Quantitative determination of sulfated glycopeptide by two-dimensional electrophoresis on cellulose acetate membrane

Quantitative determination of the sulfated glycoproteins present in tissue and secretion fluid was performed. After digestion of the specimen with pronase in order to convert glycoproteins to glycopeptides, the sulfated glycopeptides were separated from a mixture of acidic glycans (glycosaminoglycans, sialoglycopeptides and sulfated glycopeptides) by two-dimensional electrophoresis on cellulose acetate membrane [(1986) J. Biochem. Biophys. Methods 12, 239-246]. After staining with alcian blue, the spot of sulfated glycopeptide on the cellulose acetate membrane was cut out, and then only the dye bound to the sulfated glycopeptide was extracted with a 5% cetylpyridinium chloride solution at 100 degrees C for 15 min. The extract was then measured by absorbance at 615 nm using an authentic sulfated glycopeptide as a standard. This method facilitated the determination of sulfated glycopeptides, which were separated from other acidic glycans, within the range 0-25 micrograms.     

Two-dimensional electrophoresis on the layers of cellulose acetate membrane

A new type of two-dimensional electrophoresis for analysis of protein using cellulose acetate membrane has been developed. Prior to the separation, proteins in a sample are concentrated to a narrow zone on a strip of cellulose acetate according to “steady-state stacking” of isotachophoresis. Electroendosmotic counterflow on cellulose acetate membranes is advantageous for the isotachophoretic concentration of large sample volumes. The concentrated protein zone is then subjected to electrophoretic separation on the same strip. This first-dimensional separation including the concentrating process is named “concentrating electrophoresis.” Iso-electric focusing on several layers of cellulose acetate membrane is performed in the second-dimensional step. Many kinds of detection methods can be applied to the layers among which proteins are distributed. The novel two-dimensional electrophoresis takes only 5 h to perform.     

血清蛋白醋酸纤维薄膜电泳实验的改进

<血清蛋白的醋酸纤维薄膜电泳>实验采用宽膜条很好的解释了由窄膜条造成的区带弯曲问题.实验用到的一些试剂较昂贵.在保证和提高生物化学实验教学质量的前提下.以降低实验成本、减少对环境污染角度出发,采用牛血清代替人血清、改变实验条件、回收缓冲液和漂洗液等方法对实验进行了改进,改进的结果是实验稳定,重复性好,用于实验课教学可以达到预期的实验目的和要求.     

Comparative study of serum lipoproteins separated by the method of cellulose acetate membrane electrophoresis in humans and rats

The evaluation of lipoproteins in rat serum which was separated by cellulose acetate membrane electrophoresis was studied in comparison with that in human serum. In contrast to the human lipoprotein pattern, the top of the rat lipoprotein fraction exceeded the albumin fraction towards the anode. By the analysis of ultracentrifugation and post-heparin serum lipolytic activity, the characters of lipoprotein fractions electrophoretically separated in rat serum was confirmed as similar to human serum lipoprotein.     

血清蛋白醋酸纤维素薄膜电泳实验教学的优化设计

醋酸纤维素薄膜电泳（PAME）由于其微量、快速、简便、分辨力高等优点,目前已成为高校生化实验常规项目之一,并被广泛应用于血清蛋白、糖蛋白等生物大分子的临床分离与检测。但它也是在生化实验中影响因素较多、较为复杂并难以取得理想效果的实验。实验采用鸡血清蛋白代替价格昂贵的人血清蛋白进行PAME实验教学,并采取措施优化实验条件和程序,从而较好地调控了实验进程,有效地提高了教学的质量和效率。     

Rapid separation of carbonic anhydrase isozymes using cellulose acetate membrane electrophoresis

A method is described for the rapid separation of carbonic anhydrase(CA) isozymes by cellulose acetate membrane electrophoresisin which CA activity is detected using the pH-indicating dye,bromcresol purple. This method can detect bovine erythrocyteCA in a 0.3 mm³ sample applied at a concentration of 100 ngcm^–3 (total of 30 pg applied) while at higher concentrationsthree isozymes were observed. It was found, using a potentiometrictechnique, that intact cells of Anabaena flos-aquae (Cyanophyceae)and Chlorella ellipsoidea had no detectable activity while C.saccharophila and Chlamydomonas reinhardtii (Chlorophyceae)had external CA activity. CA activity of the extracts suggestedthe presence of internal CA in all species. After electrophoresisit was found that C. saccharophila and C. reinhardtii had twoisozymes while A. flos-aquae and C. ellipsoidea had only a singledetectable band. Spinach had up to five detectable isozymesthat were difficult to resolve. Incubation of spinach extractwith the CA inhibitor ClO₄^– (500 mol m^–3) inhibitedCA activity by 90% using the potentiometric technique, but afterelectrophoresis had no detectable effect. This technique isuseful in identifying isozymes that are substantially differentin electrical charge and in monitoring CA isozyme activity duringenzyme purification. Key words: Carbonic anhydrase, isozymes, cyanobacteria, microalgae, spinach     

Esterase-D polymorphism in Assam by cellulose acetate electrophoresis

Summary With the help of a simplifed and quick method, cellulose acetate electrophoresis, the phenotypes of esterase D were determined in an Assamese population. The gene frequencies of Es D¹ were 0.7263 and 0.2737 for Es D².

