Isoelectric Focusing Of Proteins Research Articles

New approaches to capillary isoelectric focusing of proteins. cIEF offers advantages in sample preparation, quantitation, and reproducibility.

New approaches to capillary isoelectric focusing of proteins. cIEF offers advantages in sample preparation, quantitation, and reproducibility.

Ampholyte‐free isoelectric focusing of proteins in cone shaped capillaries

AbstractA simple method of generating pH gradients for use in capillary isoelectric focusing was described and evaluated experimentally. In the proposed scheme, the cone shaped capillary filled with a conventional buffer is used to produce an effect of nonuniform heating which then generate temperature and corresponding pH gradients along the capillary axis. Concentration and separation of proteins by applying a simple isoelectric focusing system based on this principle is demonstrated. An inexpensive absorption imaging detection system is used to continuously monitor progress of the separation and concentration processes.

Banding Patterns of Fasciola hepatica and Fasciola gigantica (Trematoda) by Isoelectric Focusing

Liver flukes Fasciola hepatica and Fasciola gigantica are polymorphic and vary morphologically depending upon the host being parasitized. It is known also that mixed infection occurs where both species are present. A technique involving protein separation was used to distinguish the 2 species. Isoelectric focusing of soluble proteins was performed on polyacrylamide gels using whole-body proteins from adult flukes. Although many bands appeared common to both species and some were shared with host tissues, the banding patterns could be used to distinguish 1 species from the other. Soluble protein isoelectric focusing is simple, reproducible, and has very good resolution. It seems well suited to the differentiation of the 2 fluke species.

Self-incompatibility related glycoproteins of Brassica are produced and secreted by transgenic tobacco cell cultures

A Nicotiana tabacum cell line was transformed, using particle bombardment, with constructs encoding the Brassica stigma glycoproteins, S-locus glycoprotein(SLG)-6 and S-locus related glycoprotein-1, under the control of a duplicated cauliflower mosaic virus 35S promoter and the nos terminator sequence. Calli and cell suspension lines that express each of the two proteins were recovered. Using specific antibodies, the foreign proteins can be detected in the cells, the cell wall eluate and the culture medium. The characteristic band patterns representing glycoforms (and isoforms) of the transgenically produced proteins on sodium dodecyl sulfate and isoelectric focusing protein gels are very similar to those from Brassica stigmas. Partial purification of SLG from the culture medium is described and the potential of the system for studying the biochemical basis of self-incompatibility is discussed.

Isoelectric focusing of proteins in a microcapillary with universal concentration gradient detection

AbstractProteins were separated by capillary isoelectric focusing (CIEF) performed in a 5‐μm i.d., 12‐cm long uncoated capillary, and detected by an on‐column concentration gradient detector based on Schlieren optics. The narrow capillary allows the application of a 15‐kV separation voltage. The high voltage resulted in a very narrow 30‐μm wide zone for carbonic anhydrase, which corresponded to a 60‐fL zone volume. All focused proteins could be detected by the universal concentration gradient detector without derivatization in less than 7 min. On‐column mass detection limits for proteins were at the 100 amol level, which was lower than that of a UV‐vis absorption spectrometric detector. The detection volume of the detector was calculated to be 20 fL, the smallest detection volumes reported to date for optical or spectroscopic detectors. This microcapillary CIEF‐concentration gradient detector system may be a useful technique for direct analysis of proteins at the single cell level.

Isoelectric focusing of soluble proteins from Fasciola hepatica L, 1758 and Fascioloides magna B, 1875

Summary Isoelectric focusing was performed on the soluble proteins of whole-body and excretory-secretory products (esp) of Fasciola hepatica and Fascioloides magna. Adult F hepatica flukes were recovered from experimentally infected sheep and esp obtained from the flukes; portions of liver were cut and frozen at - 70 C. Fascioloides magna adults were collected from naturally infected white-tailed deer and esp obtained; portions of liver were collected from noninfected white-tailed deer. Adult flukes and their host tissues were homogenized and centrifuged; protein concentrations with their esp were determined and adjusted to < 2.50 mg/ml. Seven esp samples from F hepatica and 1 from Fascioloides magna were subjected to isoelectric focusing with the 2 species of fluke and their respective host liver homogenates. After separation, gels were stained with silver and scanned on a laser densitometer. Protein banding patterns of the 2 species of flukes were dissimilar. In the pH range of 3.5 to 9.6, the body protein had approximately 30 peaks and esp about 23 peaks in both species. Overall banding patterns of the body protein and esp of both species were distinct from those of respective host tissues. Of the peaks reported as dominant, 3 of the body protein and 2 of esp were shared between the 2 species. Fascioloides magna had more dominant peaks than F hepatica. This technique of soluble protein isoelectric focusing is simple and reproducible, and the 2 fluke species can easily be differentiated by this technique, as well as by morphologic characteristics.

