Displaying publications 1 - 20 of 226 in total

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  1. Fulltext Whole And Parotid Saliva - Protein Profiles As Separated On 5-20% SDS-Polyacrylamide Gradient gel Electrophoresis And Using Maldi-tof Mass Spectrometry
    Huq, N.L., DeAngelis, A., Rahim, Z.H.A., Ung, M., Lucas, J., Cross, K.J., et al.
    Ann Dent, 2004;11(1):-.
    MyJurnal
    The aim was to examine the protein profiles of whole and parotid saliva using Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) and MALDI-TOF mass spectrometry. The banding patterns of proteins exhibited by the unstimulated whole saliva samples on the gel remained quite constant but the intensity of the protein bands were slightly different from one sample to another. Comparison of the protein profiles of unstimulated whole saliva and stimulated parotid saliva showed almost similar banding pattern. The exception is the presence of a pink protein band in the 65-67 kD region in the stimulated parotid saliva samples which was also observed in the unstimulated whole saliva sample contributed by a cerebral palsy patient. Analysis of the saliva samples using MALDI-TOF mass spectrometry also revealed that the stimulated parotid saliva samples exhibited some peaks that were in the same region as those for the unstimulated whole saliva sample of the cerebral palsy subject. This may imply that there is ineffective control of the parotid secretion in cerebral palsy subject under unstimulated condition. The SDS-PAGE and MALDI-TOF analyses may provide more information on the profiles of the salivary proteins which could be beneficial in the diagnosis of salivary gland dysfunction.
    Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
  2. Virus-like particles of Macrobrachium rosenbergii nodavirus produced in bacteria
    Goh ZH, Tan SG, Bhassu S, Tan WS
    J Virol Methods, 2011 Jul;175(1):74-9.
    PMID: 21536072 DOI: 10.1016/j.jviromet.2011.04.021
    Macrobrachium rosenbergii nodavirus (MrNv) infects giant freshwater prawns and causes white tail disease (WTD). The coding region of the capsid protein of MrNv was amplified with RT-PCR and cloned into the pTrcHis2-TOPO vector. The recombinant plasmid was introduced into Escherichia coli and protein expression was induced with IPTG. SDS-PAGE showed that the recombinant protein containing the His-tag and myc epitope has a molecular mass of about 46 kDa and it was detected by the anti-His antibody in Western blotting. The protein was purified using immobilized metal affinity chromatography (IMAC) and transmission electron microscopic analysis revealed that the recombinant protein assembled into virus-like particles (VLPs) with a diameter of about 30±3 nm. The size of the particles was confirmed by dynamic light scattering. Nucleic acids were extracted from the VLPs and treatment with nucleases showed that they were mainly RNA molecules. This is the first report describing the production of MrNv capsid protein in bacteria and its assembly into VLPs.
    Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
  3. Use of principal component analysis for differentiation of gelatine sources based on polypeptide molecular weights
    Nur Azira T, Che Man YB, Raja Mohd Hafidz RN, Aina MA, Amin I
    Food Chem, 2014 May 15;151:286-92.
    PMID: 24423534 DOI: 10.1016/j.foodchem.2013.11.066
    The study was aimed to differentiate between porcine and bovine gelatines in adulterated samples by utilising sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) combined with principal component analysis (PCA). The distinct polypeptide patterns of 6 porcine type A and 6 bovine type B gelatines at molecular weight ranged from 50 to 220 kDa were studied. Experimental samples of raw gelatine were prepared by adding porcine gelatine in a proportion ranging from 5% to 50% (v/v) to bovine gelatine and vice versa. The method used was able to detect 5% porcine gelatine added to the bovine gelatine. There were no differences in the electrophoretic profiles of the jelly samples when the proteins were extracted with an acetone precipitation method. The simple approach employing SDS-PAGE and PCA reported in this paper may provide a useful tool for food authenticity issues concerning gelatine.
    Matched MeSH terms: Electrophoresis, Polyacrylamide Gel/methods*
  4. Two-dimensional polyacrylamide gel electrophoresis of Bali bull (Bos javanicus) seminal plasma proteins and their relationship with semen quality
    Sarsaifi K, Haron AW, Vejayan J, Yusoff R, Hani H, Omar MA, et al.
