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- 검색키워드
- 주제어 : two-dimensional gel electrophoresis (검색결과 106 건)
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Solubilization of Proteins from Human Lymph Node Tissue and Two-Dimensional Gel Storage
De Marqui, Alessandra Bernadete Trovo,Vidotto, Alessandra,Polachini, Giovana Mussi,De Mattos Bellato, Claudia,Cabral, Hamilton,Leopoldino, Andreia Machado,De Gois Filho, Jose Francisco,Fukuyama, Erica Korean Society for Biochemistry and Molecular Biol 2006 Journal of biochemistry and molecular biology Vol.39 No.2
In the present study, we compared six different solubilization buffers and optimized two-dimensional electrophoresis (2-DE) conditions for human lymph node proteins. In addition, we developed a simple protocol for 2-D gel storage. Efficient solubilization was obtained with lysis buffers containing (a) 8M urea, 4% CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate), 40 mM Tris base, 65 mM DTT(dithiothreitol) and 0.2% carrier ampholytes; (b) 5M urea, 2M thiourea, 2% CHAPS, 2% SB 3-10 (N-decyl-N, N-dimethyl-3-ammonio-1-propanesulfonate), 40mM Tris base, 65 mM DTT and 0.2% carrier ampholytes or (c) 7M urea, 2M thiourea, 4% CHAPS, 65 mM DTT and 0.2% carrier ampholytes. The optimal protocol for isoelectric focusing (IEF) was accumulated voltage of 16,500 Vh and 0.6% DTT in the rehydration solution. In the experiments conducted for the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), best results were obtained with a doubled concentration (50 mM Tris, 384 mM glycine, 0.2% SDS) of the SDS electrophoresis buffer in the cathodic reservoir as compared to the concentration in the anodic reservoir (25 mM Tris, 192 mM glycine, 0.1% SDS). Among the five protocols tested for gel storing, success was attained when the gels were stored in plastic bags with 50% glycerol. This is the first report describing the successful solubilization and 2D-electrophoresis of proteins from human lymph node tissue and a 2-D gel storage protocol for easy gel handling before mass spectrometry (MS) analysis.
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Kimata, Junko,Shigeri, Yasushi,Yoshida, Yasukazu,Niki, Etsuo,Kinumi, Tomoya Korean Society for Mass Spectrometry 2012 Mass spectrometry letters Vol.3 No.1
Artificially oxidized cysteine residues in peroxiredoxin 6 (Prx6) were detected by electrospray interface capillary liquid chromatography-linear ion trap mass spectrometry after the preparation of two-dimensional gel electrophoresis (2D-GE). We used Prx6 as a model protein because it possesses only two cysteine residues at the 47th and 91st positions. The spot of Prx6 on 2D-GE undergoes a basic (isoelectric point, pI 6.6) to acidic (pI 6.2) shift by exposure to peroxide due to selective overoxidation of the active-site cysteine Cys-47 but not of Cys-91. However, we detected a tryptic peptide containing cysteine sulfonic acid at the 47th position from the basic spot and a peptide containing both oxidized Cys-47 and oxidized Cys-91 from the acidic spot of Prx6 after the separation by 2D-GE. We prepared two types of oxidized Prx6s: carrying oxidized Cys-47 (single oxidized Prx6), and other carrying both oxidized Cys-47 and Cys-91 (double oxidized Prx6). Using these oxidized Prx6s, the single oxidized Prx6 and double oxidized Prx6 migrated to pIs at 6.2 and 5.9, respectively. These results suggest that oxidized Cys-47 from the basic spot and oxidized Cys-91 from the acidic spot are generated by artificial oxidation during sample handling processes after isoelectric focusing of 2D-GE. Therefore, it is important to make sure of the origin of cysteine oxidation, if it is physiological or artificial, when an oxidized cysteine residue(s) is identified.
