• Title/Summary/Keyword: two dimensional electrophoresis

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Purification of Bovine Pregnancy-Associated Proteins by Two-Dimensional Gel Electrophoresis

  • Hwang, Sun-Il;Lim, Jin-Kyu
    • BMB Reports
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    • v.32 no.5
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    • pp.445-450
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    • 1999
  • We purified and characterized a bovine pregnancy-associated protein in pregnant cow urine using two-dimensional gel electrophoresis. Urine from cows was collected according to their status of pregnancy and non-pregnancy. Proteins in the cow urine were fractionated with 50% ammonium sulfate prior to two-dimensional gel electrophoresis. Proteins separated on the gels were compared in terms of expression level and new expression by molecular mass and isoelectric point. We localized two pregnancy-associated protein spots on the gels at molecular masses of 24 kDa and 20 kDa and isoelectric points of 5.5 and 5.7, respectively. Likewise, two non-pregnancy specific proteins were localized at 27 kDa and 28 kDa with isoelectric points of 5.7 and 5.9, respectively. To rule out the possibility that environmental or genetic factors might influence the expression of the proteins, we demonstrated the pregnancy-associated expression of the proteins in two-dimensional gels with pregnant urine taken from cows raised in a different institute. The pregnancy-associated protein with molecular mass of 20 kDa and isoelectric point of 5.7, namely spot 2, was microsequenced and found to be highly homologous to the bovine collagen alpha 1 chain.

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Identification of Rhizobium fredii and Bradyrhizobium japonicum by Polyacrylamide Gel Electrophoresis (전기영동법(電氣泳動法)에 의(依)한 대두(大豆) 근류균(根瘤菌) Rhizobium fredii와 Bradyrhizobium japonicum의 분류(分類) 및 동정(同定))

  • Yun, Han-Dae;Cho, Moo-Je;Lee, Ke-Ho
    • Applied Biological Chemistry
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    • v.30 no.2
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    • pp.163-168
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    • 1987
  • A method, based upon the separation of cellular proteins by one-and two-dimensional electrophoresis was used for distinguishing butween Bradyrhizobium japonicum strains and Rhizobium fredii strains. Significant differences in protein pattern of one-dimensional SDS-PAGE vs-ere observed between Rhizobium fredii strains and Bradyrhizobium japonicum strains. The differences in six distinct main lands were observed among total 52 kinds of protein bands. Furthermore, the distribution of proteins in two groups by two-dimensional polyacrylamide gel electrophoresis was very different. The majority of visible proteins of Rhizobium fredii were acidic, whereas those of Bradyrhizobium japonicum were basic. In addition, amino acid composition was analyzed to detect the differences between two groups. No significant differences in amino acid composition were observed between Bradyrhizobium japonicum strains and Rhizobium fredii strains. The results indicate that one-and two-dimensional polyacrylamide gel electrophoresis were useful for identifying rhizobia isolates. One-dimensional SDS-PAGE of rhizobia proteins provided a rapid method for screening a large number of isolates, whereas two-dimensional electrophoresis was more of resolution and easiness for analyzing protein spots.

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Correlation Analysis between the Breeding Value of Carcass Traits in Hanwoo (Korean Brown Cattle), Bos Taurus, L. and Spot Intensity on Two-dimensional Gel Electrophoresis

  • Seo, Kang Seok;Shen, Y.N.;Salces, A.J.;Yoon, D.H.;Lee, H.G.;Kim, S.H.
    • Asian-Australasian Journal of Animal Sciences
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    • v.19 no.10
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    • pp.1404-1408
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    • 2006
  • In order to investigate the genetic marker associated with economic performance in Hanwoo (Korean Brown Cattle), proteomic approach was used. Breeding values were estimated from performance tested steers. The top 20 and bottom 19 steers based on carcass weight (CW), eye muscle area (EMA), backfat thickness (BF) and marbling score (MS) evaluation for one progeny testing period was used. Meat samples dissected from longissimus dorsi muscles were taken from the slaughter house and analyzed for two-dimensional gel electrophoresis. A total of 102 significant spots out of total 146 on each gel were detected and compared with the reference gel (synthetic gel) to be evaluated. Four candidate spots for marbling score were identified: 205, 84, 204 and 198. The study confirmed the relationship between breeding values of economic traits of Hanwoo cattle and spot intensity.

