• Molecules and Cells
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• 제22권1호
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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.

• Asian-Australasian Journal of Animal Sciences
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• 제16권7호
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• pp.1041-1045
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• 2003

In order to study the reaction behaviors of bovine $\alpha$-lactalbumin ($\alpha$-La), $\beta$-lactoglobulin ($\beta$-Lg), and their mixtures during heat treatment, samples were analyzed using native-polyacrylamide gel electrophoresis (Native-PAGE), sodium dodecylsulfate (SDS)-PAGE, and two-dimensional (2-D)-PAGE. The electrophoresis demonstrated that the loss of native-$\alpha$-La increased as temperature increased, and that the loss of apo-$\alpha$-La was slightly higher than that of holo-$\alpha$-La. The tests also showed that during heat treatment, a mixture of $\alpha$-La and $\beta$-Lg was less stable than $\alpha$-La alone. As such, it was assumed that $\beta$-Lg induced holo-$\alpha$-La to be less stable than apo-$\alpha$-La during heat treatment. The reaction behavior of $\alpha$-La (holo-, apo-form) during heat treatment showed similar patterns in the 2-D-PAGE electropherogram, but the mixture of $\alpha$-La and $\beta$-Lg created new bands. In particular, the results showed a greater loss of native $\alpha$-La in the holo-$\alpha$-La and $\beta$-Lg mixture than in the apo-$\alpha$-La and $\beta$-Lg mixture. Thus, it can be concluded that the holo-$\alpha$-La and $\beta$-Lg mixture was more intensively affected by heat treatment than other samples, and that free sulphydryl groups took part in the heat-induced denaturation.

• 한국동물학회지
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• 제29권2호
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• pp.86-106
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• 1986

사람의 혈액중 혈청으로써 임신의 진행에 따른 단백질의 변화 양상을 조사함에 있어서 정상인 남자, 비임신 여자, 임신한 여자 및 분만 직후 여자의 혈청을 SDS/polyacrylamide gel 전기영동과 2차원 전기영동 및 아미노산분석을 시도하였다. 각 실험 방법에 의해 분석된 결과는 다음과 같다. 1. SDS/polyacrylamide gel 전기영동에 의해 분자량 약 10,000에서 110,000 dalton 사이의 정상인 남자와 비임신 여자를 비교하였을 때의 숫적 양상은 서로 동일하였으나 bands 3 (22,000 dalton)과 6(39,000)등은 남자에 있어서 여자보다 적은 양으로 나타났다. 비임신 여자와 2주 간격으로 혈액을 채취한 임신한 여자의 혈청단백질 패턴을 비교하면 여러 band들의 양적인 증감이 관찰되었다. 임심한 여자의 단백질은 비임신에 비해 band 3(22,000)이 16주까지는 비임신의 경우와 거의 비슷한 양상을 보였으나 18주부터 임신 말기까지는 단백질의 양이 계속하여 감소되었다. 반면 bands 4(24,000), 9(69,000), 10(70,000), 12(80,000), 13(83,000), 14(86,000), 15(91,000) 및 16(94,000)은 임신이 진행됨에 따라 일반적으로 증가되나 그 이후에는 약간씩 감소하였다. 분만 직후의 혈청단백질의 패턴을 임신 말기와 비교하였을 때 bands 12(80,000), 15(91,000) 및 16(94,000)들의 양이 비교적 증가하였고 여아를 분만한 경우 남아를 분만한 경우보다 bands 4(24,000), 7(51,000) 및 10(70,000)들의 단백질 양이 많음이 관찰되었다. 2. 2차원 전기영동으로 남자와 비임신 여자의 혈청단백질 패턴을 비교하면 spot a(22,000)의 3개의 spot가 남자에 있어서는 나타나지 않았고, spotc(39,000)의 군은 남자에 있어 여자보다 농도가 매우 낮았다. 임신한 여자에 있어 albumin이 임신 10주와 12주에 매우 감소하였고 그 이후에 다시 회복되었다. 그리고 spot f(70,000)는 임신 10주에 매우 감소 되었다가 그 이후에 증가되었다. 3. 각 군들의 아미노산을 분석한 결과 임신한 여자에 있어서 전반적으로 glutamic acid, lysine, aspartic acid, leucine 및 valine이 대체로 많은 양으로 나타났고, methionine, isoleucine 및 glycine이 대체로 적은 양을 가지는 것으로 나타났다. 전체 아미노산의 양은 임신중기에 현저히 증가하였다가 말기에는 중기에 비해 감소되었다. 이상의 결과를 볼 때 임신기간 중에는 모체의 혈청을 구성하는 단백질이 특정한기에 따라 증가하므로 임신한 여자의 혈청단백질을 조사함으로써 임신의 각기에 나타나는 독특한 단백질 양상 변화를 밝혀내는 동시에, 앞으로 좀더 연구함으로써 태아의 성을 단백질 패턴으로 명확히 구별해 낼 수 있을 것으로 생각된다.

