Separation Of Water-soluble Proteins Research Articles

Extraction and Separation of Water-Soluble Proteins from Different Wheat Species by Acidic Capillary Electrophoresis

Optimization of protein extraction and a capillary zone electrophoresis method for water-soluble protein analysis in wheat is described. The optimal separation was obtained with a 50 microm i.d. x 27 cm (20 cm to detector) uncoated capillary filled with 0.1 M phosphoric acid/beta-alanine, pH 2.5, buffer containing urea (1 M), 0.05% (w/v) hydroxypropylmethylcellulose, and 20% (v/v) acetonitrile. Separation was carried out at 15 kV and 35 degrees C for 9 min. Extract stability was also investigated within 2 h from the extraction. Good visual peak parameters and a higher sensitivity can be obtained when 30% ethanol is used as an extraction medium. The method was successfully used to analyze extracts obtained from whole and refined meals of six Triticum spp. Moreover, the described methodology could be applied to the discrimination of species with different ploidy levels and to the detection of durum wheat adulteration, as well as to screen wheat collections for enzymes involved with the quality of wheat derivatives.

Capillary HPLC–ICP MS mapping of selenocompounds in spots obtained from the 2-D gel electrophoresis of the water-soluble protein fraction of selenized yeast

A method based on ICP collision-cell MS detection in capillary HPLC was developed to gain an insight into the purity and identity of selenium-containing proteins separated by 1-D and 2-D electrophoresis. The bands and spots obtained after the separation of water-soluble proteins in selenized yeast were digested with trypsin prior to chromatography. Selenium could be detected down to the subpicogram level. The method, assisted by information obtained by MALDI TOF MS on the 5000 Da cut-off fraction, permitted the purity of bands and spots to be estimated and the efficiency of tryptic digestion and the quantity of selenium present in individual peptides to be evaluated. Owing to the high sensitivity and the lack of matrix suppression effects, the method provided chromatograms with signal-to-noise ratios of 10-1000 in conditions where the common ES Q-TOF MS detection failed.

Separation of Tissue Proteins of Human Lung Carcinomas by Partial-Filling Capillary Electrophoresis

Tissue proteins from human squamous cell lung carcinomas (SQCLC) and small cell lung carcinomas (SCLC) were separated in 0.01% hydroxypylmethyl cellulose (HPMC) linear polymer sieving solutions in the inlet portion of the capillary and next to the outlet of the capillary, followed by capillary zone electrophoresis (CZE) in 40 mM phosphate buffer, pH 2.5. A proper HPMC concentration could cause a molecular sieving effect through the formation of an entangled polymer network. The migration time of the analyte in this matrix depended on the size and electrophoretic mobility of the analyte, the mesh size, and the electric field strength. In the CZE separation, the electroosmotic flow and the charge-to-size ratio of the analyte were important parameters. HPMC concentration and zone length were examined to optimize the separation. Applying this partial-filling technique to the separation of water-soluble proteins from human lung tissues, we found a greatly improved resolution and increased peak intensity. The capillary electrophoresis patterns of normal, SQCLC, and SCLC were obtained and compared for their molecular classifications.

Capillary electrochromatography with monolithic stationary phases: III. Evaluation of the electrochromatographic retention of neutral and charged solutes on cationic stearyl-acrylate monoliths and the separation of water-soluble proteins and membrane proteins

This article, which is closely related to part II, is concerned with the evaluation of the retentive properties of cationic stearyl-acrylate monoliths (i.e. cationic C 17 monoliths) over a wide range of elution conditions with various uncharged and charged solutes including proteins. The retention parameters for charged solutes including the retention factor k ∗ observed under capillary electrochromatography conditions and the velocity factor k ∗ ep, which reflects the electrophoretic process, were measured for weak, moderate and strong basic compounds. These retention parameters allowed the assessment of the respective contributions from electrophoretic and partitioning separation mechanisms. The cationic C 17 monoliths exhibited sufficient hydrophobic interactions with relatively weak basic solutes. Moderate and strong bases showed migration behaviors dominated by their relatively strong electrophoretic mobility with marginal chromatographic partitioning. At low pH, the cationic C 17 monoliths allowed the separation of proteins with minimum electrostatic interactions between proteins and the cationic sites on the surface of the stationary phase. The utility of the cationic C 17 monoliths was demonstrated in the rapid and efficient separation of two crude extracts of membrane proteins, namely galactosyl transferase and cytochrome c reductase. Short capillary columns (8.5 cm effective length) of the cationic C 17 monoliths allowed rapid and efficient separations of neutral and charged pesticides and metabolites, phenylthiohydrantoin amino acids and proteins at the time scale of seconds at relatively high flow velocity.

Disc-electrophoretic separation of water soluble proteins from scales of different forms of ichthyosis.

In extracts of scales of different forms of ichthyosis, disc-electrophoretic separation of water soluble proteins was performed. Number and position of the protein bands correspond with number and position of the bands of extracts of normal keratin and psoriatic scales. However, in comparison to normal keratin, all examined forms of ichthyosis showed in zone II the enriched bands Nos. 6 and 7. This permits a distinct differentiation from psoriasis in which in zone II the bands Nos. 9 and 10 are enriched. In congenital ichthyosiform erythroderma and in ichthyosis combined with atopic dermatitis, in zone III the bands, containing gamma globulins, are enriched as an expression of the concomittant exudative process. The protein content of scale extracts of ichthyosis is 2 to 5 times lower than the one of psoriasis.

Separation of water soluble proteins in psoriatic scales with different polyacrylamide gel concentrations and molecular weight estimations of the separated bands by disc-electrophoresis

The present investigations are concerned with disc-electrophoretic separation and molecular weight estimation of water soluble proteins in psoriatic scales.

Disk Electrophoresis of Plasmodium berghei

A method of analytic separation of soluble proteins in aqueous extracts of Plasmodium berghei is described. Extracts are prepared by sonication of suspensions of whole washed parasites obtained by saponin lysis of infected rat red cells. Separation of the extracts into reproducible electrophoretic patterns is carried out by zone electrophoresis on polyacrylamide gel. Extracts prepared in this manner contain a heterogeneous mixture of soluble protein constituents of both host and parasite origin. A minimum of 15 parasite proteins and an additional minimum of six proteins of host origin have been identified. This paper presents an analytic separation of water soluble proteins of Plasmodium berghei by the technique of disk electrophoresis. Electrophoresis is carried out in an acrylamide gel matrix of defined pore size permitting separation of components simultaneously by differences in charge and in size and shape. Application of this method to extracts of P. berghei has provided confirmation and extension of reported findings by agar immunoelectrophoretic methods that such extracts are a complex mixture of proteins (Zuckerman, 1964; Sherman, 1964).

Separation of Brain Proteins by Starch Gel Electrophoresis.

SummaryStarch-gel electrophoresis has been employed to separate the proteins in human and rat brain. This technic yielded results superior to those attained by paper or agar-gel electrophoresis. Separation of water-soluble proteins on starch-gel yielded 11–12 discrete protein bands in rat brain and 14–15 bands in human white matter. Human white matter proteins are characterized by low concentration of albumin-type proteins.