Displaying publications 1 - 20 of 226 in total

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  1. Mehrnoush A, Mustafa S, Yazid AM
    Molecules, 2011 Dec 08;16(12):10202-13.
    PMID: 22158589 DOI: 10.3390/molecules161210202
    A 'Heat treatment aqueous two phase system' was employed for the first time to purify serine protease from kesinai (Streblus asper) leaves. In this study, introduction of heat treatment procedure in serine protease purification was investigated. In addition, the effects of different molecular weights of polyethylene glycol (PEG 4000, 6000 and 8000) at concentrations of 8, 16 and 21% (w/w) as well as salts (Na-citrate, MgSO₄ and K₂HPO₄) at concentrations of 12, 15, 18% (w/w) on serine protease partition behavior were studied. Optimum conditions for serine protease purification were achieved in the PEG-rich phase with composition of 16% PEG6000-15% MgSO₄. Also, thermal treatment of kesinai leaves at 55 °C for 15 min resulted in higher purity and recovery yield compared to the non-heat treatment sample. Furthermore, this study investigated the effects of various concentrations of NaCl addition (2, 4, 6 and 8% w/w) and different pH (4, 7 and 9) on the optimization of the system to obtain high yields of the enzyme. The recovery of serine protease was significantly enhanced in the presence of 4% (w/w) of NaCl at pH 7.0. Based on this system, the purification factor was increased 14.4 fold and achieved a high yield of 96.7%.
    Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
  2. Wong SY, Lee CC, Ashrafzadeh A, Junit SM, Abrahim N, Hashim OH
    PLoS One, 2016;11(10):e0164993.
    PMID: 27741315 DOI: 10.1371/journal.pone.0164993
    Proteome analysis of the human hair remains challenging due to the poor solubility of hair proteins and the difficulty in their extraction. In the present study, we have developed a rapid extraction protocol for hair shaft protein using alkaline-based buffer. The new protocol accelerated the procedure by reducing the extraction time from at least a day to less than two hours and showed a protein recovery of 47.3 ± 3.72%. Further analyses of the extracted protein sample through sodium dodecyl sulfate polyacrylamide gel electrophoresis and Quadrupole-time-of-flight mass spectrometry analysis unveiled a total of 60 proteins, including 25 that were not previously reported. Identification of these proteins is anticipated to be crucial in helping to understand the molecular basis of hair for potential applications in the future.
    Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
  3. Tan CH, Tan KY, Tan NH
    Methods Mol Biol, 2019;1871:83-92.
    PMID: 30276733 DOI: 10.1007/978-1-4939-8814-3_5
    Snake venoms are complex mixtures of proteins and peptides that play vital roles in the survival of venomous snakes. As with their diverse pharmacological activities, snake venoms can be highly variable, hence the importance of understanding the compositional details of different snake venoms. However, profiling venom protein mixtures is challenging, in particular when dealing with the diversity of protein subtypes and their abundances. Here we described an optimized strategy combining a protein decomplexation method with in-solution trypsin digestion and mass spectrometry of snake venom proteins. The approach involves the integrated use of C18 reverse-phase high-performance liquid chromatography (RP-HPLC), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and nano-electrospray ionization tandem mass spectrometry (nano-ESI-LC-MS/MS).
    Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
  4. Tan NH, Arunmozhiarasi A, Ponnudurai G
    PMID: 1685421
    1. The biological properties of twelve samples of venoms from all four species of Dendroaspis (mamba) were investigated. 2. Dendroaspis venoms generally exhibited very low levels of protease, phosphodiesterase and alkaline phosphomonoesterase; low to moderately low level of 5'-nucleotidase and very high hyaluronidase activities, but were devoid of L-amino acid oxidase, phospholipase A, acetylcholinesterase and arginine ester hydrolase activities. The unusual feature in venom enzyme content can be used to distinguish Dendroaspis venoms from other snake venoms. 3. All Dendroaspis venoms did not exhibit hemorrhagic or procoagulant activity. Some Dendroaspis venoms, however, exhibited strong anticoagulant activity. The intravenous median lethal dose of the venoms ranged from 0.5 microgram/g mouse to 4.2 micrograms/g mouse. 4. Venom biological activities are not very useful for the differentiation of the Dendroaspis species. The four Dendroaspis venoms, however, can be differentiated by their venom SDS-polyacrylamide gel electrophoretic patterns.
    Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
  5. Tan NH, Ponnudurai G, Mirtschin PJ
    Toxicon, 1993 Mar;31(3):363-7.
    PMID: 8470140
    The biological properties of adult and juvenile inland taipan (Oxyuranus microlepidotus) snake venoms were examined. The enzymatic activities, intravenous median lethal dose and procoagulant activity of the juvenile venom samples were not significantly different from those of the adult venom samples. Also, the juvenile and adult venoms exhibited similar electrophoretic patterns, indicating that they possessed similar protein composition.
    Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
  6. Tan NH, Ponnudurai G
    Comp. Biochem. Physiol., B, 1992 May;102(1):103-9.
    PMID: 1526113
    1. Examination of the polyacrylamide gel electrophoretic (PAGE) and SDS-PAGE patterns of snake venoms shows that these patterns are useful for species differentiation (and hence identification) for snakes of certain genera but have only limited application for snakes from some other genera, due either to the marked individual variations in the venoms or the lack of marked interspecific differences within the same genus. 2. There is no substantial intersubspecific difference in the electrophoretic patterns of the venoms. 3. In general there are no common characteristics in the electrophoretic patterns of the venom at the generic level because of the wide variations in the electrophoretic patterns of venoms of snakes within the same genus. 4. At the familial level, the venoms of Elapidae exhibited SDS-PAGE patterns distinct from those of Crotalidae.
    Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
  7. Su YC, Wan KL, Mohamed R, Nathan S
    Microbes Infect., 2008 Oct;10(12-13):1335-45.
    PMID: 18761419 DOI: 10.1016/j.micinf.2008.07.034
    Burkholderia pseudomallei is the etiological agent of melioidosis, a severe infectious disease of humans and animals. The role of the bacterium's proteins expressed in vivo during human melioidosis continues to remain an enigma. This study's aim was to identify B. pseudomallei target proteins that elicit the humoral immune response in infected humans. A small insert genomic expression library was constructed and immunoscreened to identify peptides that reacted exclusively with melioidosis patients' sera. Sero-positive clones expressing immunogenic peptides were sequenced and annotated, and shown to represent 109 proteins involved in bacterial cell envelope biogenesis, cell motility and secretion, transcription, amino acid, ion and protein metabolism, energy production, DNA repair and unknown hypothetical proteins. Western blot analysis of three randomly selected full-length immunogenic polypeptides with patients' sera verified the findings of the immunome screening. The patients' humoral immune response to the 109 proteins suggests the induction or significant upregulation of these proteins in vivo during human infection and thus may play a role in the pathogenesis of B. pseudomallei. Identification of B. pseudomallei immunogens has shed new light on the elucidation of the bacterium's pathogenesis mechanism and disease severity. These immunogens can be further evaluated as prophylactic and serodiagnostic candidates as well as drug targets.
    Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
  8. Riazi M, Abdul Rani B, Fairuz A, Zainul FZ
    Trop Biomed, 2010 Aug;27(2):241-53.
    PMID: 20962722 MyJurnal
    There is a need for identification of new infection markers against common Leptospira isolates in Malaysia. To achieve this goal, seven-day-old cultures of Leptospira interrogans serogroup Icterohemorrhagiae (L44) and Leptospira interrogans serogroup Javanica (L55) were used for antigen preparation by sequential extraction method using 40 mM Tris, 8M Urea and 2M thiourea. Immunoblot analysis of the antigens were performed using serum samples from 46 local patients with confirmed acute leptospirosis, 28 patients with other infections and 14 healthy controls. The patients serum samples used in this study contained heterologous antibody against a number of different leptospira serovars. A strong IgM reactivity to a broad diffuse band of 10-15 kDa was observed. Combining results using L44 and L55 antigens showed sensitivity of 80.4% and specificity of 95.2% for detection of leptospirosis. Proteinase K and periodate treatment indicated that the band is likely to be lipopolysaccharide (LPS) in nature. This study showed that the 10-15 kDa antigen could potentially be useful for serodiagnosis of acute leptospirosis in Malaysia.
    Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
  9. Bakar AF, Alitheen NB, Keong YS, Hamid M, Ali SA, Ali AM
    Hybridoma (Larchmt), 2009 Jun;28(3):199-203.
    PMID: 19519247 DOI: 10.1089/hyb.2007.0531
    Hybridoma clone C3A8, which is a fusion product between splenic lymphocytes of Balb/c mice immunized with MCF7 breast carcinoma cells and SP2/0 myelomas, was produced and characterized. A stable clone that secreted IgM monoclonal antibody (MAb) with kappa light chain was obtained through limiting dilutions. Cell-ELISA screening, flow cytometry analysis, and immunofluorescence staining revealed that the MAb C3A8 had bound specifically and strongly to MCF7 and HT29 but cross reacted weakly or not on HeLa cell line. The MAb C3A8 reacted positively with paraffin-embedded tissues of human breast and colon cancers but there were no positive reactions on normal tissues. Western blot analysis showed the MAb recognized a 55 kDa protein, which was present in the extract of MCF7 and HT29 cell lines. Our results demonstrated that MAb C3A8 could be used for basic and clinical research of breast and colon cancers.
    Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
  10. Ismail NF, Hamdan S, Mahadi NM, Murad AM, Rabu A, Bakar FD, et al.
    Biotechnol Lett, 2011 May;33(5):999-1005.
    PMID: 21234789 DOI: 10.1007/s10529-011-0517-8
    L-Asparaginase II signal peptide was used for the secretion of recombinant cyclodextrin glucanotransferase (CGTase) into the periplasmic space of E. coli. Despite its predominant localisation in the periplasm, CGTase activity was also detected in the extracellular medium, followed by cell lysis. Five mutant signal peptides were constructed to improve the periplasmic levels of CGTase. N1R3 is a mutated signal peptide with the number of positively charged amino acid residues in the n-region increased to a net charge of +5. This mutant peptide produced a 1.7-fold enhancement of CGTase activity in the periplasm and significantly decreased cell lysis to 7.8% of the wild-type level. The formation of intracellular inclusion bodies was also reduced when this mutated signal peptide was used as judged by SDS-PAGE. Therefore, these results provide evidence of a cost-effective means of expression of recombinant proteins in E. coli.
    Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
  11. Murugan P, Han L, Gan CY, Maurer FH, Sudesh K
    J Biotechnol, 2016 Dec 10;239:98-105.
    PMID: 27746304 DOI: 10.1016/j.jbiotec.2016.10.012
    Bacterial polyhydroxyalkanoates (PHA) are expensive partly due to the recovery and purification processes. Thus, many studies have been carried out in order to minimize the cost. Here we report on the use of mealworm, which is the larva of mealworm beetle (Tenebrio molitor) to recover PHA granules from Cupriavidus necator. Mealworms were shown to readily consume the freeze-dried C. necator cells and excrete the PHA granules in the form of whitish feces. Further purification using water, detergent and heat resulted in almost 100% pure PHA granules. Comparison with chloroform extraction showed no signs of reduction in the molecular weight and dispersion of the PHA molecules. Scanning electron microscopy and dynamic light scattering measurements revealed that the biologically recovered PHA granules retained their native spherical morphology. The PHA granules were subjected to a battery of tests to determine their purity and properties in comparison to the chloroform extracted PHA. This study has demonstrated the possibility of using mealworms as a biological agent to partially purify the PHA granules.
    Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
  12. Mohd Khalizan Sabullah, Azlan Jualang Gansau, Mohd Rosni Sulaiman, Fisal Ahmad
    MyJurnal
    Observations on the effects of copper on the liver proteome of Puntius javanicus based on the
    one dimensional PAGE was carried out. The liver was dissected from each fish, which was
    separately treated with different concentrations of copper sulfate ranging from 0.1 to 5.0 mg/L.
    The livers were extracted and one dimensional PAGE was performed under nonreducing
    (native) and reducing (SDS)-PAGE. Several bands were resolved in the native PAGE with
    probable candidates for the effect of copper observed showing an increased in the expression
    and downregulation strongly associated with increasing copper concentrations. This study
    showed that high concentrations of copper significantly alters P. javanicus liver at the proteome
    level, and preliminary screening based on one dimensional PAGE is considered rapid and
    simple to assess the toxicity effect of copper before more advanced and extensive assesment
    with a second dimensional PAGE is carried out.
    Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
  13. Müller-Sienerth N, Shilts J, Kadir KA, Yman V, Homann MV, Asghar M, et al.
    Malar J, 2020 Jan 17;19(1):31.
    PMID: 31952523 DOI: 10.1186/s12936-020-3111-5
    BACKGROUND: Malaria remains a global health problem and accurate surveillance of Plasmodium parasites that are responsible for this disease is required to guide the most effective distribution of control measures. Serological surveillance will be particularly important in areas of low or periodic transmission because patient antibody responses can provide a measure of historical exposure. While methods for detecting host antibody responses to Plasmodium falciparum and Plasmodium vivax are well established, development of serological assays for Plasmodium knowlesi, Plasmodium ovale and Plasmodium malariae have been inhibited by a lack of immunodiagnostic candidates due to the limited availability of genomic information.

    METHODS: Using the recently completed genome sequences from P. malariae, P. ovale and P. knowlesi, a set of 33 candidate cell surface and secreted blood-stage antigens was selected and expressed in a recombinant form using a mammalian expression system. These proteins were added to an existing panel of antigens from P. falciparum and P. vivax and the immunoreactivity of IgG, IgM and IgA immunoglobulins from individuals diagnosed with infections to each of the five different Plasmodium species was evaluated by ELISA. Logistic regression modelling was used to quantify the ability of the responses to determine prior exposure to the different Plasmodium species.

    RESULTS: Using sera from European travellers with diagnosed Plasmodium infections, antigens showing species-specific immunoreactivity were identified to select a panel of 22 proteins from five Plasmodium species for serological profiling. The immunoreactivity to the antigens in the panel of sera taken from travellers and individuals living in malaria-endemic regions with diagnosed infections showed moderate power to predict infections by each species, including P. ovale, P. malariae and P. knowlesi. Using a larger set of patient samples and logistic regression modelling it was shown that exposure to P. knowlesi could be accurately detected (AUC = 91%) using an antigen panel consisting of the P. knowlesi orthologues of MSP10, P12 and P38.

    CONCLUSIONS: Using the recent availability of genome sequences to all human-infective Plasmodium spp. parasites and a method of expressing Plasmodium proteins in a secreted functional form, an antigen panel has been compiled that will be useful to determine exposure to these parasites.

    Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
  14. Ramasamy B, Nadarajah VD, Soong ZK, Lee HL, Mohammad SM
    Trop Biomed, 2008 Apr;25(1):64-74.
