The apparatus devised consists of two buffer vessels, two Ag(AgCl)KCl-electrodes, two electrode vessels, a horizontal plate, a cover, and a supporter frame-work for filterpaper.
A few basic experiments were performed by means of this apparatus.
The buffer used was veronal-acetate buffer at pH8.6.
Results:
1) Veronal-buffer was found incomparably best adapted for filterpaper-electrophoretic analysis of blood serum.
2) 15 hours' electrophoresis under the current of 2mA or so was found to be most appropriate.
3) 0.02cc of serum was sufficient for the purpose.
4) The same buffer could be used 5 times in succession without any practical disadvantage.
5) The result obtained was approximately identical with that obtained by the use of the classical Tiselius apparatus.
6) The serum values obtained in 10 normal persons averaged Alb. 59.0%, α₁-Glob. 3.7%, α₂-Glob. 5.9%, β-Glob. 10.8% and γ-Glob. 20.6%.

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Using a laminated dry battery for portable radio use as an electrical source, human serum and eggwhite solution are paperelectrochromatographically developed.
In case when paper of the size of 2×20cm is used, current required was only 0.2mA with our circuit and arrangement. Fairly good patterns were obtained and it was verified that a laminated dry battery is never worse than a high-class rectifier as far as proteins are separated.

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By the application of the crossing paper electrophoresis, antigen-antibody reactions could be detected on the filter paper. Antiserum was applied on a line on the anodic side of the filter paper and antigen was applied on a line drawn obliquely to the former on the cathodic side. By the electrophoresis, the antiserum was separated into its components and crossed over by the antigenic proteins. Antigen-antibody precipitates occurred in the zone of γ-globulin of the antiserum.
The line of precipitate formed by the reaction of egg albumin with its homologous antiserum was dissolved and moved, if excess of egg albumin had crossed with it. The antiserum obtained in the earlier stage of immunization, however, showed a line of complex, which was not dissolved by the excess of egg albumin. The complex was suspected not to be precipitate. Thus it was inferred that the anti-egg albumin antibody of the earlier stage would be “incomplete”, which would become complete precipitin afterwards.
The cross reactions of the anti-hen's egg albumin with heterogenous antigens, egg albumins of duck, goose, guinea hen, and quail, could also be deteced.
By the crossing paper electrophoresis of bovine serum and its rabbit antiserum, at least 6 lines of pricipitate were detected. Two-dimensional application of the method was also carried out: The antiserum alone was first separated on a line in the first dimension. Then the antigenic bovine serum was applied on a line vertical to the first one. And the second electrophoresis was carried out to the 2nd direction vertical to the first one. The proteins of bovine serum migrated into the zone of γ-globulin of the antiserum to form lines of precipitate. By the two-dimensional technique, however, the sensibility of the method could not be raised. A comparison with the technique of Garbar's immunoelectrophoresis was made.
The cross reactions of the anti-bovine serum rabbit antiserum with heterogenous antigens, human, swine and goat serum, were detected by the method.

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1) The daily change of the electrophoretic pattern of the silkworm blood from larval to pupal stage was examined. Three components (b₃, b₂, b₁) were present in the blood of the early period of the 5th instar as shown in the previous paper (AIZAWA, 1955), while from 4-5th day of the 5th instar to pupal stage, there appeared two components, which were densely stained (Figs. 1, 3). In the latter case, when the electrophoresis was performed with the diluted blood, three protein components could be clearly detected (Fig. 2). b₃ is the fastest moving component and it seems to be albumin. b₂ and b₁ are probably globulin. b₃ component decreased with the pupal age and finally disappeared, at earlier time in male than in female.
2) Comparison of the electrophoretic patterns was made by veronal, phosphate, citrate, borate or tris (hydroxymethyl) aminomethane buffer with the varying ion concentration and pH. The separation of the protein fractions with veronal buffer (pH 8.6, I=0.05) was better than with other buffers.
3) Comparison of staining, was examined using bromphenol blue, solar blue black and amido black. All dyes were suitable for the purpose. By sudan black staining, b₃ component was slightly stained (Fig. 4).
4) Paper strips were cut off from the electrophoresis paper of the jaundice-diseased blood at the regions of b₃, b₂+b₁ and the starting line as shown in Fig. 5. The virus was extracted with the distilled water and the injection experiments were performed. The virus amount was highest in b₂+b₁ fractions and it decreased in the fraction from near the starting line. The virus activity was scarecely shown in the fraction of b₃.
5) Any difference, except for quantitative one was not observed in the electrophoretic patterns of the blood between the normal and diseased larvae (both nuclear and cytoplasmic polyhedroses). On the contrary, the pattern of Galleria mellonella is, however, different from that of Galleria-adapted silkworm jaundice virus (Aizawa, unpub.).