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Zusammenfassung In einer Stichprobe aus Assam wurde mit Hilfe einer einfachen und schnellen Methode, der Cellulose-Acetat-Elektrophorese, die Bestimmung der Esterase D-Phänotypen durchgeführt. Die Genfrequenzen wurden für Es D¹ zu 0,.7263 und für Es D² zu 0.2737 bestimmt.

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Supported by the Deutsche Forschungsgemeinschaft, the Stiftung VW and the Fonds der Chemischen Industries.     

Solubilization of membrane proteins for two-dimensional gel electrophoresis: identification of sarcoplasmic reticulum membrane proteins

Solubilization of membrane proteins for two-dimensional electrophoresis (2DE) is very difficult. In this study, we report the use of 1,2-diheptanoyl-sn-glycero-3-phosphatdiyl choline (DHPC) as a detergent to solubilize integral membrane proteins for 2DE. Rat ventricular microsomal fractions enriched with sarco(endo)plasmic reticulum (SR) membrane proteins were used as a model system. Compatibility of DHPC with a high concentration of urea increases the solubility of proteins compared with sulphobetaines or ASB-14. Peptide mass analysis assisted in the identification of key SR membrane proteins including SR Ca(2+) ATPase and other membrane proteins, which have not previously been reported on 2DE. These results suggest that DHPC is a better detergent for solubilizing membrane proteins and may be useful in generating proteomic maps for most complex organelles including SR.     

A method for the two-dimensional electrophoresis of leaf proteins

Proteins extracted from green leaves of tobacco were subjected to analysis by two-dimensional electrophoresis. It was found that electrophoretic separations were unsatisfactory when leaf extracts were analyzed directly without prior extraction of pigments, phenols, and lipids by acetone treatment. The plant pigments and several phenolic compounds present in leaves presumably interacted with the proteins and created charge heterogeneity, streaking, and other artifacts. It was found that these problems could be overcome by treatment of leaf extracts with acetone followed by solubilization and electrophoresis of the acetone-treated proteins. Leaf extracts were prepared by grinding deribbed leaf disks in a buffer containing 5 mm potassium carbonate, 9.5 m urea, 0.5 dithiothreitol, 2% Nonidet P-40 detergent, 500 μg/ml l-lysine, and 2% Ampholines. The extracts, after centrifugation to remove cell debris and insoluble material, were treated several times with ice-cold acetone. The acetone-precipitated proteins were treated with nucleases, reprecipitated with cold acetone, and then resuspended in the above grinding buffer. The presence of l-lysine and Ampholines were required for good electrophoretic separations. The resuspended proteins were subjected to two-dimensional electrophoresis and proteins detected by staining and or fluorography. At least 300 distinct proteins could be recognized in radioactive samples. The method gives reproducible patterns even after repeated freezing and thawing of the samples.     

Esterase-D polymorphism in Assam by cellulose acetate electrophoresis.

With the help of a simplified and quick method, cellulose acetate electrophoresis, the phenotypes of esterase D were determined in an Assamese population. The gene frequencies of Es D(1) were 0.7263 and 0.2737 for Es D(2).     

A novel system for the two-dimensional electrophoresis of membrane proteins.

Membrane proteins were resolved in two dimensions by a novel technique that uses discontinuous electrophoresis in both directions. After electrophoresis in the first direction in chloral hydrate, the membrane proteins were further resolved by a novel system that used organic-base dodecyl sulphates to stack and then resolve them. This latter system has several advantages over conventional electrophoresis in sodium dodecyl sulphate, notably that it avoids the production of artifacts generated by other systems.     

Silver stain for proteins on a cellulose acetate membrane

A rapid and sensitive silver staining method to detect proteins on a cellulose acetate membrane has been established. This method is achieved by modification of the silver-based color staining for detection of proteins in polyacrylamide gels [D. W. Sammons, L. D. Adams, and E. E. Nishizawa, Electrophoresis 2, 135-141 (1981)] and applied to our new type of two-dimensional electrophoresis for analysis of proteins on a cellulose acetate sheet [T. Toda, T. Fujita, and M. Ohashi, Anal. Biochem. 119, 167-176 (1982)]. Maximal sensitivity of silver stain for proteins on a cellulose acetate membrane can be obtained by an optimal balance between deposition of silver on the protein and on the background. Certain kinds of proteins are colored red, orange, or grayish-blue. The silver stain is 20-80 times more sensitive than Coomassie blue and some spots are visualized reproducibly by silver only. Densitometric evaluation of standard proteins stained with silver and Coomassie blue is also demonstrated. The method takes only 50 min to perform and is sensitive, simple, and reproducible.     

Bidimensional electrophoresis of factor VIII antigen on cellulose acetate

It is described two-dimensional immunoelectrophoresis using cellulose acetate as supporting medium compared with agarose gel method. The results show that the determination of the FVIIIR: Ag on cellulose acetate is a technique more simple and rapid than agarose gel method and many free from the technical failure.     

Preparation of membrane proteins for analysis by two-dimensional gel electrophoresis

In order to separate hydrophobic membrane proteins, we have developed a novel two-dimensional electrophoresis system. For the iso-electric focusing, agarose was used as a supporting matrix and n-dodecyl-beta-D-maltopyranoside was used as a surfactant. In combination with a previously developed Tris/MES electrophoresis system in the second dimension, distinct spots were reproducibly detected from hydrophobic membrane proteins whose grand average hydropathicity (GRAVY) exceed 0.3. In contrast to the immobilized pH gradient system, c-type heme was also visualized in this system.