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  • 標題: SDS

検索結果 26件

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s15-s19
Activation of myosin by phosphorylation has been implicated in regulation of smooth muscle contractions. The ability to analyze myosin phosphorylation is crucial for better understanding of the regulatory mechanisms of smooth muscle contraction. In myosin phosphorylation analysis, it is important to quantify the ratio between phosphorylated and unphosphorylated myosin because those can have opposite effects on contractions. For this reason, urea/glycerol-PAGE and 2-D electrophoresis in combination with Coomassie blue staining or Western blotting are commonly used to separate and quantify myosin based on its phosphorylated state. However, those conventional techniques are not sensitive enough to analyze minute smooth muscles, such as terminal resistant arterioles, partly because of a poor separation in electrophoresis with a small sample. Recently we overcame this problem by introducing a phos-tag technique, and achieved picogram sensitivity. In this article, the sensitive phosphorylation quantification method and application of phos-tag electrophoresis to smooth muscle physiology research are reviewed.
  
今後の展望1 中性条件で泳動を行う改良型 Phos-tag SDS-PAGEを用いたリン酸化タンパク質解析
  • 木下 恵美子, 木下 英司, 小池 透
  • 生物物理化学
  • Vol. 56 (2012) No. Suppl_1
  • 公開日: 2012年01月18日
s41-s44
We describe an improved Phos-tag SDS-PAGE (Zn2+-Phos-tag SDS-PAGE) using a dizinc(II) complex of Phos-tag acrylamide in conjunction with a neutral-pH gel system buffered with Bis-Tris hydrochloride (Bis-Tris-HCl) to detect shifts in the mobility of phosphorylated proteins. Our alternative technique (Mn2+-Phos-tag SDS-PAGE) using a polyacrylamide-bound Mn2+-Phos-tag and a conventional Laemmli's buffer system under alkaline pH conditions has limitations for separating certain phosphoproteins. The major improvements and utilities were demonstrated by visualizing novel up-shifted bands of commercially available pepsin, recombinant substrate protein tau treated in vitro with tyrosine kinases, and endogeneous β-catenin in whole cell lysates. Additionally, the Zn2+-Phos-tag SDS-PAGE gels showed better long-term stability than the Mn2+-Phos-tag SDS-PAGE gels. We can thus present a simple, convenient, and more reliable "in-house" gel system for phosphate-affinity SDS-PAGE.
  
ホウ素化合物MPBAを用いた電気泳動法による糖タンパク質の分析
  • 相京 祐一, 大石 正道
  • 生物物理化学
  • Vol. 58 (2014) No. 2
  • 公開日: 2014年10月31日
33-35
Phos-tagを用いたCdk5制御サブユニットp35のin vivoリン酸化の定量的解析
  • 久永 眞市
  • 生物物理化学
  • Vol. 56 (2012) No. Suppl_1
  • 公開日: 2012年01月18日
s9-s13
Phosphorylation is a major post-translational modification widely used in regulation of many cellular processes. Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase activated by activation subunit p35. Cdk5-p35 regulates various neuronal activities such as neuronal migration, synaptic activity, and cell death. The kinase activity of Cdk5 is regulated by proteolysis of p35: proteasomal degradation causes downregulation of Cdk5 whereas cleavage of p35 by calpain causes overactivation of Cdk5. Phosphorylation of p35 determines the proteolytic pathway. We have previously identified Ser8 and Thr138 as major phosphorylation sites using metabolic labeling of cultured cells followed by 2D-phosphopeptide mapping and phospho-specific antibodies. However, these approaches cannot determine the extent of p35 phosphorylation in vivo. Here we report the use of Phos-tag SDS-PAGE to reveal the in vivo phosphorylation states of p35. Using Phos-tag acrylamide, electrophoretic mobility of phosphorylated p35 was delayed because it is trapped at Phos-tag sites. We constructed phosphorylation-dependent banding profiles of p35 and Ala substitution mutants at phosphorylation sites co-expressed with Cdk5 in COS-7 cells. Using the standard banding profiles, we assigned respective bands of endogenous p35 with combinations of phosphorylation states, and quantified Ser8, Ser91 and Thr138 phosphorylation. This is the first quantitative and site-specific measurements of phosphorylation of p35, demonstrating the usefulness of Phos-tag SDS-PAGE for analysis of phosphorylation states of in vivo proteins.
  
