• Title/Summary/Keyword: gradient gel

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Proteome Analysis of Amniotic Fluid by gradient 2-D PAGI (Gradient 2-D PAGE를 이용한 양수 프로테옴 분석)

  • 이은희;김재찬;변상요
    • KSBB Journal
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    • v.18 no.1
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    • pp.35-38
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    • 2003
  • Analysis of proteome in amniotic fluid was performed by 2-D PAGE (polyacrylamide gel electrophoresis). Proteins in amniotic fluid were separated by centrifugation and solubilized in buffer solution for IEF, using an IPG strip of pH 4-7L. Both a homogeneous slab gel of 12.5% and a gradient gel of 8-18%, were used. After 2-D PAGE, spots were stained with silver nitrate and picked up for in-gel digestion. Digested peptides were analyzed by MALDI-TOF and proteins were further identifical. More protein spots were detected in the gradient gels and a protein not previously reported was identified.

Bacterial Community Analysis during Composting of Garbage using Denaturing Gradient Gel Electro-phoresis (Denaturing Gradient Gel Electrophoresis를 이용한 음식물 쓰레기 퇴비화 세균 군집 분석)

  • Ryu Hee Wook;Cho Kyung-Suk
    • Microbiology and Biotechnology Letters
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    • v.33 no.3
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    • pp.226-230
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    • 2005
  • The microbial community during composting of gargage was analyzed using 16S rDNA PCR - DGCE (denaturing gradient gel electrophoresis). Pseudomonas spp. was found throughout the process, and thermophilic Bacillus spp. was dominated at the thermophilic stage. Six thermophilic bacteria were isolated and identified as B. caldoxylolyticus, B. thermoalkalophilus, and B. thermodenitrificans.

Detection of Mutated DNA Fragment by the Heteroduplex Analysis at the Temperature Gradient Gel (온도 기울기(temperature gradient) 젤에서 Heteroduplex Analysis 기법을 이용한 돌연변이 DNA의 검출)

  • 조용석;구미자;박귀근;박영서;강종백
    • Environmental Mutagens and Carcinogens
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    • v.18 no.2
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    • pp.83-88
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    • 1998
  • To detect the mutation in a given sequence, there are variety of methods developed by use of the gel electrophoresis. One of the methods, TGGE (Temperature Gradient Gel Electrophoresis), is a popular technique because it can detect mutations in DNA fragment with ease and at low cost. This study used 200 bp BamHI-digested DNA fragment containing the human $\varepsilon$-globin promoter which was mutated[$\varepsilon$ F1*(-141), GATA- I*(-163), and GATA-1* & $\varepsilon$F1]. This BamHI-digested DNA fragment was directly used to detect the mutated DNA fragment on 50% denaturant gel with temperature gradient of 45$^{\circ}C$ through $53^{\circ}C$. In agreement with the theoretical result of MELTSCAN program (Brossette and Wallet, 1994) the mobilities of mutated DNA fragments were shown to be nearly distinguished on the temperature gradient gel. In contrast to the above result the heteroduplex analysis under the temperature gradient condition was shown to detect the mutated DNA fragments through the heteroduplex formation between strands of mutated DNA and wild-type DNA.

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Korean Paddy Soil Microbial Community Analysis Method Using Denaturing Gradient Gel Electrophoresis (Denaturing gradient gel electrophoresis를 이용한 한국의 논 토양 미생물 다양성 분석 방법)

  • Choe, Myeongeun;Hong, Sung-Jun;Lim, Jong-Hui;Kwak, Yunyoung;Back, Chang-Gi;Jung, Hee-Young;Lee, In-Jung;Shin, Jae-Ho
    • Journal of Applied Biological Chemistry
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    • v.56 no.2
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    • pp.95-100
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    • 2013
  • Soil microbes are important integral components of soil ecosystem which have significant and diverse role in organic matter decomposition, nitrogen cycling, and nitrogen fixation. In this study an effective denaturing gradient gel electrophoresis (DGGE) method was employed for paddy soil microbial diversity survey. For optimum paddy soil microbial DNA extraction, different methods such as Lysis buffer, skim milk bead, sodium phosphate buffer, Epicentre Soil Master DNA extraction kit (Epicentre, USA) and Mo Bio Power Soil DNA kit (MO BIO, USA) methods were utilized. Among all the method, using Mo Bio Power Soil kit was most effective. DGGE analysis of Bacteria was carried out at 6% polyacylamide gel and 45-60% denaturing gradient in the optimal conditions. Whereas DGGE analysis of fungi was done at 6% polyacrylamide gel and 45-80% denaturing gradient in the optimal conditions. By applying the above assay, it was found that variation within the microbial community of paddy soil occurs by a factor of time. DGGE assay used in this study through for a variety of soil microbial analysis suggests the potential use of this method.

