metronidazole (15%). Unstimulated saliva samples were collected, transferred to a Falcon tube, and stored at 37°C. The sets were partially immersed in microtubes containing 300 μL of saliva and were incubated at 37°C ± 1°C for 7 days. Microbial infiltration was evaluated (37 bacterial species and 5 species of Candida). The results were analyzed with Wald-Type, ANOVA, and multiple comparisons analysis between groups. Results: After comparing the quantity of microorganisms, both gel-treated groups (no MN gel and MN gel) had more significant microorganism presence than the control group (P < .001), and no significant result was found between the no MN gel and MN gel groups (P > .05). Regarding the bacteria found, the most common were Aggregatibacter actinomycetemcomitans, Prevotella melaninogenica, Bacteroides fragilis, and Candida tropicalis. Conclusion: Within the limitations of this study, it was concluded that the gel containing metronidazole used in this study was not effective in preventing the infiltration of microorganisms through the Morse taper implant-abutment interface.

Supplementary Table 2 from Vemurafenib Resistance Signature by Proteome Analysis Offers New Strategies and Rational Therapeutic Concepts

Abstract: <p>Comparison of protein expression in A375 and M24met by 2D-gel electrophoresis. Corresponding proteins which are assigned in the 2-D gel in Figure 2. Accession numbers are from the Uniprot database. Vol indicates the volume identified by 2D-gel electrophoresis n.d =not determined</p>

Electrophoresis of RNA

Abstract: Electrophoresis is a chromatography technique by which a mixture of charged molecules is separated according to size when placed in an electric field. The accurate determination of the size of RNA species is just as important as a deduction of the molecular weight of any other macromolecules subjected to electrophoresis. This is accomplished by comparing the electrophoresis of an RNA sample against molecular weight standards. The successful electrophoresis of RNA is accomplished in two parts: RNA is denatured prior to loading the gel; and during the electrophoresis, conditions that support and maintain the denatured state are established. These steps will ensure that like species of RNA comigrate, and the best possible resolution is achieved when gels are poured thinly and run at low voltage. The choice of denaturing system and gel matrix is determined primarily by the size range of the RNAs to be separated and the most commonly used RNA denaturants for agarose gel electrophoresis are formaldehyde and urea. Several gel-staining techniques are used for the visualization of nucleic acids in agarose gels such as ethidium bromide, SYBR green, SYBR gold, SYBR safe, GelStar, silver staining, acridine orange, and methylene blue. The proper maintenance and use of electrophoresis gel boxes and power supplies are of utmost importance for the safety of all.

Assessment of the effect of cell transplantation on DNA repair in rat hepatocytes exposed to carbon tetrachloride (CCl4) by DNA comet assay

Abstract: Introduction. The effect of carbon tetrachloride (freon-10, asordin, hladon-10) is an organochlorine compound with the chemical formula CCl4 and the subsequent transplantation of fetal liver cells (FLC) on DNA degradation and repair in rat hepatocytes by means of alkaline single-cell gel electrophoresis (DNA comet assay) was assessed. Material and methods. Acute toxic damage to the rat liver was simulated by a single oral administration to female Wistar rats of CCl4 in an oil solution at a dose of 3000 mg/kg. As a protective agent, a suspension of FLC of E19 rat fetuses was used. Quantitative assessment of the degree of damage to the nuclear DNA of liver cells was performed by DNA comet assay on days 1, 3, 5, 7 and 16 of the experiment. Results. Intravenous injections of fetal liver cells 6 h after exposure to CCl4 induces DNA repair processes in rat hepatocytes in 5-7 days and led to a decrease in the intensity of nuclear DNA damage. The trend toward a decrease in the number of undamaged hepatocytes continued on the 16th day of the experiment, and, therewith, the enhancement of reparative processes after FLC injection revealed itself in in a significant decrease in the number of hepatocytes with a high intensity of nuclear DNA damage. Limitations. To prevent unwanted death of animals in the group, studies were limited to a dose of 3000 mg/kg of CCl4 in oil solution. Conclusion. The method of alkaline single-cell gel electrophoresis (DNA comet assay) allowed quantitative assessment of the degrees of genome damage and repair. The obtained positive results suggest that FLC exert a protective effect of the structure of the DNA of rat liver role after acute exposure to CCl4.