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Eun Kyung Cho(조은경),Young Ju Choi(최영주) 한국생명과학회 2008 생명과학회지 Vol.18 No.5
고온에 노출된 식물의 단백질 보호에 있어서 HSP70의 효과를 연구하기 위해 2차 전기영동 법이 시행되었다. 본 논문에서는 고온에서 유도되는 담배 HSP70인 NtHSP70-1 (AY372069)에 대한 식물 HSP70의 고온내성을 분석하였다. 우선, NtHSP70-1이 항상 발현되는 형질전환 식물과 vector만을 지닌 형질전환 식물, 그리고 antisense 형태의 형질전환 식물이 제조되었다. 이들 세 종류의 형질전환체들은 western blot 분석법을 사용하여 확인되었고, NtHSP70-1에 대한 sense형태의 형질전환체들은 고온에 대한 내성이 있는 것으로 보였다. 45도에서 2시간 동안 고온처리 후 26도에서 5일 동안 유지한 세 종류의 형질전환체들과 고온처리 하지 않은 이들 형질전환체들에 대한 2차 전기영동 분석에서 잠정적으로 28개의 단백질들에 대해 비교한 결과 이들 단백질의 발현량에 차이가 있음을 확인 할 수 있었다. NtHSP70-1이 항상 발현되는 형질전환체들에서는 이들 단백질들이 유지되거나 향상된 반면, 다른 종류의 형질전환체들에서는 이들 단백질들이 감소되거나 사라졌다. 이 결과들은 NtHSP70-1이 고온에서 단백질을 보호하는 분자 chaperone으로써 기능을 함으로써 식물의 고온내성에 중요한 역할을 할 것으로 시사하고 있다. The effect of heat shock protein 70 (HSP70) on soluble proteins extracted from seedlings exposed to high temperature was examined by two-dimensional gel electrophoresis (2-DE). NtHSP70-1 (AY372069), which is an HSP70 that is induced by heat stress in Nicotiana tabacum, was studied to analyze the protective role of plant HSP70. First, transgenic tobacco plants that constitutively expressed elevated levels of the tobacco HSP70, NtHSP70-1, and transgenic plants containing either the vector alone or the NtHSP70-1 gene in the antisense orientation were constructed. Plants with altered NtHSP70-1 levels were subjected to immunoblotting, and seedlings with enhanced levels of NtHSP70-1 in their transgenic sense lines were observed to show tolerance to heat stress. In the analysis using 2-DE, 28 putative proteins were differentially expressed in the comparison of three kinds of transgenic seedlings after high temperature/recovery treatment (45℃, 2 hr; 26℃, 5 day) with the seedlings under normal condition (26℃). In transgenic sense seedlings, the proteins were maintained or increased, whereas proteins in the transgenic seedlings, containing either the vector alone or the NtHSP70-1 in the antisense orientation, were present in decreased amounts or had disappeared. These results suggested that NtHSP70-1 as molecular chaperone protecting proteins from aggregation induced by heat stress may play an important role in acquisition of thermotolerance.
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( Junko Kimata ),( Yasushi Shigeri ),( Yasukazu Yoshida ),( Etsuo Niki ),( Tomoya Kinumi ) 한국질량분석학회 2012 Mass spectrometry letters Vol.3 No.1
Artificially oxidized cysteine residues in peroxiredoxin 6 (Prx6) were detected by electrospray interface capillary liquid chromatography-linear ion trap mass spectrometry after the preparation of two-dimensional gel electrophoresis (2D-GE). We used Prx6 as a model protein because it possesses only two cysteine residues at the 47th and 91st positions. The spot of Prx6 on 2D-GE undergoes a basic (isoelectric point, pI 6.6) to acidic (pI 6.2) shift by exposure to peroxide due to selective overoxidation of the active-site cysteine Cys-47 but not of Cys-91. However, we detected a tryptic peptide containing cysteine sulfonic acid at the 47th position from the basic spot and a peptide containing both oxidized Cys-47 and oxidized Cys-91 from the acidic spot of Prx6 after the separation by 2D-GE. We prepared two types of oxidized Prx6s: carrying oxidized Cys-47 (single oxidized Prx6), and other carrying both oxidized Cys-47 and Cys-91 (double oxidized Prx6). Using these oxidized Prx6s, the single oxidized Prx6 and double oxidized Prx6 migrated to pIs at 6.2 and 5.9, respectively. These results suggest that oxidized Cys-47 from the basic spot and oxidized Cys-91 from the acidic spot are generated by artificial oxidation during sample handling processes after isoelectric focusing of 2D-GE. Therefore, it is important to make sure of the origin of cysteine oxidation, if it is physiological or artificial, when an oxidized cysteine residue(s) is identified.
