• Title/Summary/Keyword: Two Dimension Electrophoresis

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Two-Dimensional Electrophoretic Analysis of Rice Seed Proteins (쌀 종자 단백질의 2차원 전기영동적 분석)

  • Yoon, Hye-Hyun;Kim, Seung-Ho
    • Applied Biological Chemistry
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    • v.32 no.2
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    • pp.85-90
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    • 1989
  • High resolution two-dimensional (2-D) electrophoresis with isoelectrofocusing in the first dimension and electrophoresis in sodium dodecyl sulfate in acrylamide gradient gels in the second dimension has been used to produce maps of proteins, extracted from rice seeds with 2% sodium dodecyl sulfate/5% 2-mercaptoethanol. Six rice cultivars-three Japonica types and three Tongil(high-yielding) types-at six maturities were studied. Composite map was constructed and more than 300 polypeptide spots were counted in the pH range of $5.2{\sim}8.3$ and molecular range of $20,000{\sim}100,000$. Vast differences were observed between varieties and between maturities in the maps.

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Comparative Proteomic Analysis of Changes in the Bovine Whey Proteome during the Transition from Colostrum to Milk

  • Zhang, Le-Ying;Wang, Jia-Qi;Yang, Yong-Xin;Bu, Deng-Pan;Li, Shan-Shan;Zhou, Ling-Yun
    • Asian-Australasian Journal of Animal Sciences
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    • v.24 no.2
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    • pp.272-278
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    • 2011
  • Bovine whey protein expression patterns of colostrum are much different from that of milk. Moreover, bovine colostrum is an important source of protective, nutritional and developmental factors for the newborn. However, to our knowledge, no research has been performed to date using a comparative proteomic method on the changes in the bovine whey proteome during the transition from colostrum to milk. This study therefore separated whey protein of days 1, 3, 7 and 21 after calving using two dimension electrophoresis. Differentially expressed proteins at different collection times were identified using high-performance liquid chromatography in tandem with mass spectrometry (LC/MS) and validated by enzyme-linked immunosorbent assay (ELISA) in order to understand the developmental changes in the bovine whey proteome during the transition from colostrum to milk. The expression patterns of whey protein of days 1 and 3 post-partum were similar except that immunoglobulin G was down-regulated on day 3, and four proteins were found to be down-regulated on days 7 and 21 compared with day 1 after delivering, including immunoglobulin G, immunoglobulin M, albumin, and lactotransferrin, which are involved in immunity and molecule transport. The results of this study confirm the comparative proteomic method has the advantage over other methods such as ELISA and immunoassays in that it can simultaneously detect more differentially expressed proteins. In addition, the difference in composition of milk indicates a need for adjustment of the colostrum feeding regimen to ensure a protective immunological status for newborn calves.

Two-dimensional gel Electrophoresis of Helicobacter pylori for Proteomic Analysis

  • Jung, Tae-Sung;Kang, Seung-Chul;Choi, Yeo-Jeong;Jeon, Beong-Sam;Park, Jeong-Won;Jung, Sun-Ae;Song, Jae-Young;Choi, Sang-Haeng;Park, Seong-Gyu;Choe, Mi-Young;Lee, Byung-Sang;Byun, Eun-Young;Baik, Seung-Chul
    • The Journal of the Korean Society for Microbiology
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    • v.35 no.2
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    • pp.97-108
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    • 2000
  • Two-dimensional gel electrophoresis (2-DE) is an essential tool of proteomics to analyse the entire set of proteins of an organism and its variation between organisms. Helicobacter pylori was tried to identify differences between strains. As the first step, whole H. pylori was lysed using high concentration urea contained lysis buffer [9.5 M Urea, 4% CHAPS, 35 mM Tris, 65 mM DTT, 0.01% SDS and 0.5% Ampholite (Bio-Rad, pH 3-10)]. The extract ($10\;{\mu}g$) was rehydrated to commercially available immobilised pH gradient (IPG) strips, then the proteins were separated according to their charges as the first dimensional separation. The IPG strips were placed on Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) to separate according to molecular mass of the proteins as the second dimension. The separated protein spots were visualised by silver staining in order to compare different expression of proteins between strains. Approximately 120 spots were identified in each mini-protein electrophoresised gel, furthermore about 65 to 75 spots were regarded as identical proteins in terms of pI value and molecular weight between strains used. In addition, distinct differences were found between strains, such as 219-1, Y7 and Y14, CH150. Two representative strains were examined using strips which had pH range from 4 to 7. This strips showed a number of isoforms which were considered large spots on pH range 3-10. Furthermore, the rest of spots on pH 4-7 IPG strips appeared very distinctive compared to broad range IPG strips. 2-DE seems to be an excellent tool for analysing and identifying variations between H. pylori strains.

