• Title/Summary/Keyword: Capillary gel electrophoresis

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Fast Microchip Electrophoresis Using Field Strength Gradients for Single Nucleotide Polymorphism Identification of Cattle Breeds

  • Oh, Doo-Ri;Cheong, Il-Cheong;Lee, Hee-Gu;Eo, Seong-Kug;Kang, Seong-Ho
    • Bulletin of the Korean Chemical Society
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    • v.31 no.7
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    • pp.1902-1906
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    • 2010
  • A microchip electrophoresis (ME) method was developed using a programmed field strength gradients (PFSG) for the single nucleotide polymorphism (SNP) based fast identification of cattle breeds. Four different Korean cattle (Hanwoo) and Holstein SNP markers amplified by allele-specific polymerase chain reaction were separated in a glass microchip filled with 0.5% poly(ethyleneoxide) ($M_r$ = 8 000 000) by PFSG as follows: 750 V/cm for 0 - 14 s, 166.7 V/cm for 14 - 31 s, 83.3 V/cm for 31 - 46 s, and 750 V/cm for 46 - 100 s. The cattle breeds were clearly distinguished within 45 s. The ME-PFSG method was 7 times and 5 times faster than the constant electric field ME method and the capillary electrophoresis- PFSG method, respectively, with a high resolving power ($R_s$ = 5.05 - 9.98). The proposed methodology could be a powerful tool for the fast and simultaneous determination of SNP markers for various cattle breeds with high accuracy.

Analysis of Androgen Receptor Gene by Capillary Gel Eelectrophoresis (모세관 젤 전기영동을 이용한 안드로젠 수용체 유전자 분석)

  • Kim, Yong-Seong;Baek, Seung-Gwon;Gang, Chung-Mu;Gang, Dae-Cheon;Lee, Su-Man;Choe, Byeong-Ok;Jeong, Gi-Hwa;Choe, Gyu-Seong
    • Journal of the Korean Chemical Society
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    • v.50 no.1
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    • pp.37-45
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    • 2006
  • gel electrophoresis (CGE) with poly(ethylene oxide) has been applied to the measurement of CAG repeat number in Androgen receptor (AR) gene related to male infertility. Non-linear regression analysis was performed using the standard X174 RF/Hae III, 100bp step ladder DNA in order to investigate the exact number of CAG repeat. For 79 Korean infertile males and 89 controls, CAG repeats at exon 1 in AR gene was compared and analyzed by CGE. It turned out that CAG repeat numbers were 24.972.6 range, 17-29) for the infertile male, and 23.992.4 range, 18-29) for the control, respectively. P value (0.018) was less then 0.05, meaning that the result was statistically meaningful.

Purification and Characterization of Angiotensin I Converting Enzyme lnhibitory Peptides from Enzymatic Hydrolysate of Cod Liver Protein (대구의 간 단백질의 효소적 가수분해물로부터 안지오텐신 I 전환효소 저해 펩타이드의 분리.정제 및 특성)

  • 최영일;박표잠;최정호;변희국;정인철;문성훈;김세권
    • Journal of Life Science
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    • v.10 no.2
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    • pp.140-149
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    • 2000
  • In order to utilize marine processing waste which would normally be discarded, cod liver protein was hydrolysed by ${\alpha}$-chymotrysin, and the hydrolysate was investigated for the new angiotensin I converting enzyme (ACE) inhibitor. Thy hydrolysate was separated into three major types, with molecular weight cut-off (MWCO) values less than 10 kDa, 5 kDa and 1 kDa of ultrafiltration membranes, respectively. ACE inhibitory peptides were isolated from the fractions passed through MWCO 1 kDa membrane, and purified by using ion-exchange chromatography on a SP-Sephadex C-25 column, gel filtration on a Sephadex G-15 column, and HPLC on an ODS column. The purity was identified with capillary electrophoresis. The amino acid sequences of two peptides were Met-Ile-Pro-Pro-Tyr-Tyr (IC50=10.9 ${\mu}$M) and Gly-Leu-Arg-Asn-Gly-Ile (IC50=35.0 ${\mu}$M)

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A Comprehensive Identification of Synaptic Vesicle Proteins in Rat Brains by cRPLC/MS-MS and 2DE/MALDI-TOF-MS