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The first two-dimensional reference map of the fission yeast, Schizosaccharomyces pombe proteins
Sun, Namkyu,Jang, Jaewon,Lee, Seunghee,Kim, Sunkyung,Lee, Seunghyun,Hoe, Kwang-Lae,Chung, Kyung-Sook,Kim, Dong-Uk,Yoo, Hyang-Sook,Won, Misun,Song, Kyung Bin WILEY-VCH Verlag 2005 Proteomics Vol.5 No.6
<P>Cytosolic proteins of Schizosaccharomyces pombe were separated by two-dimensional (2-D) gel electrophoresis, to construct the first 2-D reference map. In the pI range 4–7, more than 500 spots were detected by silver staining, and 70 different proteins corresponding to 111 spots were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and tandem mass spectrometry, where necessary. In the pI range 6–9, approximately 330 spots were detected, and 31 proteins corresponding to 38 spots were identified by mass spectrometry. More than 50% of the identified proteins were involved in amino acid, carbohydrate or nucleotide metabolism, and energy production. A second large group of identified proteins comprises heat shock and other stress related proteins and chaperones.</P>
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Protein Expression Analysis of Halobacillus dabanensis $D-8^T$ Subjected to Salt Shock
Feng De Qin,Zhang Bo,Lu Wei Dong,Yang Su Sheng The Microbiological Society of Korea 2006 The journal of microbiology Vol.44 No.4
To investigate the mechanism of salt tolerance of gram-positive moderately halophilic bacteria, two-dimensional gel electrophoresis (2-D PAGE) was employed to achieve high resolution maps of proteins of Halobacillus dabanensis $D-8^T$. Approximately 700 spots of proteins were identified from these 2-D PAGE maps. The majority of these proteins had molecular weights between 17.5 and 66 kDa, and most of them were distributed between the isoelectric points (pI) 4.0 and 5.9. Some protein spots were distributed in the more acidic region of the 2-D gel (pI <4.0). This pattern indicated that a number of proteins in the strain $D-8^T$ are acidic. To understand the adaptation mechanisms of moderately halophilic bacteria in response to sudden environmental changes, differential protein profiles of this strain were investigated by 2-D PAGE and $Imagemaster^{TM}$ 2D Platinum software after the cells were subjected to salt shock of 1 to 25% salinity for 5 and 50 min. Analysis showed 59 proteins with an altered level of expression as the result of the exposure to salt shock. Eighteen proteins had increased expression, S proteins were induced, and the expression of 33 proteins was down-regulated. Eight of the up-regulated proteins were identified using MALDI-TOF/MS and MASCOT, and were similar to proteins involved in signal transduction, proteins participating in energy metabolism pathways and proteins involved in stress.
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Protein Expression Analysis of Halobacillus dabanensis D-8T Subjected to Salt Shock
De Qin Feng,Bo Zhang,Wei Dong Lu,Su Sheng Yang 한국미생물학회 2006 The journal of microbiology Vol.44 No.4
To investigate the mechanism of salt tolerance of gram-positive moderately halophilic bacteria, two-dimensional gel electrophoresis (2-D PAGE) was employed to achieve high resolution maps of proteins of Halobacillus dabanensis D-8T. Approximately 700 spots of proteins were identified from these 2-D PAGE maps. The majority of these proteins had molecular weights between 17.5 and 66 kDa, and most of them were distributed between the isoelectric points (pI) 4.0 and 5.9. Some protein spots were distributed in the more acidic region of the 2-D gel (pI <4.0). This pattern indicated that a number of proteins in the strain D-8T are acidic. To understand the adaptation mechanisms of moderately halophilic bacteria in response to sudden environmental changes, differential protein profiles of this strain were investigated by 2-D PAGE and ImagemasterTM 2D Platinum software after the cells were subjected to salt shock of 1 to 25% salinity for 5 and 50 min. Analysis showed 59 proteins with an altered level of expression as the result of the exposure to salt shock. Eighteen proteins had increased expression, 8 proteins were induced, and the expression of 33 proteins was down-regulated. Eight of the up-regulated proteins were identified using MALDI-TOF/MS and MASCOT, and were similar to proteins involved in signal transduction, proteins participating in energy metabolism pathways and proteins involved in stress.
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벼종자 미랑 단백질의 프로테오믹스 연구를 위한 글루테린 저장 단백질의 제거방법
우선희,김세영,김태선,조성우,조건,정근욱,김선림,조용구,김홍식,송범헌,이철원,정승근,박영목 韓國作物學會 2006 Korean journal of crop science Vol.51 No.1S
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. Abundant proteins often cause problems in proteome study. Glutelin family proteins (hereafter referred to glutelin) are present in rice proteome sample as over-whelming constituents with very high abundance. In order to increase the number of identified proteins in rice proteome study, we developed a newly improved method for sample preparation through the removal of glutelin. When the protein samples from rice seed were extracted by the conventional trichloroacetic acid (TCA) extraction method, glutelin accounts for about 60% of total rice seed proteins in SDS gels. Using our new water extraction method, glutelin consists of only about 10% of total proteins. After analyzing on a two-dimensional gel electrophoresis (2-DE), 937 protein spots were detected using the conventional TCA extraction method. On the other hand, 1240 proteins could be seen using the new water extraction method. The selectivity for non-glutelin and less abundant protein by the water extraction method was also confirmed by ESI-Q/TOF mass spectrometry analysis. Thus, the new water extraction method developed here can be efficiently used to study the proteome analysis of rice storage seed
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