    PMID: 18600206
    Vegetative proteins from Malaysian strains of Bacillus thuringiensis israelensis strains (Bt 11, Bt 12, Bt 15, Bt 16, Bt 17, Bt 21 and Bt 22) and Bacillus sphaericus H-25 strains (Bs 1 and Bs 2) were screened for haemolytic, cytotoxic and larvicidal activity. SDS-PAGE profiles of the Bacillus thuringiensis strains studied consistently showed major bands of 33-37 kDa and 47 kDa. Bt 16 also showed two bands of 66 kDa and 45 kDa similar to the previously reported binary vegetative protein, Vip1Ac (66 kDa) and Vip 2Ac (45 kDa). Both the Bacillus sphaericus strains showed a 35 kDa band that was similiar to a previously reported vegetative protein, the Mtx2 protein. Bs 2 also contains a 37 kDa band, similar to another vegetative protein, the Mtx 3 protein. With the exception of Bt 17 and Bt 21, vegetative proteins from all Bacillus thuringiensis and Bacillus sphaericus strains were highly haemolytic to human erythrocytes, causing more than 75% haemolysis at the highest concentration of 200 microg/ml. High haemolytic activity was associated with high cytotoxic activity with most of the haemolytic strains being indiscriminately cytotoxic to both CEM-SS (human T lymphoblastoid) and HeLa (human uterus cervical cancer) cell lines. Interestingly, the less haemolytic vegetative proteins from Bt 17 and Bt 21 demonstrated cytotoxic activity comparable to that of the highly haemolytic vegetative proteins. Bt 21 displayed toxicity towards both cell lines while Bt 17 was more toxic towards CEM-SS cells. Bioassay against Aedes aegypti and Culex quinquefasciatus larvae revealed that vegetative proteins from the Bacillus thuringiensis strains had activity against both species of larvae but vegetative proteins from Bacillus sphaericus were weakly larvicidal towards Cx. quinquefasciatus only.
    Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
  15. Ho CW, Tan WS, Chong FC, Ling TC, Tey BT
    J Microbiol Biotechnol, 2009 Apr;19(4):416-23.
    PMID: 19421000
    Hepatitis B core antigen (HBcAg) is an important serological marker used in the diagnosis of hepatitis B virus (HBV) infections. In the current study, a fast and efficient preparative purification protocol for truncated HBcAg from Escherichia coli disruptate was developed. The recombinant HBcAg was first captured by anion exchange expanded bed adsorption chromatography integrated with a cell disruption process. This online capture process has shortened the process time and eliminated the "hold-up" period that may be detrimental to the quality of target protein. The eluted product from the expanded bed adsorption chromatography was subsequently purified using size-exclusion chromatography. The results showed that this novel purification protocol achieved a recovery yield of 45.1% with a product purity of 88.2%, which corresponds to a purification factor of 4.5. The recovered HBcAg is still biologically active as shown by ELISA test.
    Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
  16. Leow TC, Rahman RN, Basri M, Salleh AB
    Extremophiles, 2007 May;11(3):527-35.
    PMID: 17426920
    A thermoalkaliphilic T1 lipase gene of Geobacillus sp. strain T1 was overexpressed in pGEX vector in the prokaryotic system. Removal of the signal peptide improved protein solubility and promoted the binding of GST moiety to the glutathione-Sepharose column. High-yield purification of T1 lipase was achieved through two-step affinity chromatography with a final specific activity and yield of 958.2 U/mg and 51.5%, respectively. The molecular mass of T1 lipase was determined to be approximately 43 kDa by gel filtration chromatography. T1 lipase had an optimum temperature and pH of 70 degrees C and pH 9, respectively. It was stable up to 65 degrees C with a half-life of 5 h 15 min at pH 9. It was stable in the presence of 1 mM metal ions Na(+), Ca(2+), Mn(2+), K(+) and Mg(2+ ), but inhibited by Cu(2+), Fe(3+) and Zn(2+). Tween 80 significantly enhanced T1 lipase activity. T1 lipase was active towards medium to long chain triacylglycerols (C10-C14) and various natural oils with a marked preference for trilaurin (C12) (triacylglycerol) and sunflower oil (natural oil). Serine and aspartate residues were involved in catalysis, as its activity was strongly inhibited by 5 mM PMSF and 1 mM Pepstatin. The T(m) for T1 lipase was around 72.2 degrees C, as revealed by denatured protein analysis of CD spectra.
    Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
  17. Omidvar V, Siti Nor Akmar A, Marziah M, Maheran AA
    Plant Cell Rep, 2008 Sep;27(9):1451-9.
