Free flow and capillary isoelectric focusing of bacteria from the tomatoes plant tissues

The means of the preconcentration and preseparation of selected species or pathovars of bacteria directly from the plant tissue suspension by free flow isoelectric focusing are introduced here. After the focusing, the resulting fraction of microorganisms, native or dynamically modified by the non-ionogenic tenside on the basis of pyrenebutanoate, was separated by capillary isoelectric focusing and/or cultivated and positively identified by gas chromatographic analysis of fatty acid methyl esters. Simultaneously, capillary isoelectric focusing with UV and fluorometric detection was used for the rapid estimation of unknown isoelectric points of the examined plant pathogenic species of genus Clavibacter, Xanthomonas and Pseudomonas prior to the preconcentration and preseparation. The microorganisms were of different origin, native and/or dynamically modified by the non-ionogenic tenside.     

改性明胶沉降剂的等电点及其分布的研究

改性明胶已经被广泛地用作感光乳剂的沉降剂。然而对其沉降作用的机理,人们却还了解得很少。改性明胶的等电点,是影响改性明胶沉降的重要因素之一。我们采用等电聚焦的新技术,经过适当的改变,成功地测定了改性明胶的等电点及其分布。所得等电点分布的数值,能较好地说明改性胶为什么只有在确定的pH范围内才能进行沉降。     

Estimation of biotinylated lectin by isoelectric focusing

Concanavalin A (Con A) was biotinylated to various degrees using N-biotinyl-omega-aminocaproic-acid-N-hydroxy succinimide ester as the biotinylation reagent, and then analyzed by isoelectric focusing using PhastGel IEF 3-9. The isoelectric points of biotinylated ConAs were found to decrease with increasing concentration of the biotinylation reagent. Analysis by isoelectric focusing followed by dot blotting clearly indicated that the biotinylated ConA with an isoelectric point lower than that of the original ConA by 2.2 +/- 0.6 had the strongest binding activity for ovalbumin.     

Direct MALDI-MS Analysis of Proteins Isolated by Liquidphase Isoelectric Focusing Electrophoresis

A liquid-phase isoelectric focusing electrophoresis system(Rotofor) was used as the prefractionation tool for the sample preparation in the MALDI-MS analysis of a protein mixture. Each fraction collected was then directly subjected to MALDI-TOF-MS analysis. By this approach, we are able to resolve two types of hemoglobins, A and C, which cannot be successfully separated by means of the traditional SDS-PAGE method.     

Analysis of histones by on-line capillary zone electrophoresis-electrospray ionisation mass spectrometry

Clotting factor IX preparations from human plasma (pdFIX) have been characterized using electrophoretic methods like sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectric focusing and two-dimensional polyacrylamide gel electrophoresis. Factor IX prior to and after activation with factor XIa was separated by one- and two-dimensional polyacrylamide gel electrophoresis and on isoelectric focusing gels. The main differences between the band patterns of the two pdFIX preparations are due to their purity. Vitronectin was identified by immunological techniques as major accompanying plasma protein, separated from factor IX and characterized by isoelectric focusing and two-dimensional polyacrylamide gel electrophoresis.     

Carrier ampholyte‐free isoelectric focusing on a paper‐based analytical device for the fractionation of proteins

Isoelectric focusing plays a critical role in the analysis of complex protein samples. Conventionally, isoelectric focusing is implemented with carrier ampholytes in capillary or immobilized pH gradient gel. In this study, we successfully exhibited a carrier ampholyte‐free isoelectric focusing on paper‐based analytical device. Proof of the concept was visually demonstrated with color model proteins. Experimental results showed that not only a pH gradient was well established along the open paper fluidic channel as confirmed by pH indicator strip, the pH gradient range could also be tuned by the catholyte or anolyte. Furthermore, the isoelectric focusing fractions from the paper channel can be directly cut and recovered into solutions for post analysis with sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and matrix‐assisted laser desorption/ionization‐time‐of‐flight mass spectrometry. This paper‐based isoelectric focusing method is fast, cheap, simple and easy to operate, and could potentially be used as a cost‐effective protein sample clean‐up method for target protein analysis with mass spectrometry.     