Figure S4 from Induced Telomere Damage to Treat Telomerase Expressing Therapy-Resistant Pediatric Brain Tumors

Abstract: <p>A, cell growth kinetics of HFF, HFF+hTERT, Saos2, and U2OS cells. B, relative cell growth. C, percent viability of HFF+hTERT and U2OS cells treated with 3 µM 6-thio-dG for 3 days followed by 10 days post-drug removal. D, agarose gel electrophoresis (0.7%) of genomic DNA extracted from HFF+hTERT (lanes 2, 3), U2OS (lanes 4, 5), and HFF (lanes 6, 7) cells treated with DMSO or 3 µM 6-thio-dG treated for 3 days. Lane 1, molecular weight marker, lambda phage DNA/HindIII, is indicated. Gel was run two independent times.</p>

Plasmid and Total Protein Analyses of Extended Spectrum Beta Lactamase Producing Bacteria from Periplaneta americana (Coackroaches)

Abstract: Background: The importance of plasmid-encoded Extended Spectrum Beta Lactamase (ESBL) organisms in the dissemination of multi-drug resistance and complicated infections are contributory factors to treatment failure. Despite these, there is a paucity of information regarding plasmid-borne associated infection from primary sources including cockroaches while studies correlating total protein and plasmid-encoded ESBL are also limited especially in cockroaches. The objective of this study was therefore aimed at determining the presence of plasmid in the ESBL organisms from cockroaches as well as their total protein profiles. Materials and Methods: Bacterial isolation, characterization and molecular identification were carried out in our previous work following standard recommended techniques. These molecularly identified organisms which were delineated into twenty-two (22) were further subjected to plasmid and total protein analyses using alkaline lysis and Sodium Dodecyl Sulfate Poly Acrylamide Gel Electrophoresis (SDS-PAGE) respectively. Results: Results obtained identifies 18(81.8%) of the ESBL isolates to be harbouring plasmids of different molecular weights (2-21.5kbp). The total protein profile of the plasmid-encoded ESBL and the non-plasmid encoded ESBL reveals differential protein expression patterns except for two non- plasmid encoded ESBL isolates that have similar patterns as that of plasmid-encoded ESBL organisms (isolates 2 and 3). Conclusion: There is a need to monitor the primary sources of infection in the epidemiological distribution of resistance. Improving environmental sanitation through proper disinfection of carrier insects may be a means to curtailing such spread.

JoVE Video Dataset

Abstract: The brain secretome consists of proteins either actively secreted or shed from the cell surface by proteolytic cleavage in the extracellular matrix of the nervous system. These proteins include growth factor receptors and transmembrane proteins, among others, covering a broad spectrum of roles in the development and normal functioning of the central nervous system. The current procedure to extract the secretome from cerebrospinal fluid is complicated and time-consuming, and it is difficult to isolate these proteins from experimental animal brains. In this study, we present a novel protocol for isolating the brain secretome from mouse brain slice cultures. First, the brains were isolated, sliced, and cultured ex vivo. The culture medium was then filtered and concentrated for isolating proteins by centrifugation after a few days. Finally, the isolated proteins were resolved using sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently probed for purity characterization by western blot. This isolation procedure of the brain secretome from ex vivo brain slice cultures can be used to investigate the effects of the secretome on a variety of neurodevelopmental diseases, such as autism spectrum disorders.

Comparison of collagens extracted from swim bladder and bovine Achilles tendon

Abstract: Collagen is a type of natural biopolymer material, which is widely used in tissue engineering and medicine owing to its exceptional properties such as biodegradability, biocompatibility, hemostatic properties, and low immunogenicity. Collagens from different sources can differ in type, structure, and function. In this study, collagen was extracted from swim bladder and bovine Achilles tendon by acid-enzyme binding method at low temperature. UV spectrum, Fourier transform infrared spectrum, sodium dodecyl sulfate–polyacrylamide gel electrophoresis, scanning electron microscope, and differential scanning calorimetry were used to characterize these two collagens. The blood compatibility and cytotoxicity of the two kinds of collagen were studied.The results showed that the collagens from the two sources belong to the characteristics of type I collagen and had biological safety. Their differences in structure and thermal stability can provide a theoretical basis for the selection of collagen in practical application.

Characterization of Low-Molecular-Weight Collagen from Korean Native Chicken Feet Hydrolyzed Using Alcalase

Abstract: The aims of this study were to optimize the preparation of low-molecular-weight collagen using a proteolytic enzyme (alcalase) derived from the feet of Korean native chickens, and to characterize the process of collagen hydrolysis. Foreign bodies from chicken feet were removed using ultrasonication at 28 kHz with 1.36 kW for more than 25 min. The hydrolytic pattern and molecular weight distribution of enzyme-treated collagen from chicken feet were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high-performance liquid chromatography, respectively. Ideally, chicken feet should be treated at 100°C for 8 h to obtain a high collagen content using hot water extraction. The collagen content of the chicken foot extract was 13.9 g/100 g, and the proportion of low-molecular-weight collagen increased with increasing proteolytic enzyme concentration and reaction time. When treated with 1% alcalase, the average molecular weight of collagen decreased rapidly to 4,929 Da within 5 h and thereafter decreased at a slower rate, reaching 4,916 Da after 7 h. Size exclusion chromatography revealed that low-molecular-weight collagen peptides of approximately 1,000–5,000 Da were obtained after hydrolysis with 1% alcalase for 1 h.

