FACE (fluorophore-assisted carbohydrate electrophoresis) is a method for the separation of fluorophore-labeled saccharides by the use of polyacrylamide gel electrophoresis. The method is characterized by high resolution, high sensitivity, and ease of use without expensive giant-sized machine such as mass spectrometry. It has been found to be useful for the analysis of oligosaccharides profiling of N-linked or O-linked glycans after releasing saccharides from glycoproteins and for sequencing oligosaccharides component. Judging from the importance of oligosaccharides in glycoproteins and the changes in oligosaccharides of cancer cell surface, the method described here is hopeful as a unique analysis tool in the field of basic medical science.

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We established a simple method for measuring HDL subfraction cholesterols by gradient polyacrylamide gel electrophoresis (gradient PAGE). Prestained human serum was separated into two major bands identical with HDL₂ and HDL₃ by the gradient PAGE. HDL-cholesterol (HDL-C) obtained by precipitation method was divided according to HDL₂/HDL₃ ratio determined by densitometry of the electrophoretic pattern. HDL subfraction cholesterols determined by this method was well correlated with those measured by zonal ultracentrifugation (HDL₂-C: r=0.98, HDL₃-C: r=0.94, n=11). Mean serum HDL₂-C level was significantly higher in normal females than in normal males (p<0.01). HDL₃-C levels were significantly decreased in liver diseases, especially in cirrhotics. This simple electrophoretic method for quantification of HDL₂ and HDL₃ is suitable for clinical application in various pathological states.

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We examinied the differentiation of lipoprotein in human serum using polyacrylamide gradient gel (PAGG) electrophoresis to analyze the lipid components and to quantify the small, dense LDL. Electrophoretic patterns were classified into three types, which included two types reported by Krauss et al. and one new type containing equal same amounts of regular and small size LDL. We devised a direct staining method to stain triglyceride (TG) and cholesterol (Cho) in lipoprotein separated by the polyacrylamide gel electrophoresis and components of lipoprotein was quantitaivety analyzed with the densitogram. At the same time, the presence of apoproteins LP (a) and B in the lipoprotein was confirmed. As a result of the staining, we observed more amounts of Cho containing in the fraction of regular size LDL than small dense LDL, and more amounts of TG containing in the fraction of small dense LDL than regular size LDL. Apo B was detected in both fractions of regular size LDL and small dense LDL, but no LP (a) was detected.

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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.

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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.

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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 0.45_M Tris-HCl (pH 8.8), 0.1% SDS and 7_M 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.

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A vertical polyacrylamide gel slab electrophoresis apparatus, which employed a discontinuous buffer system, and spacer gel 5mm thick at the upper portion, 1mm at the lower portion and running gel 1mm in thickness, was devised.
The increased thickness of the upper portion of the spacer gel and the discontinuous buffer system made it possible to apply a large quantity of dilute sample and to obtain a sharp electrophoretogram similar to that of disc electrophoresis.
The thinness of the running gel made it possible to use ultraviolet method for detecting isozyme.
Using this method, human lactate dehydrogenase isozymes and pyruvate kinase isozymes were demonstrated in sharp bands. Furthermore, purification of human erythrocyte pyruvate kinase was performed and purity of the enzyme in each step of purification was examined with simultaneous comparison of protein and enzyme stain. These results confirmed that this apparatus was useful for the study of dilute enzyme preparations especially of unstable enzyme such as pyruvate kinase.

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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.

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Horizontal thin-layer polyacrylamide gel electrophoresis proved to provide a good resolution of urine amylase isozymes, with up to eight bands. In order to determine the source of amylase isozymes in urine, comparative studies were made on the electrophoretic mobilities of amylase bands in urine, salivary gland and pancreas homogenates obtained under the same condition. The results suggested that the amylase isozymes contained in urine consisted of those originated in pancreas and salivery glands. Storage of urine at 4°C and -18°C for 6 months resulted in no change of the mobilities of urine amylase isozymes. From these results, it seemed to be reasonable to determine the origin of each amylase isozyme on the basis of the electrophoretic mobility. Heat treatment of urine and saliva at 56°C for 6 hours caused no or slight reduction in amylase activity. The amylase activity of pancreatic juice, however, was completely inactivated by heat treatment at 56°C for 60 minutes. The mobility of each amylase isozyme did not change by the heat treatment, while, the activity of the isozyme AmyU-1 (pancreatic origin) showed marked reduction. This behavior suggests a possible physicochemical difference between the pancreatic and salivary amylases.