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Proteome Approach as a Tool for the Efficient Separation of Seed Storage Proteins from Buckwheat

  • Cho, Seong-Woo;Kwon, Soo-Jeong;Roy, Swapan Kumar;Woo, Sun-Hee
    • KOREAN JOURNAL OF CROP SCIENCE
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    • v.60 no.1
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    • pp.29-32
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    • 2015
  • Two-dimensional electrophoresis (2-DE) was executed to separate the seed storage proteins from the buckwheat. The proteins extracted from the whole seed proteins were better separated and observed in the use of lysis buffer. Using this method, the highly reproducible isoelectric focusing (IEF) can be obtained from polyacrylamide gels, and IEF from the polyacrylamide gel at all the possible pH range (5.0-8.0) was more easily separated than IPG (immobilized pH gradient) gels. The polyacrylamide gels in the first dimension in 2-DE was used to separate and identify a number of whole seed proteins in the proteome analysis. In this new apparatus using 2-DE, 27cm in length of plate coated with polyacrylamide gel was used and the experiment was further investigated under the various conditions.

MEMS Fabrication of Microchannel with Poly-Si Layer for Application to Microchip Electrophoresis (마이크로 칩 전기영동에 응용하기 위한 다결정 실리콘 층이 형성된 마이크로 채널의 MEMS 가공 제작)

  • Kim, Tae-Ha;Kim, Da-Young;Chun, Myung-Suk;Lee, Sang-Soon
    • Korean Chemical Engineering Research
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    • v.44 no.5
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    • pp.513-519
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    • 2006
  • We developed two kinds of the microchip for application to electrophoresis based on both glass and quartz employing the MEMS fabrications. The poly-Si layer deposited onto the bonding interface apart from channel regions can play a role as the optical slit cutting off the stray light in order to concentrate the UV ray, from which it is possible to improve the signal-to-noise (S/N) ratio of the detection on a chip. In the glass chip, the deposited poly-Si layer had an important function of the etch mask and provided the bonding surface properly enabling the anodic bonding. The glass wafer including more impurities than quartz one results in the higher surface roughness of the channel wall, which affects subsequently on the microflow behavior of the sample solutions. In order to solve this problem, we prepared here the mixed etchant consisting HF and $NH_4F$ solutions, by which the surface roughness was reduced. Both the shape and the dimension of each channel were observed, and the electroosmotic flow velocities were measured as 0.5 mm/s for quartz and 0.36 mm/s for glass channel by implementing the microchip electrophoresis. Applying the optical slit with poly-Si layer provides that the S/N ratio of the peak is increased as ca. 2 times for quartz chip and ca. 3 times for glass chip. The maximum UV absorbance is also enhanced with ca. 1.6 and 1.7 times, respectively.

Identification of Bovine Pregnancy-Specific Whey Proteins using Two-Dimensional Gel Electrophoresis