A Preliminary Analysis of Secreted Proteins from Bifidobacterium pseudocatanulatum BP1 by Two-Dimensional Gel Electrophoresis

  • Moon, Gi-Seong
    • Preventive Nutrition and Food Science
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    • v.13 no.4
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    • pp.366-369
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    • 2008
  • Proteins secreted from bifidobacteria are believed to play important roles in human intestines via interacting with different host cells. In this respect, proteins secreted from Bifidobacterium pseudocatanulatum BP1, which has been rarely studied, were analyzed by two-dimensional gel electrophoresis (2DE). Using this approach, approx-imately 21 protein spots on a 2DE gel were detected and 10 of these spots were identified by mass spectrometry. Five spots were identified as hypothetical proteins and the remaining 5 spots were identified as a putative iron-side-rophore binding lipoprotein, a short-chain dehydrogenase/reductase SDR, an exonuclease, cytochrome P450 hydroxylase, and a putative dehydrogenase. The identification of secreted putative iron-siderophore binding lipoprotein was highly interesting since it is an important protein that is involved in ferric iron uptake in pathogenic bacteria. This finding could accelerate studies on the probiotic effect of Bifidobacterium by explaining the competition between bifidobacteria and intestinal pathogens for ferric iron.

Presence of an Albumin-Like Protein on the Plasma Membrane of Adipocytes Isolated from Korean Cow

  • Choi, Chang-Bon;Rhee, Seung-Keun
    • BMB Reports
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    • v.31 no.6
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    • pp.542-547
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    • 1998
  • The main function of adipocytes in various species is to store nutrient energy in the form of triglycerides, and this function may he closely related with hormonal signaling through the plasma membrane of adipocytes. Using SDS-PAGE, two-dimensional gel electrophoresis, and a membrane biotinylation technique, we have identified a 55KDa protein (55K protein) from the plasma membrane fraction of adipocytes, with an isoelectric point (pI) of 8.1-8.3. However, this 55K protein was not observed with a two-dimensional gel electrophoresis carried out on plasma membrane fractions prepared from the liver, heart, and kidney tissues. An analysis of the 12 amino acids sequence at the N-terminal of the 55K protein showed that it has a similar sequence to that of bovine serum albumin.

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Electrophoretical Analysis of 36-Kilodalton Outer Membrane Protein of Vibrio vulnificus ATCC 27562

  • Moon-Soo Heo;Cho-Rok Jung
    • Journal of Life Science
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    • v.9 no.1
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    • pp.35-39
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    • 1999
  • Elecrophoreticl analysis of a 36 kDa protein was runned by SDS-PAGE, isoelectric focusing (IEF) and two dimensional electrophoresis pattern. Major 36 kDa and 25, 46, 48, 66 kDa protein were detected by Coomassie blue stain on SDS-PAGE. Major 36kDa protein was eluted for production of antiserum for serological analysis, IEF and two dimensional electrophoresis. Isoelectric point of 36kDa was aout pH 8.5. Two dimensional electrophoresis of eluted 36kDa showed one point on the gel. Anti-36 kDa serum made by newzilland rabbit for serological test. In ELISA, final titer of antibody was 100×{TEX}$2^5}${/TEX} : 1. Neutralize ability of serum was examined by slide agglutination test and colonization test in rat. Anti-36 kDa serum agglutinated whole cell of V. vulnificus were inhibited colonization on intestine in rat. Accordingly In this paper contain some electrophoretical analysis and serological test of a 36 kDa OMP of V. vulnificus.

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Enhanced Proteomic Analysis of Streptomyces peucetius Cytosolic Protein Using Optimized Protein Solubilization Protocol

  • Lee, Kwang-Won;Song, Eun-Jung;Kim, June-Hyung;Lee, Hei-Chan;Liou, Kwang-Kyoung;Sohng, Jae-Kyung;Kim, Byung-Gee
    • Journal of Microbiology and Biotechnology
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    • v.17 no.1
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    • pp.89-95
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    • 2007
  • Improvements in the dissolution of proteins in two-dimensional gel electrophoresis have greatly advanced the ability to analyze the proteomes of microorganisms under a wide variety of physiological conditions. This study examined the effect of various combinations of chaotropic agents, a reducing agent, and a detergent on the dissolution of the Streptomyces peucetius cytosolic proteins. The use of urea alone in a rehydration buffer as a chaotropic agent gave the proteome a higher solubility than any of the urea and thiourea combinations, and produced the highest resolution and clearest background in two-dimensional gel electrophoresis. Two % CHAPS, as a detergent in a rehydration buffer, improved the protein solubility. After examining the effect of several concentrations of reducing agent, 50 mM DTT in a rehydration buffer was found to be an optimal condition for the proteome analysis of Streptomyces. Using this optimized buffer condition, more than 2,000 distinct and differentially expressed soluble proteins could be resolved using two-dimensional gel electrophoresis with a pI ranging from 4-7. Under this optimized condition, 15 novel small proteins with low-level expression, which could not be analyzed under the non-optimized conditions, were identified. Overall, the optimized condition helped produce a better reference gel for Streptomyces peucetius.