    Theriogenology, 2015 Oct 1;84(6):956-68.
    PMID: 26119476 DOI: 10.1016/j.theriogenology.2015.05.035
    The present study evaluated the relationship between Bali bull (Bos javanicus) seminal plasma proteins and different semen quality parameters. Semen samples from 10 mature Bali bulls were evaluated for conventional semen parameters (general motility, viability, and normal morphology), sperm functionality (acrosome reaction, sperm penetration rate, sperm penetration index), sperm kinetics (computer-assisted semen analysis parameters such as sperm velocity), and sperm morphology (acrosome and membrane integrity). Frozen-thawed semen with higher sperm motility, viability, acrosome integrity, and membrane integrity (P < 0.05) are consistently higher in acrosome reaction and sperm penetration assay. Three bulls showed the highest, four bulls displayed the medium, and the remaining three bulls showed the lowest for all sperm parameters and SPA. The proteome maps of seminal plasma from high-quality and low-quality Bali bulls were also established. Seminal plasma of both high-quality and low-quality Bali bulls was subjected to two-dimensional SDS-PAGE with isoelectric point ranged from 3 to 10 and molecular weight from 10 to 250 kDa. Approximately 116 spots were detected with Blue Silver stain, and of these spots, 29 were selected and identified by MALDI-TOF/TOF-MS/MS. A majority of the proteins visualized in the seminal plasma two-dimensional maps was successfully identified. An essential group of the identified spots represented binder of sperm 1 (BSP1), clusterin, spermadhesin, tissue inhibitor of metalloproteinases 2 (TIMP-2), and phospholipase A2 (PLA2). Other proteins found in high abundance included seminal ribonuclease, serum albumin, cationic trypsin, and peptide similar to β2 microglobulin. Thus, a reference map of Bali bull seminal plasma proteins has been generated for the very first time and can be used to relate protein pattern changes to physiopathologic events that may influence Bali bull reproductive performance.
    Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
  5. Toxoplasma gondii: serological characterization and immunogenicity of recombinant surface antigen 2 (SAG2) expressed in the yeast Pichia pastoris
    Lau YL, Fong MY
    Exp Parasitol, 2008 Jul;119(3):373-8.
    PMID: 18457835 DOI: 10.1016/j.exppara.2008.03.016
    The full length surface antigen 2 (SAG2) gene of the protozoan parasite Toxoplasma gondii was cloned and intracellularly expressed in the Pichia pastoris expression system. The molecular weight of the expressed recombinant SAG2 (36 kDa) was much larger than the native SAG2 (22 kDa). This discrepancy in size was due to hyperglycosylation, as deglycosylation assay reduced the size of the recombinant SAG2 to 22 kDa. Despite being hyperglycosylated, the recombinant SAG2 reacted strongly with pooled anti-Toxoplasma human serum, pooled anti-Toxoplasma mouse serum and a SAG2-specific monoclonal antibody. The glycosylated recombinant SAG2 was further evaluated in Western blot and in-house enzyme-linked immunosorbent assay (ELISA) using 80 human serum samples, including confirmed early acute (IgM positive, IgG negative; n=20), acute (IgM positive, IgG positive; n=20) and chronic (IgM negative, IgG positive; n=20) toxoplasmosis patients, and toxoplasmosis negative control patients (n=20). Results of the Western blot showed that the recombinant SAG2 reacted with all 60 samples of the toxoplasmosis cases but not with the Toxoplasma-negative samples. The sensitivity of in-house ELISA was 80%, 95% and 100% for early acute, acute and chronic patients' serum samples, respectively. Vaccination study showed that serum from mice immunised with the glycosylated recombinant SAG2 reacted specifically with the native SAG2 of T. gondii. The mice were significantly protected against lethal challenge with live T. gondii RH strain tachyzoites (P<0.01) and their survival time was increased compared to controls. Therefore, the present study shows that the P. pastoris-derived recombinant SAG2 was specific and suitable for use as antigen for detecting anti-Toxoplasma IgG and IgM antibodies. The vaccination study showed that recombinant SAG2 protein was immunoprotective in mice against lethal challenge.
    Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
  6. The rubber elongation factor of rubber trees (Hevea brasiliensis) is the major allergen in latex
    Czuppon AB, Chen Z, Rennert S, Engelke T, Meyer HE, Heber M, et al.
    J Allergy Clin Immunol, 1993 Nov;92(5):690-7.
    PMID: 8227860
    BACKGROUND: Allergy to latex-containing articles is becoming more and more important because it can result in unexpected life-threatening anaphylactic reactions in sensitized individuals.

    METHODS: A protein of 58 kd with an isoelectric point of 8.45 was purified from raw latex and from latex gloves and identified as the major allergen, completely blocking specific IgE antibodies in the serum of latex-sensitized subjects. The allergen is a noncovalent homotetramer molecule, in which the 14.6 kd monomer was identified, by amino acid composition and sequence homologies of tryptic peptides, to be the rubber elongation factor found in natural latex of the Malaysian rubber tree.

    RESULTS: Competitive immunoinhibition tests showed that the starch powder covering the finished gloves is the airborne carrier of the allergen, resulting in bronchial asthma on inhalation. The purified allergen can induce allergic reactions in the nanogram range.

    CONCLUSION: The identification of the allergen (Hev b I) may help to eliminate it during the production of latex-based articles in the future.

    Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
  7. The evolution of two-dimensional gel electrophoresis - from proteomics to emerging alternative applications
    Lee PY, Saraygord-Afshari N, Low TY
    J Chromatogr A, 2020 Mar 29;1615:460763.
    PMID: 31836310 DOI: 10.1016/j.chroma.2019.460763
    Two-dimensional gel electrophoresis (2-DE) is a technique that has been widely applied in a variety of proteomics studies. It is capable of resolving complex protein mixtures into individual protein spots based on their isoelectric point and molecular weight, enabling large-scale analysis of protein expression patterns for deciphering their changes in different biological conditions. 2-DE is a powerful tool that empowers researchers to perform differential qualitative and quantitative proteome analysis and is particularly advantageous for characterizing protein isoforms and post-translationally modified proteins. Despite its popularity as the workhorse for proteomics in the past few decades, it has been gradually displaced by the more sophisticated and high-performance mass spectrometry-based methods. However, there are several variations of the 2-DE technique that have emerged as promising approaches that shine new light on specific niches that 2-DE could still contribute. In this review, we first provide an overview of the applications of 2-DE, its merits and pitfalls in the current proteomic research arena, followed by a discussion on several alternative approaches for potential future applications.
    Matched MeSH terms: Electrophoresis, Polyacrylamide Gel/trends
  8. Fulltext The detection and characterization of pathogenic Leptospira and the use of OMPs as potential antigens and immunogens
    Gebriel AM, Subramaniam G, Sekaran SD
    Trop Biomed, 2006 Dec;23(2):194-207.
    PMID: 17322822 MyJurnal
    The detection of leptospires in patient blood in the first week of the disease using PCR provides an early diagnostic tool. PCR using two sets of primers (G1/G2 and B64-I/B64-II) tested with samples seeded with 23 leptospiral strains from pathogenic and non-pathogenic strains was able to amplify leptospiral DNA from pathogenic strains only. Of the 39 antibody negative samples collected from patients suspected for leptospirosis, only 1 sample (2.6%) was PCR positive. Using LSSP-PCR, the G2 primers allowed the characterization of Leptopira species to 10 different genetic signatures which may have epidemiological value in determining species involved in outbreaks. Leptospiral outer membrane proteins from three strains were purified and reacted against patients sera and gave rise to different profiles for pathogenic and non-pathogenic strains. Lymphocytes of mice injected with OMPs proliferated and released IFN(-3) when stimulated in vitro using Leptospira OMP as antigens. This suggests that an immune response could be established using leptospiral OMPs as a putative vaccine. OMPs were also used in a Dot-ELISA to detect antibodies against Leptospira pathogens in humans.
    Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
  9. The biological properties of venoms of some American coral snakes (Genus micrurus)
    Tan NH, Ponnudurai G
    Comp. Biochem. Physiol., B, 1992 Mar;101(3):471-4.