  • Han, Rong-Xun;Choi, Su-Min;Kim, Myung-Youn;Quan, Yan Shi;Kim, Baek-Chul;Diao, Yun Fei;Koqani, Reza;Park, Chang-Sik;Jin, Dong-Il
    • Reproductive and Developmental Biology
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    • v.32 no.4
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    • pp.255-261
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    • 2008
  • The early diagnosis of bovine pregnancy is an essential component of successful reproductive planning on farms, because lack of bovine pregnancy over the long term results in reproductive failure and low milk yield-the latter of which is a special concern on dairy farms. This study was designed to identify early pregnancy-specific whey proteins in bovine, by comparing milk samples collected from cattle during pregnancy (Days 30 and 50) and from non-pregnant cattle. In this study, differentially expressed proteins in five pregnant and five non-pregnant Holstein dairy cattle were investigated and compared, using proteomics analysis. The first dimension was applied to a pH $3.0{\sim}10.0$ strip, by loading a 2-mg milk protein sample. After the second-dimension separation was performed, the gels were stained with colloidal Coomassie brilliant blue. The stained gels were scanned and the images were analyzed, to detect variations in protein spots between non-pregnant and pregnant cattle milk protein spots, using ImageMaster, this was followed by analysis with MALDI TOF-MS. Analysis of the 2-DE gel image resulted in a total of approximately $500{\sim}600$ protein spots, of which 12 spots were differentially expressed, six spots were up-regulated, and four spots were down-regulated; two spots were identified as pregnancy-specific proteins. These proteins were identified as lactoferrin, NA-DH dehydrogenase subunit 2, albumin, serum albumin precursor and transferrin. Our results via 2-D PAGE analysis revealed composite profiles of several milk proteins related to early bovine pregnancy, implying the possible use of these milk proteins in the early detection of bovine pregnancy.

Light/Dark Responsiveness of Kinetin-Inducible Secondary Metabolites and Stress Proteins in Rice Leaf

  • Cho, Kyoung-Won;Kim, Dea-Wook;Jung, Young-Ho;Shibato, Junko;Tamogami, Shigeru;Yonekura, Masami;Jwa, Nam-Soo;Kubo, Akihiro;Agrawal, Ganesh Kumar;Rakwal, Randeep
    • Journal of Crop Science and Biotechnology
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    • v.10 no.2
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    • pp.112-116
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    • 2007
  • Kinetin(KN) is an inducer of rice(Oryza sativa L.) defense/stress responses, as evidenced by the induction of inducible secondary metabolite and defense/stress protein markers in leaf. We show a novel light-dependent effect of KN-triggered defense stress responses in rice leaf. Leaf segments treated with KN(100 ${\mu}M$) show hypersensitive-like necrotic lesion formation only under continuous light illumination. Potent accumulation of two phytoalexins, sakuranetin and momilactone A(MoA) by KN that peaks at 48 h after treatment under continuous light is completely suppressed by incubation under continuous dark. Using two-dimensional gel electrophoresis we identified KN-induced changes in ribulose-1, 5-bisphosphate carboxylase/oxygenase, energy- and pathogenesis-related proteins(OsPR class 5 and 10 members) by N-terminal amino acid sequencing and mass spectrometry. These changes were light-inducible and could not be observed in the dark(and control). Present results provide a new dimension(light modulation/regulation) to our finding that KN has a potential role in the rice plant self-defense mechanism.

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Influence of CO2 concentration on carbon concentrating mechanisms in cyanobacteria and green algae: a proteomic approach

  • Ramanan, Rishiram;Vinayagamoorthy, Nadimuthu;Sivanesan, Saravana Devi;Kannan, Krishnamurthi;Chakrabarti, Tapan
    • ALGAE
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    • v.27 no.4
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    • pp.295-301
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    • 2012
  • Carbon concentrating mechanisms play a vital role in photosynthesis in microalgae and cyanobacteria especially in the proper functioning of Rubisco and assimilation of carbon via the Calvin cycle. This study evaluates the role of carbon dioxide on carbon concentrating mechanism (CCM) in a cynaobacteria, Spirulina platensis and a microalga, Chlorella sp. 786. The study organisms were grown in both atmospheric (control sample, 0.035%) and high (exposed sample, 10%) $CO_2$ concentrations. Second dimension (2D) electrophoresis revealed a huge difference in the protein profiles of both organisms suggesting the induction of CCM related proteins in the sample maintained at atmospheric $CO_2$ concentration and the repression of CCM related proteins in the sample maintained at 10% $CO_2$. Liquid chromatography-mass spectroscopy analysis revealed the presence of two important $C_i$ transporter proteins in the control sample of S. platensis, namely ferredoxin-$NADP^+$ reductase and ATP binding cassette (ABC) transport system protein. These proteins were only expressed in the control sample and were downregulated or not expressed at all in the exposed sample. Consequently, this study conclusively proves that CCMs are only inducted at low $CO_2$ concentrations and are not functional at high $CO_2$ concentration.