Comparative Proteomics Analysis of Colorectal Cancer

  • Wang, Jun-Jiang;Liu, Ying;Zheng, Yang;Lin, Feng;Cai, Guan-Fu;Yao, Xue-Qing
    • Asian Pacific Journal of Cancer Prevention
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    • v.13 no.4
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    • pp.1663-1666
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    • 2012
  • Background and Objective: Protein expression in colon and rectal cancer (CRC) and paired normal tissues was examined by two-dimensional gel electrophoresis (2-DE) to identify differentially expressed proteins. Materials and Methods: Five fresh colorectal cancer and paired adjacent normal tissues were obtained and differentially expressed protein spots were determined using PDQuest software, with identification on the basis of MALDI-TOF mass spectra. Results: Compared with normal colorectal mucosa, protein abnormal expression of 65 spots varying more than 1.5 times were found in 2-DE gels from colorectal cancer samples (P<0.05); forty-two proteins were up-regulated and 23 were down-regulated; twelve protein spots were identified using mass spectrometry, of which 8 were up-regulated, includimng HSPB1and Annexin A4, while 4 were down-regulated, the results being consistent with Western blot findings. Conclusions: Two-dimensional electrophoresis reference maps for CRC tissues and adjacent normal mucosa (NMC) were established and 12 differentially expressed proteins were identified. Up-regulated HSPB1 and Annexin A4 may play many important roles in the pathogenesis of colorectal cancer.

Evaluation of a Pretreatment Method for Two-Dimensional Gel Electrophoresis of Synovial Fluid Using Cartilage Oligomeric Matrix Protein as a Marker

  • Kong, Min-Kyung;Min, Byoung-Hyun;Lee, Pyung-Cheon
    • Journal of Microbiology and Biotechnology
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    • v.22 no.5
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    • pp.654-658
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    • 2012
  • Osteoarthritis (OA) is the most common rheumatic pathology. One of the major objectives of OA research is the development of early diagnostic strategies such as those using proteomic technology. Synovial fluid (SF) in OA patients is a potential source of biomarkers for OA. The efficient and reliable preparation of SF proteomes is a critical step towards biomarker discovery. In this study, we have optimized a pretreatment method for twodimensional gel electrophoresis (2DE) separation of the SF proteome, by enriching low-abundance proteins and simultaneously removing hyaluronic acid, albumin, and IgG. SF samples pretreated using this optimized method were then evaluated by 1DE and 2DE separation followed by immunodetection of cartilage oligomeric matrix protein (COMP), a known OA biomarker, and by the identification of 3 proteins (apolipoprotein, haptoglobin precursor, and fibrinogen D fragment) that are related to joint diseases.

Protein Separation in Functional Rice Grains Using Two-Dimensional Gel Electrophoresis

  • Chung, Soo Im;Lee, Sang Chul;Kan, Mi Young
    • KOREAN JOURNAL OF CROP SCIENCE
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    • v.58 no.4
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    • pp.388-392
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    • 2013
  • The proteins from functional rice cultivars (Nogwonchalbyeo, Giant embryonic, Arhyangchalbyeo, and Goamibyeo) and general white rice were extracted and separated using two-dimensional (2D) gel electrophoresis. A wide variation in the molecular weight (MW) and pH range of the expressed proteins in rice samples were observed. The green-kerneled rice (Nogwonchalbyeo) exhibited proteins with MW of 9-57 kDa and appeared at a pH range of 4-7. The Giant embryonic contained proteins with MW of 31-63 kDa and a pH range of 5-6. The aromatic glutinous rice (Arhyangchalbyeo) showed proteins with MW of 24-28 and pH of 5.8-6.8. The high-amylose rice (Goamibyeo) exhibited proteins with MW of 3-63 and pH of 5.2-5.6. The identified proteins uniquely found and highly expressed in each cultivar may have a significant role on rice functionality. The results illustrate that the 2D gel electrophoresis is a valuable method in the determination of the protein expression profiles in functional rice grains and may be useful in the identification of specific marker proteins associated with the functional property of rice.