エミューと他鳥種間の卵白成分の電気泳動像の比較
  • 横濱 道成, 佐山 勇輔, 小川 博
  • 生物物理化学
  • Vol. 53 (2009) No. 2
  • 公開日: 2009年12月04日
41-42
To prospect any value of animal resource of the emu as new domestic fowl, we analyzed albumen components in emu and nine other birds by SDS-polyacrylamide electrophoresis. The electrophoresis pattern of emu was quite different from that of chicken and other birds. Proteins with identical mobilities to chicken ovalbumin and lysozyme, major allergic proteins in chicken albumen, were not found in emu albumen. Emu egg could be promising as an alternative food for allergic patients against chicken albumen.
  
Cellulose acetate 膜およびSDS-polyacrylamide gel 電気泳動法によるイヌの尿タンパク質に関する研究
  • 七條 喜一郎, 竹内 崇, 鈴木 實, 斎藤 俊之
  • 生物物理化学
  • Vol. 37 (1993) No. 6
  • 公開日: 2009年03月31日
389-392
121-128
SDS-電気泳動と immuno-blot 法を用いて, I型およびII型コラーゲンの混在試料中のII型コラーゲンの定量法について検討した.
蛋白染色法では, 多量のI型コラーゲンを含むII型コラーゲン混在試料を用いた場合, 全α1鎖の染色性が理論値よりも低下するという問題を有するものの, I型コラーゲンが0.24μg以下, II型コラーゲンが0.51μg以下においては10%以内の誤差で混在試料中のII型コラーゲンの定量が可能であった.
一方, immuno-blot 法では, 1.5ngのII型コラーゲンを検出でき, II型コラーゲンのみ含む試料を用いた場合, 3ngから75ngの範囲内で高い定量性を示した. また, 240ngのI型コラーゲンを含む混在試料を用いた場合でも, 免疫染色時間を短縮することにより, 600ng以下のII型コラーゲンを特異的に定量できた.
  
還元蛋白試料のSDS-PAGE-銀染色の際に生ずる問題点
  • 仙田 晴美, 長谷川 和子, 菅野 浩
  • 生物物理化学
  • Vol. 32 (1988) No. 4
  • 公開日: 2009年03月31日
197-202
The presence of 2-mercaptoethanol or dithiothreitol in the protein denaturing solution used to prepare samples for SDS-PAGE gives rise to artifacts by silver staining of the gel. Addition of iodoacetamide to the reduced protein sample prevents the appearance of artifacts. Silver staining is strengthened by the dithiothreitol treatment but weakened by subsequent treatment with iodoacetamide. High concentration of dithiothreitol also inhibited development of silver staining. These effects of iodoacetamide or high concentration of dithiothreitol were observed more pronouncedly with the silver-staining method of Merril et al.4) than of Guevara.5) Mobility of the protein having intramolecular disulfide bonds is more or less slowed down depending on the concentration of dithiothreitol used. From these results it is concluded that the most suitable concentration of dithiothreitol is in the range of 40 to 80mM from the viewpoints of sensitivity of silver staining, completion of mobility shift, and sharpness of developed protein bands.
  
145-152
The method of fine separation of alpha chains of collagen types I, III, and V by means of delayed reduction of collagen type III with thioglycolic acid (TG) during electrophoresis was investigated. This method is simple and rapid because it does not necessitate an interruption of electrophoresis for the reduction of type III collagen. Furthermore, precise quantitation of alpha chains of collagen type I [alpha1 (I)] and type III [alpha1 (III)] can be achieved even with small sample quantities which contain other types of collagen because the mobility of alpha1 (III) is determined by varying the time TG addition to running buffer for the delayed reduction.
  