Appropriate Electrophoresis Techniques and Isozymes to Identification of Barley Cultivars (보리품종 구분에 적합한 전기영동법과 효소)

  • Son, Eung-Ryong;Lee, Yong-Se;Yoon, Kyung-Eun;Ha, Yong-Woong
    • KOREAN JOURNAL OF CROP SCIENCE
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    • v.30 no.4
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    • pp.405-411
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    • 1985
  • The buffer soluble proteins were extracted from six cultivars of barley grains and analyzed by various electrophoresis; 7.5% polyacrylamide slab gel, 2-30% polyacrylamide porosity gradient tube gel, isoelectric focusing (pH4-9) and starch gel electrophoresis. The proteins, esterase, acid phosphatase, malate dehydrogenase, glutamate dehydrogenase and leucine aminopeptidase were investigated to find out the best method to differentiate barley cultivars. The result were that protein and esterase bands in 2-30% polyacrylamide porosity gradient tube gel electrophoresis and protein bands in 7.5% polyacrylamide slab gel electrophoresis showed typical varietal differences. Therefore, those methods were suitable for differentiation of barley cultivars. It was difficult to differentiate the cultivars by the other methodes and patterns of the other enzymes.

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Separation of X-and Y-Bearing Spermatozoa IV. Separtion of bull spermatozoa by the combination of density Gradient Centrifugation and Sephadex Gel filtration (X-정자와 Y-정자의 분류에 관한 연구 IV. Sephadex Gel여과법과 Percoll중층원심분류법의 병용에 의한 우 정자의 분류)

  • 이주영;정길생;김종배
    • Korean Journal of Animal Reproduction
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    • v.12 no.3
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    • pp.141-147
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    • 1988
  • These experiments were carried out to develop new techniques for in vitro separation of x-and Y-bearing spermatozoa. The results obtained in these experiments were summarized as follows: 1. Following centrifugation of discontinuous percoll density gradient, populatin of spermatozoa increased progressively from low to high density. The highest concentration of spermatozoa was observed at the 4th fraction which included 36.6% of spermatozoa. 2. As increasing percoll concentration, the higher motility index was obtained and the highest motility index(74.2) was obtained at the 5th fraction. 3. The percentage of B-body bearing spermatozoa following percoll density gradient centrifugation was decreased from 39.7% to 25.6%. 4. The sperm population following chromatography by sephadex gel and percoll density gradient centrifugation was decreased in 1st, 5th and 6th fractions but the reverse was turn for 2nd, 3rd and 7th fractions, and the highest sperm concentration was observed at the 7th fraction which included 37.4% of spermatozoa. 5. Motility index of spermatozoa was increased from 77.6 to 79.4 after the sephadex gel filtration, however it was decreased at all fractions after percoll density gradient centrifugation. The lowest motility index(33.2) was obtained from the 7th fraction. 6. The rate of B-body bearing spermatozoa was shown the trend to decrease by the sephadex gel filtration and the trend was accelerated by the percoll density gradient centrifugation. The lowest percentage of B-body bearing spermatozoa, 12.0% was obtained from the 5th fraction.