Preparative isoelectric focusing of reduced wheat gluten proteins

Proteins extracted from gluten of the bread wheat cultivar Fiorello 2 in the presence of 2-mercaptoethanol or dithiothreitol were separated by isoelectric focusing in a free solution in a pH 3-10 gradient containing 50% v/v 1-propanol or urea. The collected fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in 10% gels (high and medium molecular weight glutenin subunits) and 16% gels (low molecular weight gliadins). The isoelectric focusing pattern of gluten polypeptides in 50% v/v 1-propanol was comparable to that obtained on two-dimensional gel electrophoresis, based on isoelectric focusing and polyacrylamide gel electrophoresis or nonequilibrium pH gradient electrophoresis and polyacrylamide gel electrophoresis. A similar isoelectric focusing pattern was also observed when 3M urea was used as solvent. New gluten polypeptides, similar in mobility to the high molecular weight subunits of glutenin were detected at acidic pH.     

Comparison between isoelectric focusing methods for the detection of orosomucoid phenotypes

Orosomucoid (ORM) polymorphism was investigated by different methods including isoelectric focusing in acid pH ranges followed by silver staining, print immunofixation of desialyzed ORM, fixation using a lectin from the sea-weed Codium tomentosum, isoelectric focusing followed by immunofixation in miniaturized gels and isoelectric focusing in immobilized pH gradients. Population genetics studies were carried out in Galicia (NW Spain) and two new ORM variants were found.     

On-line coupling of capillary isoelectric focusing with transient isotachophoresis-zone electrophoresis: a two-dimensional separation system for proteomics

A microdialysis junction is employed as the interface for on-line coupling of capillary isoelectric focusing with transient isotachophoresis-zone electrophoresis in a two-dimensional separation system. Capillary isoelectric focusing not only provides high-resolution separation of tryptic peptides based on their differences in isoelectric point, but also potentially allows the analysis of low-abundance proteins with a typical concentration factor of 50-100 times. Carrier ampholytes, employed for the creation of a pH gradient during focusing, are further utilized as the leading electrolyte in the second separation dimension, transient isotachophoresis-zone electrophoresis. Many peptides which have the same isoelectric point would most likely have different charge-to-mass ratios, and thus different electrophoretic mobilities in zone electrophoresis. Two-dimensional separation of proteolytic peptides is demonstrated using standard proteins, including cytochrome c, ribonuclease A, and carbonic anhydrase II. The maximum peak capacity is estimated to be around approximately 1600 and can be significantly increased by simply increasing the capillary column length and manipulating the range of pH gradient in isoelectric focusing. In addition to enhanced separation efficiency and resolution, this two-dimensional electrokinetic separation system permits sensitive and comprehensive analysis of peptide fragments, especially when integrated with electrospray ionization mass spectrometry for peptide/protein identification.     

Carrier Ampholyte Isoelectric Focusing Based Two-Dimensional Electrophoresis in Preliminary Screening of Differential Proteomics Analysis

Two-dimensional electrophoresis is a current method for separating complex protein mixtures of a given sample in different states. In this study an improved carrier ampholyte isoelectric focusing method has been evaluated for its capacity for preliminary screening of expressional proteomics subjects. In comparison with current carrier ampholyte isoelectric focusing, this method showed enough resolution power to display major expressional changes in proteomic samples and demonstrated it can be used as a substitution for the immobiline based isoelectric focusing method.     

Separation of human alloalbumin variants by isoelectric focusing

A technique for the separation of human alloalbumin variants by means of isoelectric focusing in the presence of 8M urea and 60 mM L-serine is described. The potential usefulness of this technique in the detection and classification of genetic heterogeneity at the albumin locus is demonstrated by the differentiation of three human alloalbumin variants of European origin.     