  • Lee, Won-Kyu;Kim, Hye-Jung;Min, Hye-Ki;Kang, Un-Beom;Lee, Cheol-Ju;Lee, Sang-Won;Kim, Ick-Young;Lee, Seung-Taek;Kwon, Oh-Seung;Yu, Yeon-Gyu
    • Bulletin of the Korean Chemical Society
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    • v.28 no.9
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    • pp.1499-1509
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    • 2007
  • Proteomic analyses of synaptic vesicle fraction from rat brain have been performed for the better understanding of vesicle regulation and signal transmission. Two different approaches were applied to identify proteins in synaptic vesicle fraction. First, the isolated synaptic vesicle proteins were treated with trypsin, and the resulting peptides were analyzed using a high-pressure capillary reversed phase liquid chromatography/tandem mass spectrometry (cRPLC/MS/MS). Alternatively, proteins were separated by two-dimensional gel electrophoresis (2DE) and identified by matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF/MS). Total 18 and 52 proteins were identified from cRPLC/MS-MS and 2DE-MALDI-TOF-MS analysis, respectively. Among them only 2 proteins were identified by both methods. Of the proteins identified, 70% were soluble proteins and 30% were membrane proteins. They were categorized by their functions in vesicle trafficking and biogenesis, energy metabolism, signal transduction, transport and unknown functions. Among them, 27 proteins were not previously reported as synaptic proteins. The cellular functions of unknown proteins were estimated from the analysis of domain structure, expression profile and predicted interaction partners.

Action of atmospheric pressure non-thermal plasma on the biomolecules and bio-organism

  • Attri, Pankaj;Park, Ji Hoon;Kumar, Naresh;Ali, Anser;Kim, In Tae;Lee, Weontae;Choi, Eun Ha
    • Proceedings of the Korean Vacuum Society Conference
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    • 2015.08a
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    • pp.66.1-66.1
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    • 2015
  • Plasma medicine is an upcoming research area that has attracted the scientists to explore more deeply the utility of plasma. So, apart from the treating biomaterials and tissues with plasma, we have studied the effect of plasma with different feeding gases on modification of biomolecules. Additionally, we have checked the action of nanosecond pulsed plasma on the biomolecules. We have checked the plasma action on proteins ((Hemoglobin (Hb) Myoglobin (Mb) and lysoenzyme), calf thymus DNA and amino acids. The structural changes or structural modification of proteins and DNA have been studied using circular dichroism (CD), dynamic light scattering (DLS), gel electrophoresis, protein oxidation test, UV-vis spectroscopy and 1D NMR, while Liquid Chromatograph/Capillary Electrophoresis-Mass Spectrometer(LC/CE-MS) based qualitative bio-analysis have been used to study the modification of amino acids. We have also shown the effect of NaCl and ionic liquid on the formation of OH radicals using electron spin resonance and fluorescence techinques.

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Purification and Characterization of Antioxidative Peptides from Enzymatic Hydrolysate of Cod Teiset Protein (대구고니 단백질의 효소적 가수분해물로부터 항산화성 펩타이드의 분리${\cdot}$정제 및 특성)

  • KIM Se-Kwon;CHOI Yong-Ri;PARK Pyo-Jam;CHOI Jeoung-Ho;MOON Sung-Hoon
    • Korean Journal of Fisheries and Aquatic Sciences
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    • v.33 no.3
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    • pp.198-204
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    • 2000
  • In order to utilize by-products which would normally be discarded in marine processing plants, cod teiset protein was hydrolyzed and antioxidative actiTity of the hydrolysate was investigated. AntioxidatiTe peptide was isolated using ultrafiltration membrane, ion-exchange chromatography on a SP-Sephadex C-25 column, gel filtration on a Sephadex G-15 column, high performance liquid chromatography on an ODS column, and capillary electrophoresis chromatography. Antioxidative activities of the cod teiset hydrolysate were compared with ${\alpha}-tocopherol$, one of the commercial antioxidant. The hydrolysate passed through a membrane with molecular weight cut-off (MWCO) 1 kDa was shown the strongest antioxidative activity, and the activity was higher $10{\%}$ as compared with ${\alpha}-tocopherol$. In addition, the peptide isolated by ion-exchange chromatography, gel filtration, and HPLC, respectively, was higher $53{\%}$ as compared with ${\alpha}-tocopherol$, and the amino acid sequence was Ser-Asn-Pro-Glu-Trp-Ser-Trp-Asn.

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The Effect of Root Exudate and Chemotaxis on Host Recognition in Soybean-Bradyrhizobium Symbiosis (대두(大豆)-근류균(根瘤菌) 공생(共生)에서 뿌리분비물(分泌物)과 화학주성(化學走性)이 숙주인식(宿主認識)에 미치는 영향(影響))