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Analysis of Vaginal Lactic Acid Producing Bacteria in Healthy Women

  • Nam, Hye-Ran;Whang, Kyung-Hee;Lee, Yeon-Hee
    • Journal of Microbiology
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    • v.45 no.6
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    • pp.515-520
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    • 2007
  • Vaginal lactic acid-producing bacteria of 80 pre-menopausal women were studied by isolation on Blood and DeMan-Rogosa-Sharpe agar, PCR with group-specific primers for Lactobacillus-denaturing gradient gel electrophoresis (DGGE), and PCR with specific primers for V3 region in 16S rRNA-temporal temperature gel electrophoresis (TTGE). Conventional isolation method on media detected only one lactobacillus (Lactobacillus brevis) while TTGE detected only Lactobacillus sp. DGGE detected seven Lactobacillus species; L. coleohominis, L. crispatus, L. iners, L. reuteri, L. rhamnosus, L. vaginalis, and Leuconostoc lactis. L. acidophilus and L. gasseri, which are prevalent in Western women, were not detected in Korean women. Furthermore, L. rhamnosus, Leuc. lactis, L. coleohominis, and Weissella cibaria, which were not previously reported in the vaginal microbiota of Korean women, were detected. The five most prevalent LABs in vaginal microbiota in Korean women were L. iners, Enterococcus faecalis, L. crispatus, Leuc. lactis, and W. cibaria.

Further Purification of Radioprotective Ginseng Protein Fraction by Gel Filtration (Gel filtration에 의한 한방사선 인삼단백 분획의 정제)

  • 김춘미;박경애
    • Journal of Ginseng Research
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    • v.13 no.2
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    • pp.254-259
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    • 1989
  • A radioprotective ginseng protein fraction was obtained from Korean white ginseng powder by the following isolation and purification procedures: Tris-HCI buffer extraction, 70% ammonium sulfate fractionation, CM-rellulosr column chromatography, heat inactivation and Sephadex G-75 column chromatography. This fraction was further purified by Sepharose 4B and Sephadex G-150 column chromatographies. Three fractions obtained were subjected to Native-PAGE and SDS-PAGE using gradient gels and the silver staining method. Molecular weights of the native proteins and their subunits were estimated.

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Rapid Purification of Glucose-6-Phosphate Dehydrogenase by Affinity Chromatography (Affinity Chromatography를 이용한 Glucose-6-Phosphate Dehydrogenase의 신속한 정제방법 개발)

  • 이한수;임정빈
    • Korean Journal of Microbiology
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    • v.21 no.4
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    • pp.221-228
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    • 1983
  • An improved procedure for the rapid purification of glucose-6-phosphate dehydrogenase from extracts of Saccharomyces cerevisiae was developed by using affinity chromatography. Among six affinty media tested, $NADP^+ -agarose$ and Affi-gel Blue were more effective than others (i.e., Affi-gel Red, AMP-agarose, ATP-agarose, and $NAD^+ -agarose$). Conditions to desorb the enzyme bound to the affinity media were examined to increase the purity as well as yield. The best result was obtained when the column was developed with a linear gradient of KCl (0-1.0M). In case of Affi-gel Blue, introduction of $NAD^+$ (15mM) washing step prior to the salt gradient was most effective to remove $NAD^+ -binding$ proteins. For a large scale preparation of G-6-P dehydrogenase higher recovery was obtained by Affi-gel Blue than $NADP^+ -agarose$, however, the purity of the enzyme was decreased by 10 times if the former was used as the affinity medium. The capacity of Affi-gel Blue for G-6-P dehydrogenase was found to be 5 times higher than that of $NADP^+ -agarose$. Furthermore Affi-gel Blue could be reused repeatedly and its preparation is relatively easier and less expensive than $NADP^+ -agarose$.

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Electrophoretic Mobilities of the Potato Spindle Tuber Viroid RNA Molecules in the Urea-Gradient Gels (감자 걀쪽바이로드(PSTV) RNA 분자의 요소농도기울기겔에서 전기영동적 이동성에 관하여)

  • 이재열
    • Korean Journal of Microbiology
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    • v.25 no.1
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    • pp.23-27
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    • 1987
  • Low molecular weight plant ribonucleic acids including viroid-RNA molecules which are soluble in 2M lithium chloride were electrophoresed in the 0M to 8M urea-gradient polyacrylamide gel. Although the linear viroid-RNA molecules migrated at a similarrate across the urea-gradient gel under the denaturing temperature, the circular viroid-RNA molecules moved more rapidly at low urea-gradient region than at high urea-gradient region. Consequently, the migration of the circular viroid-RNA molecules showed a sudden shift across the band of linear forms in the midrange of the urea-gradient gels. Electrophoretic mobilities of the circular viroid-RNA molecules seemed to depend mainly on the concentration of urea in the denaturing urea-gradient gels.

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