    PMID: 1582185
    1. The biological properties of nine venom samples from six taxa of Micrurus were investigated. The venoms exhibited low protease, phosphodiesterase and 5'-nucleotidase activities, moderate to strong phospholipase A and hyaluronidase activities, variable L-amino acid oxidase activity and were devoid of arginine ester hydrolase and thrombin-like activities. Some venom samples exhibited strong acetylcholinesterase activity. Venoms of M. c. dumerili and M. frontalis exhibited exceptionally high alkaline phosphomonoesterase activity while two of the M. f. fulvius venom samples tested exhibited strong hemorrhagic activity in mice. 2. The polyacrylamide gel electrophoretic patterns of the venoms indicate that most of the Micrurus venom proteins are basic proteins. All Micrurus venoms tested exhibited similar SDS-polyacrylamide gel electrophoretic patterns, with an intense low mol. wt protein band. 3. The Micrurus venoms appear to exhibit biological properties similar to other elapid venoms found in Asia and Africa. There are, however, no common characteristics in the biological properties of the venoms examined at the generic level.
    Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
  10. The biological properties of venoms from juvenile and adult taipan (Oxyuranus scutellatus) snakes
    Tan NH, Armugam A, Mirtschin PJ
    Comp. Biochem. Physiol., B, 1992 Nov;103(3):585-8.
    PMID: 1458834
    1. The biological properties of four venom pooled samples from adult taipan (Oxyuranus scutellatus) snakes and one pooled venom sample from six juvenile taipan snakes (11 months old) were compared. 2. The intravenous LD50 (median lethal dose), procoagulant activity and enzymatic activities of the juvenile venom were not significantly different from those of the adult venoms. 3. The juvenile and adult venoms exhibited similar polyacrylamide gel electrophoretic (PAGE) and SDS-PAGE patterns, indicating that they possessed a similar protein composition. 4. The results suggest that there is no significant age-dependency in the biological properties of taipan venom.
    Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
  11. Temporal variation of Malaysian rotavirus electropherotypes
    Rasool N, Othman RY, Adenan MI, Hamzah M
    J Clin Microbiol, 1989 Apr;27(4):785-7.
    PMID: 2470775
    An analysis of rotavirus electropherotypes circulating in Kuala Lumpur, Malaysia, over 7 years showed that all except 1 of the 360 electropherotypes encountered were characteristic of group A rotaviruses. The long electropherotype predominated annually, and there was a rarity of short electropherotypes. Extensive genome variability and cocirculation of different electropherotypes were observed annually. A sequential appearance of the predominant electropherotype was observed in all years of the study, except for 1985 and 1988, when one electropherotype predominated throughout the study periods. There was no shift in the predominant electropherotype over a 6-year period.
    Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
  12. Fulltext Tear proteomics in young Malays with dry eyes
    Mohd Ali, B., Nguan, D.K.C., Bashirah, I., Chan, K.M.
    International Medical Journal Malaysia, 2013;12(2):39-43.
    MyJurnal
    Changes in tear protein concentrations may reflect ocular surface health. This study analyzes changes in tear protein concentrations of young Malays with dry eye (DE) and determines its association with the clinical findings. Methods: Subjects were screened using McMonnies questionnaire (MDEQ) and flourescein tear break up time (TBUT). Total tear protein concentration (TTPC) was determined using Bradford's technique and specific tear protein (sIgA, lysozyme, lactoferrin and human serum albumin (HSA)) concentrations were determined using SDS-PAGE. Parametric and nonparametric tests were used to compare means between groups. Spearman correlation was used to determine the association between variables measured. Results: A total of 42 subjects (21 DE and 21 NDE) were included. Mean MDEQ score for DE was 16.00±1.48 and NDE was 8.47±3.47. Mean TBUT for DE was 3.47±0.47s and NDE was 4.98±0.43s. Mean TTPC for DE and NDE was 9.84±2.40mg/ml and 8.96±1.84mg/ml respectively. Mean sIgA, lysozyme, lactoferrin and HSA for DE was 0.54±0.10mg/ml, 1.68±0.17mg/ml, 1.47±0.25mg/ml, 0.06±0.03mg/ml and for NDE was 0.57±0.09mg/ml, 2.04±0.19mg/ml, 1.75±0.23mg/ml, 0.06±0.03mg/ml accordingly. Significant differences were noted in MDEQ score (p=0.01), TBUT (p=0.01), lactoferrin (p=0.01) and lysozyme (p=0.01) but not in TTPC (p=0.19), HSA (p=0.74) and sIgA (p=0.24) between groups. Significant correlations were noted between TBUT with lactoferrin (r=0.02, p=0.02) and lysozyme (r=0.63, p=0.01) and between MDEQ score with lactoferrin (r=-0.34, p=0.02) and lysozyme (r=-0.64, p=0.01). Conclusions: There are changes in specific tear protein in dry eye patients, which correlate well with clinical results. Tear protein analysis may play an important role in the diagnosis of the dry eye.
    Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
  13. Subtractive Protein Profiling of Salmonella typhimurium Biofilm Treated with DMSO
    Yahya MFZR, Alias Z, Karsani SA
    Protein J, 2017 08;36(4):286-298.
    PMID: 28470375 DOI: 10.1007/s10930-017-9719-9
    Salmonella typhimurium is an important biofilm-forming bacteria. It is known to be resistant to a wide range of antimicrobials. The present study was carried out to evaluate the effects of dimethyl sulfoxide (DMSO) against S. typhimurium biofilm and investigate whole-cell protein expression by biofilm cells following treatment with DMSO. Antibiofilm activities were assessed using pellicle assay, crystal violet assay, colony-forming unit counting and extracellular polymeric substance (EPS) matrix assay whilst differential protein expression was investigated using a combination of one dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis, tandem mass spectrometry and bioinformatics. Treatment with 32% DMSO inhibited pellicle formation, biofilm viability, biofilm biomass and several important components of EPS matrix. Subtractive protein profiling identified two unique protein bands (25.4 and 51.2 kDa) which were present only in control biofilm and not in 32% DMSO-treated biofilm. In turn, 29 and 46 proteins were successfully identified from the protein bands of 25.4 and 51.2 kDa respectively. Protein interaction network analysis identified several biological pathways to be affected, including glycolysis, PhoP-PhoQ phosphorelay signalling and flagellar biosynthesis. The present study suggests that DMSO may inhibit multiple biological pathways to control biofilm formation.
    Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
  14. Structure-informed detection and quantification of peptides in food and biological fluids
    Agyei D, Pan S, Acquah C, Bekhit AEA, Danquah MK
    J Food Biochem, 2019 01;43(1):e12482.
    PMID: 31353495 DOI: 10.1111/jfbc.12482
    Peptides with biological properties, that is, bioactive peptides, are a class of biomolecules whose health-promoting properties are increasingly being exploited in food and health products. However, research on targeted techniques for the detection and quantification of these peptides is still in its infancy. Such information is needed in order to enhance the biological and chemometric characterization of peptides and their subsequent application in the functional food and pharmaceutical industries. In this review, the role of classic techniques such as electrophoretic, chromatographic, and peptide mass spectrometry in the structure-informed detection and quantitation of bioactive peptides are discussed. Prospects for the use of aptamers in the characterization of bioactive peptides are also discussed. PRACTICAL APPLICATIONS: Although bioactive peptides have huge potential applications in the functional foods and health area, there are limited techniques in enhancing throughput detection, quantification, and characterization of these peptides. This review discusses state-of-the-art techniques relevant in complementing bioactive detection and profiling irrespective of the small number of amino acid units. Insights into challenges, possible remedies and prevailing areas requiring thorough research in the extant literature for food chemists and biotechnologists are also presented.
    Matched MeSH terms: Electrophoresis, Polyacrylamide Gel/methods
  15. Structural proteins of Getah virus isolates from Japan and Malaysia
    Srivastava AK, Igarashi A
    Acta Virol., 1986 Mar;30(2):126-30.