    PMID: 18563415 DOI: 10.1007/s00299-008-0565-2
    The promoter of the oil palm metallothionein-like gene (MT3-A) demonstrated mesocarp-specific activity in functional analysis using transient expression assay of reporter gene in bombarded oil palm tissue slices. In order to investigate the tissue-specific expression of polyhydroxybutyrate (PHB) biosynthetic pathway genes, a multi-gene construct carrying PHB genes fused to the oil palm MT3-A promoter was co-transferred with a construct carrying GFP reporter gene using microprojectile bombardment targeting the mesocarp and leaf tissues of the oil palm. Transcriptional analysis using RT-PCR revealed successful transcription of all the three phbA, phbB, and phbC genes in transiently transformed mesocarp but not in transiently transformed leaf tissues. Furthermore, all the three expected sizes of PHB-encoded protein products were only detected in transiently transformed mesocarp tissues on a silver stained polyacrylamide gel. Western blot analysis using polyclonal antibody specific for phbB product confirmed successful translation of phbB mRNA transcript into protein product. This study provided valuable information, supporting the future engineering of PHB-producing transgenic palms.
    Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
  18. Hashim OH, Hassan H
    Immunology, 1991 Jun;73(2):235-8.
    PMID: 2071167
    Three bacterial species of Clostridium (septicum, tertium and sporogenes) were identified to produce extracellular proteases cleaving IgA to Fab and Fc fragments, as demonstrated by SDS-PAGE and immunoelectrophoretic procedures. These enzymes acted on monometric IgA1 paraproteins and normal serum IgA1 but had no activity on IgA2 paraproteins and intact secretory IgA1 from human colostrum. Their action on polyclonal serum IgA1 suggested the absence of neutralizing anti-clostridial IgA protease activity. Although the enzymes were shown not to act on secretory IgA1, they were, however, able to digest free alpha-heavy chains of the dimeric IgA molecules. Susceptibility of the alpha-heavy chain to the proteases was more likely due to the change to a more accessible conformation than because of the absence of neutralizing anti-enzymic activity.
    Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
  19. Shintani M, Minaguchi K, Suzuki K, Lim KA
    Biochem Genet, 1990 Apr;28(3-4):173-84.
    PMID: 2383244
    Three new variants of acidic proline-rich proteins (At, Au, Aw) were found in human parotid saliva by isoelectric focusing and basic gel electrophoresis. Electrophoretic comparison of the purified proteins and their tryptic peptides suggested minor charge and size differences from other acidic PRPs. Genetic and biochemical studies indicate that the At and Aw proteins are allelic products of the PRH1 locus. Gene frequencies of the At productive allele (PRH1(6)) in Japanese, Chinese, and Malays were 0.008, 0.012, and 0.004, respectively. The Au protein was also found in Japanese (2 in 746 samples), Chinese (1 in 215 samples), and Malays (1 in 220 samples), however, the Aw protein was found only in one Japanese (n = 746). These three proteins were not found in 106 Indian subjects.
    Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
  20. Lee Y, Roslan R, Azizan S, Firdaus-Raih M, Ramlan EI
    BMC Bioinformatics, 2016 Oct 28;17(1):438.
    PMID: 27793081
    BACKGROUND: Biological macromolecules (DNA, RNA and proteins) are capable of processing physical or chemical inputs to generate outputs that parallel conventional Boolean logical operators. However, the design of functional modules that will enable these macromolecules to operate as synthetic molecular computing devices is challenging.

    RESULTS: Using three simple heuristics, we designed RNA sensors that can mimic the function of a seven-segment display (SSD). Ten independent and orthogonal sensors representing the numerals 0 to 9 are designed and constructed. Each sensor has its own unique oligonucleotide binding site region that is activated uniquely by a specific input. Each operator was subjected to a stringent in silico filtering. Random sensors were selected and functionally validated via ribozyme self cleavage assays that were visualized via electrophoresis.

    CONCLUSIONS: By utilising simple permutation and randomisation in the sequence design phase, we have developed functional RNA sensors thus demonstrating that even the simplest of computational methods can greatly aid the design phase for constructing functional molecular devices.

    Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
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