Possibilities of micro gel isoelectric focusing were discussed considering; 1) the advantages of gel electrophoresis, 2) the advantages of gel isoelectric focusing using carrier-ampholytes, and 3) the advantages of micro gel electrophoresis system for the rapid separation of functional proteins under non-denaturing conditions. The importance to develop liquid handling systems in sub-microliter level was stressed for the future separation systems of high-molecular-mass protein complexes, in which the 2-D gel size would be around 1 cm × 1 cm and all the separation processes should be automated.

抄録全体を表示

immobiline gel 等電点電気泳動によるヒト Transferrin の microheterogeneity を検討した. 精製したヒト血清TFをpH5.0～6.0の範囲で immobiline gel による等電点電気泳動すると異なる等電点を持つ4つの分画に分離, 泳動された. 各々の等電点は, 酸性側からA: pI5.15～5.19, B: pI5.26～5.30, C: pI5.37～5.42, D: pI5.50～5.54であった. また, 6M Urea 電気泳動からA-D分画は, 全て diferric TFであることが確認された. 精製TFのA-D分画の割合は, A:B:C:D=1:3:5:1であった. 細胞表面に存在するTF receptor へのA-D分画の結合は, 阻害実験によって調べると著明な差は見られなかった. 正常ヒト血清を直接等電点電気泳動し, 抗血清中で immunofixation を行なうとA～D分画に相当する沈降線が得られたので, 正常ヒト血清中にも同じ鉄含量で等電点を異にするTF分画が存在する事が示唆された.

抄録全体を表示

We studied on the effect of various detergents on the pH gradient formed by Ampholine and also on the effect on migration rate of soluble proteins in the presence of detergents.
Nonionic detergents (Emulgen 108, Triton X-100, Brij 35) affected scarcely the pH gradient formation, and the migration rate of proteins was affected but not remarkably. As SDS (anionic detergent) concentration increased, the slope of the pH gradient decreased and the protein peak was transported cathodically.

抄録全体を表示

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.

抄録全体を表示

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.

抄録全体を表示

運動性 Aeromonas の分類学的研究の一環として, 等電点電気泳動法により菌体可溶性蛋白分画の分析を試みた. その成績を要約すると以下のとおりである. 泳動によって得られた主要バンドについて検討したところ, A. hydrophila は等電点pI6.5 (band a), pI 6.0(band b), pI5.7 (band c) に特徴的なバンドが認められた. A. sobria の特徴は, A. hydrophila のバンドaを欠くがバンドb, cと同一のpIを示すバンドが認められた. A. caviae では, A. hydrophila およびA. sobria に認められたaあるいはb, cのバンドは認められず, pI5.8 (band d) およびpI5.4 (band e) に特有のバンドが認められた. このように3菌種間にそれぞれ特徴的なpIを示すバンドが認められ, 3菌種の鑑別の補助手段として有用である.

抄録全体を表示

A new type apparatus was designed for carrier free isoelectric focusing which could be connected to high-performance liquid chromatography. The merits of this apparatus are as follows:
1) The electroosmosis was low due to the plastic quality of electrophoresis tubing instead of the glass tubing.
2) An amount of sample could be increased by increasing the number of tubings for electrophoresis.
3) For fractionation, this apparatus was connected by pumping-out device with peristaltic pump to a UV detector and a fraction collector.
For simplification and quick operation of this apparatus, we studied on basic conditions and on the separation of proteins by isoelectric focusing using carrier free electrophoresis. With incorporation of prefocusing process at high voltage, whole operation time could be shortened to one hour or less.

抄録全体を表示

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.

抄録全体を表示

Cellulose acetate membrane isoelectric focusing (CAIEF) of streptolysin O from Group A streptococci and direct detection method of hemolytic activity by red blood cell agar layering after focusing are described. Eleven hemolytic components from concentrated culture filtrate were detected which had pI values of 4.8, 5.0, 5.3, 5.4, 5.6, 5.9, 6.4, 6.6, 6.9, 7.2 and 7.4. Sensitivity of the direct detection method of hemolytic activity was examined with pI 6.5 hemolytic component prepared by preparative gel isoelectric focusing. This method was extremely sensitive, which was possible to detect 0.4 HD₅₀ of streptolysin O. These methods were applied to the analysis of the fractions obtained by preparative polyacrylamide or Sephadex gel isoelectric focusing of concentrated culture filtrate. Reproducibility of pI value in CAIEF was comparable to that in polyacrylamide gel isoelectric focusing. The method is inexpensive and enables easy detection of hemolytic toxin.

抄録全体を表示