    PMID: 2873729
    Purified preparations of Getah virus strains have been analysed by sodium-dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE) to reveal their structural proteins. Two envelope proteins (E1 and E2) and core protein (C) were found with the prototype AMM2021 strain both under reducing and nonreducing conditions, while separation of E1 and E2 was observed only under nonreducing conditions for 3 strains isolated in Japan. Limited digestion by Staphylococcus aureus V8 protease revealed difference in the peptide patterns of E1 between AMM2021 and Japanese isolates. Mobility of E1 and E2 was slower for the virus grown in BHK21 cells compared with the virus grown in Aedes albopictus cells, indicating host-controlled modification on the envelope glycoproteins.
    Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
  16. Statistical optimization of green fluorescent protein production from Escherichia coli BL21(DE3)
    Chew FN, Tan WS, Boo HC, Tey BT
    Prep Biochem Biotechnol, 2012;42(6):535-50.
    PMID: 23030465 DOI: 10.1080/10826068.2012.660903
    An optimized cultivation condition is needed to maximize the functional green fluorescent protein (GFP) production. Six process variables (agitation rate, temperature, initial medium pH, concentration of inducer, time of induction, and inoculum density) were screened using the fractional factorial design. Three variables (agitation rate, temperature, and time of induction) exerted significant effects on functional GFP production in E. coli shake flask cultivation and were optimized subsequently using the Box-Behnken design. An agitation rate of 206 rpm at 31°C and induction of the protein expression when the cell density (OD(600nm)) reaches 1.04 could enhance the yield of functional GFP production from 0.025 g/L to 0.241 g/L, which is about ninefold higher than the unoptimized conditions. Unoptimized cultivation conditions resulted in protein aggregation and hence reduced the quantity of functional GFP. The model and regression equation based on the shake flask cultivation could be applied to a 2-L bioreactor for maximum functional GFP production.
    Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
  17. Site-Directed Mutagenesis of Cytochrome P450 2D6 and 2C19 Enzymes: Expression and Spectral Characterization of Naturally Occurring Allelic Variants
    Dong AN, Pan Y, Palanisamy UD, Yiap BC, Ahemad N, Ong CE
    Appl Biochem Biotechnol, 2018 Sep;186(1):132-144.
    PMID: 29524040 DOI: 10.1007/s12010-018-2728-0
    Genetic polymorphism of the cytochrome P450 (CYP) genes particularly affects CYP2D6 and CYP2C19 to a functionally relevant extent, and it is therefore crucial to elucidate the enzyme kinetic and molecular basis for altered catalytic activity of these allelic variants. This study explored the expression and function of the reported alleles CYP2D6*2, CYP2D6*10, CYP2D6*17, CYP2C19*23, CYP2C19*24, and CYP2C19*25 with respect to gene polymorphisms. Site-directed mutagenesis (SDM) was carried out to generate these six alleles. After DNA sequencing, the CYP2D6 and CYP2C19 wild types alongside with their alleles were each independently co-expressed with NADPH-CYP oxidoreductase (OxR) in Escherichia coli. The expressed proteins were analyzed using Western blotting, reduced carbon monoxide (CO) difference spectral scanning, and cytochrome c reductase assay. Results from Western blot revealed the presence of all CYP wild-type and allelic proteins in E. coli membrane fractions. The reduced CO difference spectra scanning presented the distinct peak of absorbance at 450 nm, and the cytochrome c reductase assay has confirmed that spectrally active OxR was expressed in each protein preparation. As a conclusion, the results obtained from this study have proven the CYP variants to be immunoreactive and spectrally active and are suitable for use to examine biotransformation and interaction mechanism of the enzymes.
    Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
  18. Fulltext Single-step purification of the recombinant green fluorescent protein from intact Escherichia coli cells using preparative PAGE
    Chew FN, Tan WS, Ling TC, Tey BT
    Electrophoresis, 2009 Sep;30(17):3017-3023.
    PMID: 19685471 DOI: 10.1002/elps.200900246
    Mechanical and non-mechanical breakages of bacterial cells are usually the preliminary steps in intracellular protein purification. In this study, the recombinant green fluorescent protein (GFP) was purified from intact Escherichia coli cells using preparative PAGE. In this purification process, cells disruption step is not needed. The cellular content of E. coli was drifted out electrically from cells and the negatively charged GFP was further electroeluted from polyacrylamide gel column. SEM investigation of the electrophoresed cells revealed substantial structural damage at the cellular level. This integrated purification technique has successfully recovered the intracellular GFP with a yield of 82% and purity of 95%.