Design, Expression and Characterization of Lactiscin—a Novel Broad-Spectrum Peptidic Bacteriocin

Abstract: Abstract Bacteria-derived antimicrobial peptides known as peptidic bacteriocins offer a promising alternative to traditional antibiotics in the face of the emergence of multidrug-resistant bacteria. Here, a nucleotide sequence of the gene encoding Lactococcus lactis- derived peptidic bacteriocin designated as lactiscin selectively identified from the GenBank® database was synthesized with an added 6⋅His sequence and cloned into Escherichia coli . Upon low-temperature expression at 16°C, the His-tagged peptide could be produced in both soluble form and insoluble inclusions. Efficient purification of the soluble His-tagged peptide was achieved via immobilized-Ni 2+ affinity chromatography (IMAC) and its estimated molecular mass of ~ 13.4 kDa was determined by tricine-sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified peptide was highly active against both Gram-positive and Gram-negative bacteria as it exhibited a minimal inhibitory concentration of 0.45 mg/mL, 0.15 mg/mL, 0.35 mg/mL and 0.45 mg/mL against. Escherichia coli, Vibrio parachemolyticus, Staphylococcus aureus and Micrococcus luteus , respectively. In addition, the lactiscin peptide still retained antimicrobial activity over a pH range of 3.0–12.0 and heat stability of 100°C for 30 minutes. A membrane integrity study revealed that this peptidic bacteriocin was able to induce E. coli membrane permeabilization in a concentration-dependent manner, albeit it showed a negligible toxic effect on erythrocytic cells. Gel retardation assay demonstrated that the lactiscin bacteriocin could suppress the migration of genomic DNA extracted from pathogenic bacteria, suggesting the presence of bacteriocin-responsive binding genomic. Our findings of lactiscin—a novel broad-spectrum bacteriocin would be a valuable additive for the application of food industry as a potential bio-preservative.

Extraction & Purification of Plasmid from Some Spices of E. coli in Different Ways

Abstract: This study involved the use of multiple methods to separate plasmids from bacterial cell DNA for some isolates of pathogenic E. coli through several steps, starting with the analysis of the bacterial cell using lysozymes to remove the outer wall, followed by centrifugation to isolate plasmids found in the solution from the rest of the proteins and other forms of DNA. Many sequential methods were used to separate plasmids. The first method used was the basal denaturation sodium hydroxide-based, which led to the denaturation of the chromosomal DNA without affecting the plasmid DNA, followed by the addition of sodium acetate, which led to the preservation of the shape and structure of the plasmid DNA. Second, using cesium chloride gradient density to isolate the protein cell components and the rest of the DNA forms. The different densities of these components led to the appearance of sequential bundles depending on their different molecular weights. Ethidium bromide, which gave the plasmid bundles a fluorescent dye, was added using ultraviolet rays. The last purification method was using the boiling method using a water bath. Plasmid samples extracted from the previous methods were taken to perform the purification and separation process using the high electrophoresis method. Akarose gel was used to separate the high molecular weight protein fragments. Standard proteins and plasmids were migrated to determine the volumes of purified plasmids.

In vitro antibacterial effect of three intra canal medicaments with or without electrophoresis on Enterocomlus faecalis at different dentin depths

Abstract: Abstract Objective: Elimination of microorganisms from the root canal system plays a fundamental role in successful endodontic treatment. This study aimed to assess the antibacterial effect of three intra canal medicaments with/without electrophoresis on Enterococcus faecalis (E faecalis) at different dentin depths. Materials and methods: This study evaluated 81 sound extracted single-rooted, single-canal human teeth. After root canal preparation, the teeth were sterilized, and E faecalis was inoculated into the root canals for 2 weeks. The teeth were then randomly divided into 9 groups (n=9) of (I) calcium hydroxide (CH) without electrophoresis, (II) CH with electrophoresis, (III) Cupral without electrophoresis, (IV) Cupral with electrophoresis, (V) silver nanoparticles (AgNPs) with CH without electrophoresis, (VI) AgNPs with CH and with electrophoresis, (VII) electrophoresis alone, (VII) negative control group, and (IX) positive control group. The medicaments were applied in the root canals and the teeth were incubated for 4 weeks. In electrophoresis groups, 15 mA electric current was applied for 6 min, and the teeth were then incubated. Then, dentin chips were collected from three different dentin depths by peeso reamers, and cultured on agar and thioglycolate culture media. Results: No significant difference was noted in antimicrobial activity of tested materials at different depths (P>0.05). A significant difference was noted in elimination of microbial biofilm between groups with and without electrophoresis at depths 2 and 3 (P<0.05). Conclusion: Electrophoresis resulted in greater penetration of medicaments into dentinal tubules, and enhanced their antimicrobial efficacy in deeper areas of dentinal tubules. Clinical Relevance: Increasing the penetration of intra canal medicaments into the dentin increases their antimicrobial effect.