Rapid high voltage isoelectric focusing of proteins in rod gels.

A rapid procedure of isoelectric focusing (IEF) of proteins in polyacrylamide rod gels (i.d., 1.1 mm; length, 7.5 cm) is described. The time required for IEF can be reduced to 0.5 h by using high voltages up to 3000 V in the presence or absence of urea in the gels. When used as the first dimension of a two-dimensional technique for IEF sodium dodecyl sulphate electrophoresis, high voltage IEF gives smaller protein spots on the second dimension gel, associated with an increase in resolution. The method has been tested by a two-dimensional separation of an eye sample of the goodeid fish Xenotoca eiseni.

Effect of Tryptic or Peptic Digestion or Mechanical Isolation on the Extraction of Proteins, Antigens, and Ribonucleic Acids from Sarcocystis muris Bradyzoites

Various methods have been used for the isolation of bradyzoites of Sarcocystis species. Using Sarcocystis muris as a model, the effect of 3 methods, trypsin digestion, pepsin digestion, and mechanical isolation, on the subsequent extraction of S. muris proteins, antigens, and RNA was examined. Although some quantitative differences were found among the proteins, antigens, and RNA extracted after the 3 isolation methods, qualitative differences were not evident. The overall isoelectric focusing protein profile showed approximately 30 bands in the pH range of 3-9 and was dominated by 5 bands with pI values of approximately 5.3, 6.3, 6.8, 7.0, and 7.4. In experimentally infected mice, antibodies were recorded from 35 days postinoculation (PI) until the end of examination (130 days PI). The S. muris RNA appeared to consist of at least 10 subunits in the range of 200-4,200 nucleotides. A Toxoplasma gondii DNA fragment specific for small subunit ribosomal RNA (rRNA) hybridized mainly to the S. muris 1,900-nucleotide subunit and a range of smaller subunits, and a probe specific for large subunit rRNA hybridized mainly to the 4,200-nucleotide subunit and a range of smaller subunits. All 3 methods of bradyzoite isolation gave good yields of intact messenger RNAs that showed similar in vitro translation curves.

Performance of Uncoated and Coated Capillaries in Free Zone Electrophoresis and Isoelectric Focusing of Proteins

Abstract Separation of standard proteins was compared in uncoated fused silica capillaries and in capillaries coated with a linear polymer covalently attached to the capillary wall. Performance of coated versus uncoated capillaries was assessed for free zone separations and capillary isoelectric focusing. In free zone separations performed under acidic conditions, separation profiles and run-to-run reproducibility were comparable, although resolution was poor in both cases. In free zone separations performed under basic conditions, separation profiles and reproducibility were clearly improved using coated capillaries. Use of coated capillaries was essential for obtaining useful results in isoelectric focusing.

Activity, isoenzymes and purity of mushroom tyrosinase in commercial preparations

Several lots of commercial tyrosinase preparations were examined with regard to their enzyme activity, isoenzyme composition and purity. Enzyme activity toward catechol, l-dopa and tyrosine showed significant variations from lot to lot and activation by SDS. Distribution of isoenzyme forms also varied from lot to lot. Comparisons of electrophoretic and isoelectric focusing protein profiles showed considerable differences and distributions of the proteins in each sample. Tyrosinase appeared to be a minor component in each preparation when compared to a partially purified enzyme. Investigators using commercial tyrosinase should exercise caution in interpreting data due to the presence of different isoenzyme forms, their distribution in various lots, and the presence of numerous other proteins.