147-153
Quantitative analysis of protein components in the rat serum was done by a SDS-PAG electrophoretic method. The protein components were quantified by measuring the areas under peaks of the densitometric diagram obtained by scanning the electrophoretic lane in the gel. No systematic changes in the peak areas were found among lanes in the same gel and scanning positions in the same lane. Relations between the amounts of samples and the peak areas were linear in all protein components. Variations between the two measurements made twice at a 10 day interval were relatively great. It is concluded that relative amounts of proteins could be measured fairly accurately in the same gel, that the amount of a protein component could be estimated by comparing the unknown sample with a standard one, and that it would be necessary to make corrections using a standard sample to compare proteins among different gels.
  
SDS-ポリアクリルアミドゲル電気泳動による低分子量ポリペプチドの分析法
  • 堀米 恒好, 吉田 薫, 金井 祐子, 長谷川 和子, 菅野 浩
  • 生物物理化学
  • Vol. 24 (1980) No. 2
  • 公開日: 2009年03月31日
187-190
An improved method for the analysis of low molecular weight polypeptides (<molecular weight 10, 000) by SDS-polyacrylamide gel electrophoresis was described. Linear gradient polyacrylamide gels, 10-20% [1:20(w/w) bisacrylamide: acrylamide], with a stacking gel gave good resolution. Separation gels also contained 0.45M Tris-HCl (pH 8.8), 0.1% SDS and 7M urea, as well as a 0-10% linear gradient of sucrose. Polypeptides having molecular weights from 1, 400 to 100, 000 could be analyzed by this gel system. Moreover, leakage of low molecular weight polypeptides from gels in the staining procedure had been lowered by using the relatively high concentration of bisacrylmaide. This method could be applied for the analysis and detection of low molecular weight polypeptides from complex biological sources.
  
SDSゲル電気泳動および抗体を用いた物細胞骨格の研究
  • オスボーン マリー
  • 生物物理化学
  • Vol. 24 (1980) No. 2
  • 公開日: 2009年03月31日
149-170
23-26
Abnormal IgA molecule, which lacked in disulfide bridges between α chains, was detected from the sera of patients of monoclonal IgA gammopathy with albuminuria by the method of urea-SDS-PAGE performed in the reducing-agent-free condition. Throughout the procedure, protein in the specimen was kept away from any reducing agent to avoid an artificial production of half molecules during the experiment. A band of 80kDa protein, which showed immunoreactivity to both anti-α- and anti-λ-chain antibodies, was detected in sera from four patients of monoclonal IgA gammopathy accompanied with albuminuria. The molecular mass and the immunoreactivity indicated that the 80kDa protein was so-called“IgA half molecule”(α1λ1). The band of α1λ1 molecule was faint on the gel in the specimens from patients of no albuminuria. The abnormal α1λ1 molecule was eluted from the column of gel permeation chromatography into fractions corresponding to 320kDa when it was performed using urea-free buffer. The chromatographic behavior suggested the non-covalent interaction between the abnormal α1λ1 molecules in the non-denaturing buffer condition.
  
血液透析患者から得たECUM濾過液のSDS-PAGE分析
  • 本間 信幸, 菅野 浩, 下条 文武, 荒川 正昭, 伊東 義一
  • 生物物理化学
  • Vol. 30 (1986) No. 4
  • 公開日: 2009年03月31日
283-288
Serum ultrafiltrate was obtained from hemodialysis patients by using an extra corporeal ultrafiltration method equipped with a hollow fiber dialyser, and electrophoresed on the SDS-polyacrylamide gel having a high resolving power in the low molecular weight region. Among the major bands less than 40KD observed with plasma of hemodialysis patient, the bands of 29K, 28K, 25K, 22K, 16.7K and 8.9K molecular weights were also found in serum ultrafiltrate of the same patient. By an immunological method, the following bands were identified: α1-microglobulin (28K), retinol-binding protein (22K), prealbumin (16.7K) and β2-microglobulin (8.9K). As compared with serum ultrafiltrate obtained from healthy person, 28K, 25K (in some cases), 22K and 8.9K bands showed obvious increase in cases of hemodialysis patients except for one patient who showed an urine volume of 200ml/day and an electrophoretic pattern closed to that of healthy person.
  