Ti/IrO2 as anode and Zr as cathode in multicompartment electrolyzers with Immobiline membranes.

Multicompartment electrolyzers with isoelectric Immobiline membranes are used for large-scale preparative protein purification. A series of isoelectric membranes, of defined pI values, is utilized for keeping any desired species isoelectric within each compartment of the electrolyzer. It is preferable to have electrode disks of the same surface area as the membranes for a proper performance of the instrument because electrolyte solutions of low conductivity are used. The use of Pt disks would be quite expensive; we therefore propose using Zr as a cathode and Ti/IrO2 as an anode in the electrodic compartments. This pairing of electrodes seems to give the same performance as Pt wires. Also, conventional isoelectric focusing, as well as isoelectric focusing in immobilized pH gradients, both requiring a good contact area between gel and electrodes, would benefit by using flat laminae of these metals as electrodes.     

Identification of group specific component/vitamin D-binding protein (GC/DBP) mutants by isoelectric focusing in immobilized pH gradients

The six common genetic types of the group specific component/vitamin D-binding protein (GC/DBP) system are usually classified by isoelectric focusing in carrier ampholytes, followed by visualization of the GC proteins by immunoprinting with monospecific antiserum. In addition, more than 120 mutant GC types have been discovered. For their identification additional methods were necessary, including polyacrylamide gel electrophoresis, isoelectric focusing in the presence of 3 M urea as well as isoelectric focusing in immobilized pH gradients. The application of the last method is described in detail and several examples of GC/DBP mutants identified thereby are presented.     

pKa shift-associated effects in enantioseparations by cyclodextrin-mediated capillary zone electrophoresis.

Enantioselective migration of dansylated (Dns) amino acids in the presence of hydroxypropylated-beta-cyclodextrin under acidic conditions near the pI value of the analytes was investigated by means of capillary zone electrophoresis. Based on the migration data, the pH dependence of the complexation constants was evaluated, as well as the variation of the complex mobilities with pH. As a result of these data, the migration behavior in the pH region near the pI could be understood, which, in some instances, includes the reversal of migration order upon variation of selector concentration. The enantioselective pKa shifts upon complexation could be quantitated for the carboxylic and the amino group separately. pKa shifts were found in the order of 0.8 pI units, the differences between the enantiomers being up to 0.25 pH units. These data were in agreement with the pI shifts reported from isoelectric focusing experiments. The accurate determination of the pI values of the Dns amino acids makes it possible to calibrate the pI scale in isoelectric focusing in the presence of chiral selectors.     

Factor B (BF) subtyping by isoelectric focusing: methods, nomenclatures, genetics and forensic application

Usually factor B (BF) typing is performed by means of the traditional agarose gel electrophoresis. Using isoelectric focusing, the system can be extended by two common subtypes of BF F. The existence of BF F subtypes has in the meantime been confirmed by various authors and in different populations. Their inheritance has been proven by family- and mother/child analyses and molecular-genetic studies (correlation with restriction fragment length polymorphism). Different typing methods as well as different nomenclatures seem to indicate that the subtypes FA and FB (according to Geserick et al.) are identical with the Fb and Fa subtypes (according to Teng and Tan). At present, some confusion still exists for the less frequent variants and subtypes which possibly could be identified by direct comparison of the patterns. The BF system is a valuable marker in paternity testing. Its chance for exclusion of paternity in Caucasian populations has been calculated to be about 14% for agarose gel electrophoresis and increases to about 16% for BF F subtyping by isoelectric focusing. Preliminary results indicate that BF may also be used for typing of bloodstains (up to 2 weeks old).     