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A simple electrophoretic technique using thin layer polyacrylamide gel provided a good resolution of serum amylase isozymes with separation of up to 9 isozyme fractions. A strong oxidizing agent, ammonium persulfate, which had been used in polyacrylamide gel formation system, was removed by prior electrophoresis from the separating gel before sample application to avoid possible degradation of amylase molecule into subunits.
From the results of electrophoretic mobility studies of serum, pancreatic and salivary amylase isozymes, it was concluded that amylase isozymes in human serum were of pancreatic and salivary origin.
In patients with acute pancreatitis, increased amylase activities were observed on those of pancreatic origin. On the contrary, in patients with pancreatectomy, these isozyme activities disappeared from serum. In patients with mumps, amylase activities of salivary origin, which were observed in patients with pancreatectomy, increased in serum.

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平板ゲルをガラスなどで裏打ちし補強すると, これを用いた電気泳動分離ならびに染色などの操作が容易になる. しかしながら, この裏打ちにより通常のコロイド状染料の吸着に基づく染色反応は遅くなる. 本研究ではガラス基板で裏打ちしたポリアクリルアミドゲル中に泳動分離した蛋白質にヘミンを吸着させ, そのペルオキシダーゼ様の触媒活性を利用して高速に蛋白スポットを染色する方法を開発した. すなわち, このヘミン染色法と名付けた方法では, 蛋白質に吸着したヘミンが過酸化水素の存在下で2,6-キシレノールと3,3'-ジアミノベンチジンの酸化重合反応を触媒する. この反応により蛋白質分子上で染料があらたに合成され染色される. 本研究ではこの染色反応の反応条件の最適化を行い, 45分間で蛋白質が染色されることを見出した. 本論文ではヘミン染色法とクーマシーブルー染色法の比較結果についても言及する.

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Using a simple thin layer polyacrylamide gel electrophoresis, human serum alkaline phosphatase could be separated into up to 5 isozyme bands; 2 or 3 isozyme bands in normal adult's sera and 5 bands in patient's sera. They were numbered as Alp I, II, III, IV and 0 in order of decreasing anodic mobility. It was found that Alp I to IV corresponded in mobility to the major component of the respective tissue extracts, i, e, Alp I to hepatobiliary, Alp II to bone, Alp III to placental and Alp IV to intestinal alkaline phosphatase. Alp 0 was the activity remaining at the origin and was supposed to be polymers or complexes located on large particles, such as cell debris, etc.
Alp III from pregnant sera could be devided into 3 subgroups, i. e., fast (Alp III_F), intermediate (Alp III_I) and slow (Alp III_S), according to its mobility.
The activity of Alp I, sometimes with that of Alp IV, rose mainly in hepatobiliary disease, while activities of Alp II rose in bone and parathyroidal disease. Increase in activity of Alp III and 0 occurred mainly in pregnancy and malignant disease, respectively. The changes of alkaline phosphatase zymograms in pathological states gave us the key to differential diagnosis.
It was shown that the appearance of Alp IV in normal adult's serum was related to the blood group B or O with salivary secretor type.
Thus, the electrophoretic separation of alkaline phosphatase of human serum by using the simple thin layer polyacrylamide gel plate as the supporting medium was shown to give us many useful informations about clinical diagnosis. Treatments of serum before electrophoresis, such as diluting, freezing and thawing, heating and adding surface active agents, enzymes, lectins, etc., gave much more informations for the identification of alkaline phosphatase isozymes.
It was indicated from these data that the migration of alkaline phosphatase isozyme bands was strongly affected by the structure of their end polysaccharide chains.

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