Capillary Gel Electrophoresis of Proteins: Historical overview and recent advances

Abstract: This review summarizes the fundamental principles, basic methodologies, strength and weaknesses of capillary gel electrophoresis of proteins by providing both a short historical overview and highlighting new developments and applications in biopharmaceutical, biomedical as well as food and agriculture fields. The subsets of the method including native capillary gel electrophoresis, SDS capillary gel electrophoresis, capillary gel isoelectric focusing, capillary gel isotachophoresis and capillary affinity gel electrophoresis of proteins are all critically reviewed. Relevant protein labeling techniques are also addressed.

Use of the Single Cell Gel Electrophoresis Assay for the Detection of DNA-protective Dietary Factors: Results of Human Intervention Studies.

Abstract: The single cell gel electrophoresis technique is based on the measurement of DNA migration in an electric field and enables to investigate via determination of DNA-damage the impact of foods and their constituents on the genetic stability. DNA-damage leads to adverse effects including cancer, neurodegenerative disorders and infertility. In the last 25 years approximately 90 human intervention trials have been published in which DNA-damage, formation of oxidized bases, alterations of the sensitivity towards reactive oxygen species and chemicals and of repair functions were investigated with this technique. In approximately 50% of the studies protective effects were observed. Pronounced protection was found with certain plant foods (spinach, kiwi fruits, onions), coffee, green tea, honey and olive oil. Also diets with increased contents of vegetables caused positive effects. Small amounts of certain phenolics (gallic acid, xanthohumol) prevented oxidative damage of DNA; with antioxidant vitamins and cholecalciferol protective effects were only detected after intake of doses that exceed the recommended daily uptake values. The evaluation of the quality of the studies showed that many have methodological shortcomings (lack of controls, no calibration of repair enzymes, inadequate control of the compliance and statistical analyses) which should be avoided in future investigations.

Protease inhibitory activity profile of Indonesia wild swamp eel (Monopterus albus)

Abstract: A protease inhibitor is a compound that potentially could inhibit the activity of a protease or some protease enzymes. It has a significant role in fish processing to prevent quality deterioration. The study aimed to investigate the inhibitory activity of wild swamp eel (Monopterus albus) plasma fractionated with ethanol to papain enzyme. The parameters analyzed were protein content, inhibitory activity to papain enzyme, and sodium dodecyl sulphate-polyacrylamide gel electrophoresis to demonstrate the protein profile. The result showed that the amount of protein in the plasma fractionated with ethanol was decreased. The effectiveness in inhibiting papain enzyme was increased when compared to that of the crude plasma, from 65 to 128.56% per mg protein. Analysis by sodium dodecyl sulfatepolyacrylamide gel electrophoresis demonstrated that the protein complex alpha-2- macroglobulin in wild swamp eel plasma consists of two different subunits of 120 kDa and 110 kDa. This study suggested that plasma fractionated with ethanol showed inhibitory activity to papain enzyme greater than that of the crude plasma.

Synergistic effect of ultrahigh pressure and allicin on gel properties, flavor characteristics, and myosin structure of the obturator muscle of the scallop (Patinopecten yessoensis).

Abstract: The synergistic effects of the combination of ultrahigh pressure (UHP) with allicin on the gel properties, flavor characteristics, and myosin structure of scallops were investigated. The results indicated that chewiness reached maximum, uniform, and dense microstructures at B-300 MPa, and scallops with favorable gel properties. In addition, the electronic nose and tongue could clearly distinguish the olfactory and gustatory properties of scallops, and the interaction of UHP and allicin increased the variety of volatile compounds in scallops, which mainly included 1-hydroxy-2-propanone, 1-hexenal, 2-butanone-D, and 1-octen-3-ol. The main performance was fruit aroma and a plantlike aroma and mushroomlike odor. UHP and allicin changed the microenvironment of tryptophan residues, and allicin formed larger aggregates by forming disulfides with myosin. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis results could show that myosin had low degradation in B-300 MPa. Thus, comprehensively viewed, UHP and allicin play a role in gel formation of myosin from obturator muscle at 300 MPa, whereas allicin and myosin form disulfides as the main factor of myosin gelation. PRACTICAL APPLICATION: To enhance the diversity of scallop preparation methods and improve the quality of the obtained product, UHP and allicin treatment result in scallops with satisfactory chewiness and flavor, which provides application prospects for scallop processing.