傾けたフィルムにアガロース溶液を流すことにより楔型のゲルを簡単に作製できることを墨汁を含ませたゲルのデンシトメトリーにより確認し, このゲルを用いるゾーン電気泳動と等電点電気泳動を行った. ゾーン電気泳動ではゲルの厚いほうに向かって電気泳動するとゾーンの濃縮効果により, 速く泳動する成分のゾーンの分解能が改良されることを理論的に示すとともに血清蛋白質の電気泳動 (pH8.6, μ0.045のバルビタール-N-メチル-D-グルカミン緩衝液) のプレアルブミンとアルブミン分画について実証した. 等電点電気泳動法では平板ゲルに比べて, 分画像は薄いところでは拡大, 厚いところでは縮小されることを, 広域両性担体 (Sepaline, pH3.5-10, 富士写真フイルム) を用いる血清蛋白質の等電点電気泳動により示した. すなわち楔型のゲルを用いることにより平板ゲルで分画像の拡大した部分に該当するpH域の両性担体を添加するのと同じ効果がえられる.

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アガロースゲルを一次元目に用いた二次元電気泳動法を紹介し, この方法が種々の試料にも応用できるか否かについて考察した. この方法を, 筋肉, 肝臓, 腎臓, レンズにおける全タンパク質の分離分析に用いたところ, 一次元目のゲルトップに引っ掛かることなく, ほとんどすべての成分が分析でき, 分析できる等電点, 分子量の範囲は広いものであった. 植物材料, 細胞核試料以外のほとんどすべての材料で理想的な分離が可能であった. このパターンに画像解析法を適用することにより, 骨格筋タンパク質トロポニンTやトロポミオシンの多くのアイソフォームを検出し, トロポニンTとトロポニンCの間やレンズクリスタリンサブユニットの間での蓄積の相互関係を調べ, さらにはトロポミオシンアイソフォームの組織特異的分布を調べた.

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A new procedure for confirmation of focusing pattern after preparative agarose gel isoelectric focusing is described. The preparative gel was covered with a thin layer agarose gel (cover gel) from the start of focusing. A printing procedure can be omitted in this procedure since the cover gel is stained after focusing. The focused cover gel pattern is more clear than the printed cellulose acetate membrane. When the dry cover gel was used, the transcription volume was a little and the recovery of protein from the preparative gel was similar to the printing procedure with the membrane. In spite of removing carbonic acid gas with the utmost care, skewed protein bands were sometimes observed and its direction of skew was irregular. Therefore, it was assumed that there are some factors caused skewed protein bands besides carbonic acid gas.

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Analysis of serum γ-GTP isoenzymes was attempted with isoelectric focusing using agarose gel as a supporting medium. After testing various conditions to obtain electrophoretic patterns with good reproducibility, suspension of Ampholine in Agarose IEF was finally chosen as a supporting medium, and electrolysis at 6.25W/plate (length 12.5cm×width 11cm) for 1hr under cooling with circulating water was found to be most appropriate. A serum sample, 20μl, absorbed to a small piece of filter paper was applied onto the medium 1cm away from the cathode. Under these conditions, the pH gradient was linear in the range from pH 3.5 to 9.5, and serum proteins were clearly separated.
This method was applied to the separation of γ-GTP isoenzymes. γ-GTP was stained with γ-L-glutamyl-p-diethylaminoanilide. After electrophoresis, the gel was fixed with 50% saturated ammonium sulfate solution followed by removal of the ammonium sulfate, and then submitted to staining procedures. This pretreatment was found to give very clear and permanently preservable electrophoretic patterns. Serum γ-GTP was separeted into 7 isoenzyme bands by this method. The bands which consistently appeared were in pI range of 4.5-5.0, while a band at pI 5.5 was noted in the cases of bile stagnation, and a band at pI 6.5 was observed in cases with malignant tumors.

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アガロースを1次元目に用いるミクロ2次元電気泳動法を確立した. アガロースを1次元目に用いるとき, これが室温では固まりやすいためミクロ法を行うのは困難であったが, これまでの7M尿素の代わりに1Mチオ尿素5M尿素を用いると, 室温では固まらず, 4℃で固化し, 室温にもどしても固化したままにとどまることが判った. この方法をマクロとミクロで比較してみた結果, ほぼ同程度の分析が出来ることが判り, アガロースを1次元目に用いるミクロ2次元電気泳動法が行えるようになった.