Dynamics of gel isoelectric focusing with ampholytic dyes monitored by camera in real-time

The dynamics of gel isoelectric focusing were studied by using amphoteric low-molecular-mass colored substances (isoelectric point markers). The polyacrylamide gel in slab format was in direct contact with the electrodes. In addition to isoelectric focusing with a pH gradient composed of synthetic carrier ampholytes, pH gradients created by simple buffers of acetic acid, 2-(N-morpholino)ethanesulfonic acid, histidine and N,N,N',N'-tetramethylethylenediamine were applied. The progress of the electrofocusing process was monitored by a charge-coupled device camera and video recording. The gradient profile and dynamics were approximated from the positions of isoelectric point markers, which were focused both on boundaries between individual zones of simple buffers and within the zones themselves. The obtained animated records enabled the observation of the entire real focusing run within fractions of a minute, which is useful both for the understanding and optimization of the focusing.     

Interaction of the vitamin D-binding protein (group-specific component) and its ligand 25-hydroxy-vitamin D3: binding differences of the various genetic types disclosed by isoelectric focusing

The three common variants of the vitamin D binding protein, also known as group specific component (Gc), namely types 1S, 1F and 2, as well as some rare variants were studied by thin-layer polyacrylamide gel isoelectric focusing in a pH 4.5-5.4 carrier ampholyte generated pH gradient, additionally containing N-(2-acetamido)-2-aminoethanesulfonic acid (ACES). Prior to isoelectric focusing, whole serum or purified preparations of the vitamin D binding protein were incubated with 25-hydroxycholecalciferol at various ligand/protein ratios. Binding differences were found for the anodal and cathodal isoforms of Gc 1 variants and also for various allelic types. Isoforms with higher isoelectric points generally had a lower affinity for the ligand than the variants with lower isoelectric points.     

The genetic haptoglobin polymorphism: relevance of paternity assessment

A practical method for haptoglobin subtyping is described utilizing fast sample preparation by means of batch adsorption to DEAE-cellulose and subsequent isoelectric focusing of reductively cleaved samples. The expanded haptoglobin polymorphism leads to an increase of the theoretical paternity exclusion rate to approximately 33%. Hence, the system appears to be highly attractive for paternity assessment.     

Subtyping of phosphoglucomutase 1 (PGM1) in microstains of different biological materials with subsequent species differentiation without further stain material consumption

Micromethods for subtyping of phosphoglucomutase 1 (PGM 1) in small amounts of biological stain material are described, using an applicator for highly diluted stain extracts. With the aid of this applicator strip blood and semen micro-stains as well as single hair-roots could be extracted by electroelution directly on the PGM1 isoelectric focusing gel. Species differentiation was also possible either by radial immunodiffusion using the extract remaining in the applicator strip after isoelectric focusing or by interpretation of the PGM pattern itself.     

Identification of rat liver glutathione S-transferase Yb subunits by partial N-terminal sequencing after electroblotting of proteins onto a polyvinylidene difluoride membrane from an analytical isoelectric focusing gel

Rat liver glutathione S-transferases were partially purified using S-hexyl glutathione affinity chromatography, followed by native isoelectric focusing employing a pH 7-11 or pH 3-10 gradient. Proteins were excised and eluted from the gel for determination of subunit composition using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In separate experiments, isoelectric focusing gels were equilibrated with a sodium dodecyl sulfate-containing buffer at high pH, and proteins on the gel were electroblotted onto a polyvinylidene difluoride membrane, utilizing graphite plates as electrodes. The membrane-bound proteins were visualized by Coomassie Brilliant Blue staining. The protein bands were then excised from the membrane and inserted into a gas phase sequenator for direct sequencing. N-Terminal sequences thus determined were compared with published cDNA sequences. The isoelectric points (pIs) and positions on the isoelectric focusing gel of Yb1Yb1, Yb1Yb2 and Yb2Yb2 subunits were determined. We have also located on the pH 3-10 focusing gel an N-terminal blocked glutathione S-transferase which has a molecular weight similar to Yb subunits.