New Protein Extraction/Solubilization Protocol for Gel-based Proteomics of Rat (Female) Whole Brain and Brain Regions

  • Hirano, Misato;Rakwal, Randeep;Shibato, Junko;Agrawal, Ganesh Kumar;Jwa, Nam-Soo;Iwahashi, Hitoshi;Masuo, Yoshinori
    • Molecules and Cells
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    • v.22 no.1
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    • pp.119-125
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    • 2006
  • The rat is an accepted model for studying human psychiatric/neurological disorders. We provide a protocol for total soluble protein extraction using trichloroacetic acid/acetone (TCA/A) from rat (female) whole brain, 10 brain regions and the pituitary gland, and show that two-dimensional gel electrophoresis (2-DGE) using precast immobilized pH (4-7) gradient (IPG) strip gels (13 cm) in the first dimension yields clean silver nitrate stained protein profiles. Though TCA/A precipitation may not be "ideal", the important choice here is the selection of an appropriate lysis buffer (LB) for solubilizing precipitated proteins. Our results reveal enrichment of protein spots by use of individual brain regions rather than whole brain, as well as the presence of differentially expressed spots in their proteomes. Thus individual brain regions provide improved protein coverage and are better suited for differential protein detection. Moreover, using a phosphoprotein-specific dye, ingel detection of phosphoproteins was demonstrated. Representative high-resolution silver nitrate stained proteome profiles of rat whole brain total soluble protein are presented. Shortcomings apart (failure to separate membrane proteins), gel-based proteomics remains a viable option, and 2-DGE is the method of choice for generating high-resolution proteome maps of rat brain and brain regions.

S100A16 is a Prognostic Marker for Lung Adenocarcinomas

  • Saito, Keita;Kobayashi, Makoto;Nagashio, Ryo;Ryuge, Shinichiro;Katono, Ken;Nakashima, Hiroyasu;Tsuchiya, Benio;Jiang, Shi-Xu;Saegusa, Makoto;Satoh, Yukitoshi;Masuda, Noriyuki;Sato, Yuichi
    • Asian Pacific Journal of Cancer Prevention
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    • v.16 no.16
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    • pp.7039-7044
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    • 2015
  • Background: Many functional molecules controlling diverse cellular function are included in low-molecular weight proteins and peptides. Materials and Methods: To identify proteins controlling function in lung adenocarcinomas (AC), we performed two-dimensional gel electrophoresis employing tricine-SDS polyacrylamide in the second dimension (tricine 2-DE). This system was able to detect proteins under 1 kDa even with post-translational modifications. To confirm the utility of detected proteins as novel tumor markers for AC, we performed immunohistochemical analysis using 170 formalin-fixed and paraffin-embedded lung AC tissues. Results: Tricine 2-DE revealed that five proteins including S100A16 were overexpressed in lung AC-derived cells compared with lung squamous cell carcinoma, small cell carcinoma, and large cell neuroendocrine carcinoma-derived cells. Immunohistochemically, S100A16 showed various subcellular localization in lung cancer tissues and a membranous staining status was correlated with the T-factor (P=0.0008), pathological stage (P=0.0015), differentiation extent (P=0.0001), lymphatic invasion (P=0.0007), vascular invasion (P=0.0001), pleural invasion (P=0.0087), and gender (P=0.039), but not with the age or smoking history. More importantly, membranous staining of S100A16 was significantly correlated with a poorer overall survival of either stage I (P=0.0088) or stage II / III (P=0.0003) lung AC patients, and multivariate analysis confirmed that membranous expression of S100A16 was an independent adverse prognostic indicator (P=0.0001). Conclusions: The present results suggest that S100A16 protein is a novel prognostic marker for lung AC.