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The cerebrospinal fluid protein fractions were investigated by the agarose isoelectric focusing (agarose IEF) and the silver staining, for the purpose of elucidating protein abnormalities in the neural tissues in the degenerative neurological diseases, especially in motor neuron disease (MND), spinocerebellar degeneration (SCD) and Parkinson disease (PD).
In this study, we focused six bands, mainly composed of transferrin, separated by agarose IEF and the silver staining. Some of these bands showed double fractions, even in the cases of the control group consisted of the patients without organic disease. In MND, all patients had double fractions of one or more of these bands, and it was interesting that in some of them double fractions were seen on the band with the isoelectric point of 5.6. Double fractions were also seen in many of SCD (69%) and PD (91%). Cases in that there was positive in more alkaline zone than transferrin in this method, were more in disease groups (especially in MND and SCD) than in the control group.
This method combined agarose IEF with the silver staining seemed to be the excellent one as the investigating procedure for cerebrospinal fluid proteins.

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Color developmental condition in LDH isoenzyme assay using agarose gel film as medium was investigated fundamentally. Optimum conditions for this assay were pH8.6, DL-litium lactate concentration 120∼150mM/l, NAD concentration 4∼5mM/l and tricine concentration 0.1M/l. These conditions were found to be identical for those of Pol-E film method. However, optimum concentrations of diaphorase enzyme used as an intermediate electron carrier was 7, 500U/l and that of NTB was 0.5mM/l. Furthermore, the color developmental reaction in agarose gel film was a linear at least in 60 minutes. After these improvements, an assay condition with better color developmental sensibility and quantitability was obtained in comparison with Pol-E film method.

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Recently, silver staining has been used for the detection of a trace amount of protein using agarose gel electrophoresis. However, some problems, such as easy contamination of the background by nonprotein substances and necessity of complicated steps and skillful technique for staining have made it less satisfactory. A more simplified method is necessary for routine work. Therefore, we modified successfully the silver staining method published by Kerényi et al.
The present improved silver staining method gave the same sensitivity as Kerényi's method, and yet the procedure was simplified and the staining reproducibility was satisfactory. It is also useful for the detection of oligoclonal bands in cerebrospinal fluid from the patients with demyelinating diseases and various types of viral infections, when it is combined with high resolution agarose gel.

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A two-dimensional gel electrophoresis (2-DE) method that uses an agarose isoelectric focusing (IEF) gel in the first dimension (agarose 2-DE) offers advantages over the more widely used immobilized pH gradient 2-DE for separating high-molecular-mass proteins (100-500kDa) and for having a higher loading capacity (1.5mg in total). We have applied the Fluorescent 2-D differential gel electrophoresis (2-D DIGE) to our agarose 2-DE system. This allowed us to see clear differences in the 2-DE patterns from liver extracts of a diabetic model Otsuka Long-Evans Tokushima Fatty (OLETF) and its control Long-Evans Tokushima Otsuka (LETO) rats including changes in the amounts of several proteins larger than 100kDa. The combined method would increase its power to detect changes in disease proteomics.

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The electroendosmotic behavior of IsoGel and Agarose IEF was determined in a series of various wide and narrow pH range electrofocusing experiments in which glucose served as marker substance.
In IsoGel its liquid flow caused towards the anode in all wide and narrow pH range electrofocusing. The strength of segmetal liquid flow was proportional to voltage distribution across the gel. The pH gradients obtained in IsoGel when using same wide pH range ampholyte has markedly low pH values than that of Agerose IEF at acidic region.
In Agarose IEF its liquid fow behavior differed in wide and narrow pH range electrofocusing. In the case of wide pH range electrofocusing its liquid flow reversed towards the anode at pH 5.5∼6 region in the foucsed gels. The other hand, in narrow pH range electrofocusing the direction of liquid flow was differed with pH range of ampholyte. In higher pH range than Amopholine 5-7 its liquid flow coused towards the cathode and in lower pH range than Ampholine 4-6 reversed towards the anode. The direction and the streght of liquid flow in narrow pH range Agarose IEF depends on the pH distribution in the focused gel. The pH curves obtained in Agarose IEF when using same narrow pH range Ampholyte were slightly higher than that of IsoGel.

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