  • Kang, Sang Jai;Park, Woo Churl
    • Current Research on Agriculture and Life Sciences
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    • v.11
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    • pp.121-132
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    • 1993
  • This study was carried out to research the effect of the chemotaxis of Bradyrhizobium japonicum KCTC 2422 and its mutant toward soybean root exudate and to elucidate the effect of the lectin of host specificity (Host Recognition) in soybean-Bradyrhizobium symbiosis. The results obtained were as follows: The homogeneities of the purified lectins from soybean and pea seed was ascertained chromatographically and electrophoretically. Gel electrophoresis of soybean lectin in the presence of sodium dodecyl sulfate appeared a single protein band, whereas pea lectin appeared two protein bands. Soybean lectin from 2 cultivars formed immunoprecipitin arcs at same position with anti-soybean lectin rabbit IgG, but pea lectin did not form immunoprecipitin lines with anti-soybean lectin rabbit IgG. Chemotactic responses of KCTC 2422, LPN-100 and LCR-101 toward proline in capillary assays were 3.1, 1.3 and 1.0-fold above background, respectively. The chemotactic responses of KCTC 2422, LPN-100, and LCR-101 toward Paldal crude root exudate in capillary assays were 3.5, 1.4 and 1.4-fold above background, respectively. The present work shows that B. japonicum and its mutants are capable of very different responses toward root exudate fraction. The chemotactic responses of KCTC 2422 was most with neutral fraction, least with anionic fraction and intermediate with cationic fraction. The nitrogenase activity of soybean nodule was shown in 15days after inoculation with LCR-101. However, we couldn't find out the nodules when soybean was inoculated with LPN-100. From these result we can suppose that the chemotaxis of Bradyrhizobium plays inportant the role of forming the nodule (host recognition) in the soybean-B. japonicum symbiosis.

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Southern Analysis after Long-range PCR: Clinical Application in Korean Patients with Myotonic Dystrophy 1

  • Yum, Mi-Sun;Lee, Beom Hee;Kim, Gu-Hwan;Lee, Jin-Joo;Choi, Seung Hoon;Lee, Joo Yeon;Kim, Jae-Min;Kim, Yoo-Mi;Ko, Tae-Sung;Yoo, Han-Wook
    • Journal of Genetic Medicine
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    • v.10 no.1
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    • pp.33-37
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    • 2013
  • Purpose: Myotonic dystrophy 1 (DM1, OMIM 160900) is an autosomal-dominant muscular disorder caused by an expansion of CTG repeats in the 3' UTR of the DMPK gene. Variable expansions of CTG repeats preclude the accurate determination of repeat size. We tried to show the clinical and analytical validity of the application of Southern blotting after long-range PCR was demonstrated in Korean DM1 patients. Materials and Methods: The Southern blotting of long-range PCR was applied to 1,231 cases with clinical suspicion of DM1, between 2000 and 2011. PCR was performed using genomic DNA with forward 5'-CAGTTCACAACCGCTCCGAGC-3' and reverse 5'-CGTGGAGGATGGAACACGGAC-3' primers. Subsequently, the PCR fragments were subjected to gel electrophoresis, capillary transfer to a nylon membrane, hybridization with a labeled (CAG)10 probe. The correlation between clinical manifestations and the CTG repeat expansions were analyzed. Results: Among a total of 1,231 tested cases, 642 individuals were diagnosed with DM1 and the range of the detected expansion was 50 to 2,500 repeats; fourteen cases with mild DM1 ($75{\pm}14$ repeats), 602 cases with classical DM1 ($314{\pm}143$ repeats), and 26 cases with congenital DM1 ($1,219{\pm}402$ repeats). The positive and negative predictive values were 100%. The age at test requested and the CTG repeat numbers were inversely correlated (R=-0.444, P<0.01). Conclusion: This study indicates that Southern blotting after long-range PCR is a reliable diagnostic method DM1.

Development of species-specific multiplex PCR assays of mitochondrial 12S rRNA and 16S rRNA for the identification of animal species (식육감별을 위한 미토콘드리아 12S rRNA와 16S rRNA 유전자의 종 특이적 multiplex PCR 기법 개발)

  • Koh, Ba-Ra-Da;Kim, Ji-Yeon;Na, Ho-Myung;Park, Seong-Do;Kim, Yong-Hwan
    • Korean Journal of Veterinary Service
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    • v.34 no.4
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    • pp.417-428
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    • 2011
  • Species-specific PCR assay was developed for detection of cattle, sheep, goat, horse, dog, pig, chicken, duck, goose, and turkey using mitochondrial 12S rRNA and 16S rRNA as target genes. Also, an internal positive control was used to detect possible false negatives by using 18S rRNA gene. We designed species-specific primers with amplicon length of 190, 219, 350, 467, 241, 119, 171, 229, 111 and 268 bp for cattle, sheep, goat, horse, dog, pig, chicken, duck, goose, and turkey respectively. The specificity of the primers was tested against the other 10 non-target animal species and a cross-reaction was not observed. We developed two multiplex PCR assays for the simultaneous identification of Korea's major livestock species (cattle, pig, chicken and duck) and poultry species (chicken, duck, goose and turkey) from analogous samples, retaining the same specificity. The limit of detection of the multiplex PCR assay (cattle, pig, chicken and duck) ranged between 1 pg and 0.1 pg of template DNA extracts from raw meat. Applying multiplex PCR assays to DNA extracts from experimental pork/beef and pork/chicken tested raw and heat-treated ($120^{\circ}C$ for 30 min) mixtures respectively, detection limit was 0.1% level beef in pork, pork in beef and chicken in pork and 1.0% level pork in chicken. In conclusion, this assay using gel-based capillary electrophoresis would be very useful in highly sensitive and rapid identification of animal species or ingredients in minced meat and other meat products.