    Matched MeSH terms: Electrophoresis, Polyacrylamide Gel/methods*
  19. Fulltext Serological evaluation of malaria patients in Thailand: antibody response against electrophoresed antigenic polypeptides of Plasmodium falciparum
    Kano S, Onda T, Matsumoto Y, Buchachart K, Krudsood S, Looareesuwan S, et al.
    Southeast Asian J. Trop. Med. Public Health, 1998 Jun;29(2):341-3.
    PMID: 9886125
    It was reported that a 47kDa antigenic polypeptide of Plasmodium falciparum had been strongly presented by the sera from 1) imported Japanese malaria patients with severe symptoms and 2) symptomatic and parasitemic inhabitants in endemic areas in the Sudan, Malaysia and the Philippines. In the present study, we observed the reactivity of the sera from falciparum malaria patients who had been hospitalized in the Bangkok Hospital for Tropical Diseases, Faculty of Tropical Medicine, Mahidol University, and compared the antibody response against the 47kDa antigenic polypeptide according to the severity of the patients. It was observed that antibodies to this molecule were more commonly shared in sera from severer patients, although the IFAT titers against the whole P. falciparum parasite antigen were lower in the group, which suggested that this antibody against the 47kDa molecule was playing a specific role at a severe stage of the infection. Determination of the immunological features of the antigenic molecules of parasites by this type of sero-epidemiological study will provide a new assay system for evaluation of immune status of individuals in different severity and suggest a way of vaccine development.
    Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
  20. Fulltext Self-assembled insulin and nanogels polyelectrolyte complex (Ins/NGs-PEC) for oral insulin delivery: characterization, lyophilization and in-vivo evaluation
    Mudassir J, Darwis Y, Muhamad S, Khan AA
    Int J Nanomedicine, 2019;14:4895-4909.
    PMID: 31456636 DOI: 10.2147/IJN.S199507
    Introduction: Insulin is given by injection, because when administered orally, it would be destroyed by enzymes in the digestive system, hence only about 0.1% reaches blood circulation. The purpose of the present study was to use pH sensitive polyelectrolyte methyl methacrylate (MMA)/itaconic acid (IA) nanogels as carriers in an attempt to improve absorption of insulin administered orally. Methods: Insulin (Ins) was incorporated into the MMA/IA nanogels (NGs) using the polyelectrolyte complexation (PEC) method to form Ins/NGs-PEC. Several parameters, including Ins:NGs ratio, pH, incubation time and stirring rate were optimized during preparation of InsNGs-PEC. The prepared formulations were characterized in terms of particle size (PS), polydispersity index (PdI), zeta potential (ZP) and percent entrapment efficiency (% EE). Results: The optimized InF12 nanogels had a PS, PdI, ZP and %EE of 190.43 nm, 0.186, -16.70 mV and 85.20%, respectively. The InF12 nanogels were lyophilized in the presence of different concentrations of trehalose as cryoprotectant. The lyophilized InF12 containing 2%w/v trahalose (InF12-Tre2 nanogels) was chosen as final formulation which had a PS, PdI, ZP and %EE of 430.50 nm, 0.588, -16.50 mv and 82.10, respectively. The in vitro release of insulin from InF12-Tre2 nanogels in the SGF and SIF were 28.71% and 96.53%, respectively. The stability study conducted at 5±3°C for 3 months showed that lnF12-Tre2 nanogels were stable. The SDS-PAGE assay indicated that the primary structure of insulin in the lnF12-Tre2 nanogels was intact. The in-vivo study in the diabetic rats following oral administration of InF12-Tre2 nanogels at a dose of 100 IU/kg body weight reduced blood glucose level significantly to 51.10% after 6 hours compared to the control groups. Conclusions: The pH sensitive MMA/IA nanogels are potential carriers for oral delivery of insulin as they enhanced the absorption of the drug.
    Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
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