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A systematic studies of the electrophoresis technique of human serum proteins (in sodium barbiturate buffer at pH 8.6) using the open horizontal method ware carried out.
1. During electrophoresis, two kinds of the buffer flow occur on paper surface, one of which heads for the cathode on account of the electroendosmosis. The other one goes to the central part of paper from both of the anodic and cathodic buffer vessels in order to compensate for the relative dryness which occurs owing to the evaporation during electrophoresis. The apparent buffer flow on paper surface is the algebraic sums of these two kinds of buffer flow. The equlibrum points (zero points) of buffer flow are generally located on the cathodic part of the horizontal portion of paper.
2. These equlibrium points, however, displace to the central part of paper as voltage, ionic strength and electrophoresis hour incerease. It is because, in these cases, the buffer flow due to the evaporation augments more rapidly than the one due to the electroendosmosis.
3. The buffer flow on the more anodic part than the equlibrium points heads for the cathod and inversely on the more cathodic part. Their dimensions are proportional to the distances from the equlibrium points of buffer flow.
4. On account of the buffer flow on paper, 1) the electrophoretic patterns are blurred, especially on the anodic part of paper, and displaced, as a whole, to the anodic or cathodic parts, leaving some extent of tailing on paper. This is disadvantageous for the precise determination of serum fractions because one fraction may be superimposed on the other's talings.
5. As the buffer solution displaces under the continuous evaporation during electrophoresis, the ionic strength varies throughout on paper. To detect the variations of ionic strength, accumulated amounts of electrolytes on the dried papers, were examined by the high-frequency papyrography The ionic strength is proved to be small where the buffer flow is large. So, the ionic strength on paper decreases in proportion to the increases of distance from the equlibrium points of buffer flow.
6. The time-migration relationship of B. P. B, stained Alb, is not linear but of inversely S-shaped curve, the formation of which is expounded to be due to the variations of ionic strength on paper.
7. The most well separated electrophoretic patterns are obtained when serum is applied on the equlibrium points of buffer flow. It is because the applied serum migrates on the paper with relatively high ionic strength and relatively small buffer flow.
8. In drying papers, the electrophoretically separated fractions are displaced owing to the water displacement due to the evaporation.
9. The paper electrophoretic mobilities are only approximate in both of the closed and open method electrophoresis because the changes of buffer flow and ionic strength during electrophoresis, and the water displacement in drying papers do not take place uniformly on paper.

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As a part of our fundamental studies on paper electrophoresis we here present our views regarding selection of the two methods of paper electrophoresis, the Grassmann and the Flynn method. The filter paper used was Whatman No. 1, the stain, Amido Black 10 B.
Ten essentially normal human sera were used, and the results from both methods were almost identical, although differing in minor details. It is these minor differences that we wish to consider here.
(1) With the electrophoretic time being the same for both methods, the length of the electrophoretic migration was longer for the Grassmann method than for the Flynn method. This may be due to the movement of the buffer toward the center of the paper, as a result of the evaporation from the surface of the paper.
(2) The position of γ-globulin with the Flynn method was always on the negative side of the line of origin (electroosmosis), but with the Grassmann method it was consistently on the positive side. This, also, we consider is due to the flow of the buffer to compensate the evaporation loss.
(3) The protein adsorbed at the line of origin was more marked for the Flynn method than for the Grassmann method. We are now considering a way to eliminate this defect in the Flynn method. In order to observe the tailing of the albumin, therefore, the Grassmann method is more convenient.
(4) The reproducibility of both methods, as compared to free electrophoresis was not very good, owing to the evaporation from the paper and also to the sagging of the paper in the Grassmann method.
(5) From the above it is concluded that although the Grassmann method is more popularly used, the Flynn method when properly handled is far from being inferior to the former method.

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1. For the precision of densitometry, each zone of serum fractions should be geometrically so figured as to be perpendicular to the direction of electromigration on its cathodic and anodic edges and to spread bilaterally just to the both edges of a paper strip. The electrophoretic patterns suiting this request will be got when the serum is applied as a rectangularly diffused zone at the starting line. The repeated streaking method proposed by Grassmann and Hannig, and the droplets depositing method are proved to be preferable in applying the serum on paper so far as the thorough attention to avoid the disfiguring effect of the first and last droplet running out from micropipett is paid. The author, in addition, devised a special micropipett which is fitted to push out a minute amout of serum by the pressure differences of fingers and to “handwrite” a uniform streak of serum on wet paper.
2. On the paper, the migration velocities of serum fractions enhance as the water content of paper (g/cm²) increase. The increases of the tension lessen the water content of paper, subsequently leading to the slow migration velocities.
3. The distortion and disfigure of a electrophoretic pattern on a paper strip are mainly due to the differences in tension.
4. When several isolated paper strips are mounted in the same electrophoresis apparatus, the migration distance of each serum fraction on them are not generally equal, owing to the minute differences of tension among these paper strips.
5. A sheet paper with slits as shown in Fig. 6 is newly designed for paper electrophoretic use.
6. A new apparatus was devised for the facilitation of mounting a paper with slits and making the tensions of paper strips as equal as possible.
7. As each fraction on paper strips on a paper with slits has almost the same migration distance, one of these strips can be used as a guide strip for checking the eletrophoretic separation as well as for cutting off the fractions from the strips for preparative experiments. The electrophoresis using a paper with slits is especially suitable for the preparation of serum fractions. This method is named the improved preparative electrophoresis by paper srips (Yamaguchi).

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われわれの考案した交叉濾紙泳動法は,電気泳動によつて濾紙上に二物質を交叉させ,交点が単純な点であるか,それとも特別の形を呈するかによつて,二物質が附加化合物を作るか否かを検討する方法である。この方法を色素と色素,色素と蛋白などに応用して,たしかに附加化合物の検出に役立ちうることを証明することができた。
交叉濾紙泳動法を応用して実際に証明せられた附加化合物は,つぎのごとくである。色素同志では塩基性のメチレンブリューと酸性のオレンジGG,ブロム・クレゾール・グリーン,ブローム・フェノール・ブリューおよびアミドブラック10Bとの附加化合物,フクシンとブローム・クレゾール・グリーン,ブローム・フェノール・ブリュー,およびアミドブラック10Bとの附加化合物。色素と蛋白では血清アルブミンとブローム・クレゾール・グリーン,ならびにブローム・フェノール・ブリューの附加化合物,および血清グロブリンとアミドブラック,オレンジGGならびにメチレンブリューとの附加化合物である。
附加化合物の生成の証明せられなかつたのはフクシンとオレンジGG,アスパラギン酸ならびにリジンと上記色素類,アミノ酸相互間,血清蛋白と卵白アルブミン,卵白アルブミンと色素類である。

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