• 한국작물학회지
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• 제51권spc1호
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• pp.92-102
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• 2006

본 연구는 벼종자 미량 단백질의 프로테오믹스 연구를 위하여 벼종자에 고 함량으로 존재하는 벼종자 글루테린 저장 단백질을 제거하는 방법에 관한 것이다. 따라서 본 연구는, (A) 벼종자에 액체 질소를 가하고 분쇄하여 벼종자 가루를 만드는 분쇄단계; (B) 상기 분쇄된 벼종자 가루를 물에 현탁하여 현탁액을 만드는 현탁단계; (C)상기 현탁액 중 미용해 물질을 제거하는 분리단계를 포함하는, 벼종자 미량 단백질의 프로테오믹스 연구를 위한 벼종자 글루테린 저장 단백질의 제거방법에 관하여 검토하였다. 본 연구의 결과, 단순하고 신속하며 저렴하고 효율적인 방법으로 미량 비글루테린 단백질들을 용이하게 동정할 수 있을 것으로 판단되었다.

• Reproductive and Developmental Biology
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• 제32권4호
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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.

• ALGAE
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• 제26권1호
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• pp.87-96
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• 2011

Protein profiles of a common mixotrophic dinoflagellate, Prorocentrum micans, growing autotrophically and mixotrophically (fed on the cryptophyte Rhodomonas salina) were compared using two-dimensional gel electrophoresis (2-DE) to determine if they vary in different trophic modes. Approximately 2.3% of the detected proteins were differentially expressed in the different trophic modes. Twelve proteins observed only in the mixotrophic condition had lower pI value (<5) than the fifteen proteins observed only in the autotrophic condition (>5). When the internal amino acid sequences of five selected proteins differentially expressed between autotrophic and mixotrophic conditions were analyzed using matrix-assisted laser desorption time-of-flight (MALDI-TOF) mass spectrometry, two proteins that were specifically expressed in the autotrophic condition showed homology to glyceraldehyde-3-phosphatase dehydrogenase (GAPDH) and a bacterial catalase. Three mixotrophy-specific proteins showed homology to certain hypothetical proteins from an insect and bacteria. These results suggested the presence of certain gene groups that are switched on and off according to the trophic mode of P. micans.

• Journal of Microbiology and Biotechnology
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• 제19권10호
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• pp.1109-1121
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• 2009

In an attempt to discover novel biomarker proteins in type 2 diabetes prognosis, we investigated the influence of hypoglycemic extracellular polysaccharides (EPS) obtained from the macrofungus Tremella fuciformis on the differential levels of plasma proteins in ob/ob mice using two-dimensional gel electrophoresis (2-DE). The 2-DE analysis demonstrated that 92 spots from about 900 visualized spots were differentially regulated, of which 40 spots were identified as principal diabetes-associated proteins. By comparing control with EPS-fed mice, we found that at least six proteins were significantly altered in ob/ob mice, including Apo A-I, IV, C-III, E, retinol-binding protein 4, and transferrin, and their levels were interestingly normalized after EPS treatment. Western blot analysis revealed that the altered levels of the two regulatory molecules highlighted in diabetes and obesity (e.g., resistin and adiponectin) were also normalized in response to EPS. The Mouse Diabetes PCR Array profiles showed that the expression of 84 genes related to the onset, development, and progression of diabetes were significantly downregulated in liver, adipocyte, and muscle of ob/ob mice. EPS might act as a potent regulator of gene expression for a wide variety of genes in ob/ob mice, particularly in obesity, insulin resistance, and complications from diabetes mellitus.