銀染色による微量蛋白質の検出法
  • 井上 勤, 浅賀 宏昭, 田村 真弓
  • 生物物理化学
  • Vol. 30 (1986) No. 3
  • 公開日: 2009年03月31日
229-238
459-465
尿素を含むゲルを用い, 泳動途中で陰荷電還元剤チオグリコール酸を添加するSDS-ポリアクリルアミド電気泳動法をイムノブロッティング法と組み合わせることによって, 動脈壁III型コラーゲンの解析を行った. すなわち, 組織から抽出したIII型コラーゲン中で, 鎖間架橋結合をもたずに鎖間ジスルフィド結合を介して三本鎖として存在するもの, 鎖間架橋結合により二本鎖として存在するもの, および鎖間ジスルフィド結合をもたずにSDSだけで一本鎖に解離するものをそれぞれ分離定量した. これらを, それぞれIII型コラーゲンの分泌, 架橋, 分解の指標とし, ラット動脈壁および皮膚のIII型コラーゲン鎖の加齢による変化を検討した. いずれの組織でも加齢にしたがってIII型コラーゲン量が減少したが, 各組織でそれぞれの分子種の量は異なっていた. すなわち, 大動脈, 腕頭動脈では架橋が最も多かったのに対し, 皮膚では分泌が最も多く, 架橋は分解よりも少なかった.
  
SDS-ポリアクリルアミドゲル電気泳動法による妊娠, 産褥期の尿中蛋白組成の変化に関する研究
  • 黒川 達郎, 下川 浩, 東原 潤一郎, 久永 幸生, 中野 仁雄
  • 生物物理化学
  • Vol. 29 (1985) No. 3
  • 公開日: 2009年03月31日
191-195
正常妊娠における尿中蛋白組成の変化を分析するために今回の研究を施行した.
15例の正常妊婦の妊娠中の尿中蛋白組成の変化をSDS-ポリアクリルアミドゲル電気泳動法 (以下SDS-PAGEと略) により分析した.
その結果, 妊娠初期には1本あるいは2本 (分子量71×103, 94×103) の帯が認められ, 妊娠経過に伴い帯の数が増加し, 妊娠後期には7本の帯が認められた. しかし産褥1ヶ月では, 帯の数は再び2本になり, 妊娠初期と同じ像を呈した.
今回の結果より, 妊娠中の尿中蛋白の分析にSDS-PAGEが有用であることがわかった.
  
Polyacrylamide gradient gel electrophoresis と electrophoretic blotting 法による尿蛋白分析法の検討
  • 桜井 秀子, 須藤 優子, 稲毛 博実, 渡辺 孝太郎, 小山 哲夫, 成田 光陽, 東條 静夫
  • 生物物理化学
  • Vol. 30 (1986) No. 4
  • 公開日: 2009年03月31日
251-255
In order to identify urinary proteins excreted from normal subjects, we employed a new method by which molecular weight and immunochemical property of urinary proteins can be determined simultaneously. Urine was subjected to linear gradient (3∼40%) SDS-PAGE and then transferred to nitrocellulose membrane by electrophoretic blotting method. The membrane was stained with Auro Dye and blotted proteins were identified by enzyme immunoassay using specific antibodies. In this method, we identified more than 12 protein bands from urine, that is, α2-macroglobulin, tubular epithelial antigen, Tamm-Horsfall glycoprotein, IgA, IgG, transferrin, albumin, α1-microglobulin, L-chain (dimer, monomer), retinol-binding protein, and β2-microglobulin. In addition, we found native forms and some fragments of immunoglobulins. In this report, we analyzed only normal subjects.
These results suggest that this method is useful for analysis not only of glomerular or tubular proteins but also tissue antigens from urine with various kinds of renal diseases.