• Asian-Australasian Journal of Animal Sciences
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• 제20권5호
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• pp.802-810
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• 2007

Fibrobacter succinogenes, a Gram-negative, anaerobic ruminal bacterium is a major fibre digesting species in the rumen. It intensively degrades plant cell walls by an erosion type of mechanism, burrowing its way through the complex matrix of cellulose and hemicellulose with the release of digestible and undigested cell wall fragments. The enzymes involved in this process include a combination of glucanases, xylanases, arabinofuranosidase(s) and esterases. The genome of the bacterium has been sequenced and this has revealed in excess of 100 putative glycosyl hydrolase, pectate lyase and carbohydrate esterase genes, which is greater than the numbers reported present in other major cellulolytic organisms for which genomes have been sequenced. Modelling of the amino acid sequences of two glycanases, CedA and EGB, by reference to crystallized homologs has enabled prediction of the major features of their tertiary structures. Two dimensional gel electrophoresis in conjunction with mass spectroscopy has permitted the documentation of proteins over expressed in F. succinogenes grown on cellulose, and analysis of the cell surfaces of mutant strains unable to bind to cellulose has enabled the identification of candidate proteins with roles in adhesion to the plant cell wall substrate, the precursor to cellulose biodegradation.

• 한국동물학회지
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• 제26권3호
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• pp.181-192
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• 1983

이차원 전긱영동법에 의하여 A. proteus의 tD strain과 tD strain이 박테리아와 공생에 의해 유도된 xD strain의 단백질 양성을 비교하였다. Silver stain에 의해 비교 가능한 200여개의 주요 단백질 가운데 tD strain에서 분자량 45,000, 동전점 5.9의 특이성 단백질이, xD strain의 세포액과 공생낭에서는 분자량 29,000, 동전점 5.5의 공생 특이성 단백질이 탐지되었다. 공생 특이성 단백질은 아메바 고은 배양 및 실험 공생 아메바를 이용한 실험 결과 박테리아와 직접 연관 되어 있었다. 탐지된 두 특이성 단백질에 대하여 종전의 세포 핵 이식 및 배양 실험을 통해서 얻어진 결과에 비추어 이들 단백질 상호 연관 및 세포내의 기능에 관하여 논의하였다.

• 한국초지조사료학회지
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• 제42권3호
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• pp.137-145
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• 2022

Aluminum (Al) is one of the major factors adversely affects crop growth and productivity in acidic soils. In this study, the effect of Al on plants in soil was investigated by comparing the protein expression profiles of alfalfa roots exposed to Al stress treatment. Two-week-old alfalfa seedlings were exposed to Al stress treatment at pH 4.0. Total protein was extracted from alfalfa root tissue and analyzed by two-dimensional gel electrophoresis combined with MALDI-TOF/TOF mass spectrometry. A total of 45 proteins differentially expressed in Al stress-treated alfalfa root tissues were identified, of which 28 were up-regulated and 17 were down-regulated. Of the differentially expressed proteins, 7 representative proteins were further confirmed for transcript accumulation by RT-PCR analysis. The identified proteins were involved in several functional categories including disease/defense (24%), energy (22%), protein destination (9%), metabolism (7%), transcription (5%), secondary metabolism (4%), and ambiguous classification (29%). The identification of key candidate genes induced by Al in alfalfa roots will be useful to elucidate the molecular mechanisms of Al stress tolerance in alfalfa plants.