Characterization of Uromiah Lake Artemia ( Artemia uromiana) by isoelectrofocusing of isozyme patterns

1. 1. Isozyme patterns of 24 and 72 hr post-hydration Artemia uromiana (AU) nauplii and adults AU were compared with other bisexual and parthenogenetic Artemia strains using isoelectric focusing of proteins. 2. 2. Esterase (EST) and phosphoglucose isomerase (PGI) patterns reveal bisexual and parthenogenetic Artemia inhabit Uromiah Lake, Iran. 3. 3. AU genotypes were better differentiated by isozymic patterns from nauplii than from the adults. 4. 4. Unique differences exist in the isozyme patterns of both parthenogenetic AU and bisexual AU compared to other strains having similar genotypes. 5. 5. Individually isolated premature AU females became reproductive (parthenogenetic) or nonreproductive (bisexual). 6. 6. Parthenogenetic females produced 100% females and bisexual females introduced to males produced a mixture of males and females.

Experimental and theoretical dynamics of isoelectric focusing III. Transient multi-peak approach to equilibrium of proteins in simple buffers

Abstract Transient states in the isoelectric focusing of proteins in simple three-component buffer systems were examined by (i) analyzing the collected fractions from continuous flow electrophoresis at a range of operating conditions, (ii) photographing the behavior of colored proteins in capillaries, (iii) monitoring the electric field dynamics along a capillary with a potential gradient array detector and (iv) computeer simulation. The good agreement between simulation and experimental data clearly reveals how the separation dynamics of the buffer system, i.e., the formation of a natural pH gradient, produces the observed peaks and boundaries of protein during the approach to the steady state. The protein focusing dynamics are different, but characteristic, for each buffer system, with both transient double, and multiple, peaks being observed.

Experimental and theoretical dynamics of isoelectric focusing: III. Transient multi-peak approach to equilibrium of proteins in simple buffers

Transient states in the isoelectric focusing of proteins in simple three-component buffer systems were examined by (i) analyzing the collected fractions from continuous flow electrophoresis at a range of operating conditions, (ii) photographing the behavior of colored proteins in capillaries, (iii) monitoring the electric field dynamics along a capillary with a potential gradient array detector and (iv) computeer simulation. The good agreement between simulation and experimental data clearly reveals how the separation dynamics of the buffer system, i.e., the formation of a natural pH gradient, produces the observed peaks and boundaries of protein during the approach to the steady state. The protein focusing dynamics are different, but characteristic, for each buffer system, with both transient double, and multiple, peaks being observed.

Detection of IgG Oligoclonal Bands in Unconcentrated CSF by Isoelectric Focusing in Ultrathin Polyacrylamide Gel, Direct Antiserum Immunofixation and Silver Nitrate Staining

Isoelectric focusing of proteins in ultrathin polyacrylamide gel (0.4 mm), followed by direct immunofixation with monospecific antisera and silver nitrate staining, is a highly specific, sensitive and simple method for the detection of oligoclonal IgG in unconcentrated CSF samples. The ultrathin polyacrylamide gels have several advantages, i.e. significantly smaller amounts of reagents are required, and thinner gel can be more efficiently cooled, resulting in higher resolution and shorter running, washing, staining and destaining times. Direct immunofixation in the gel, a time-saving and simple step, increases the sensitivity and specificity of the method. We reduced the samples to 5-10 microliters. For the present method, the optimal concentration of IgG was 0.025-0.030 g/l. It is possible to detect oligoclonal IgG bands at an IgG concentration corresponding to the applied amount of 80-100 ng. In our testing of this method, oligoclonal bands in CSF specimens were clearly demonstrated in 33 (97%) out of 34 patients with definite multiple sclerosis, in 16 (42%) out of 38 patients with infectious diseases of the central nervous system and in 11 (18%) out of 58 patients with other neurological disorders. The method appears to be a useful alternative for the demonstration of oligoclonal IgG bands in unconcentrated CSF samples, and can be recommended for use in the CSF laboratory routine.

Carrier ampholyte-mediated oxidation of proteins in isoelectric focusing

It has been found that all commercially available carrier ampholytes (the amphoteric buffers used to create and stabilize the pH gradient in isoelectric focusing, IEF) ( e.g., Ampholine, Pharmalyte) are extensively oxidized by persulphate during gel polymerization. These oxidized species are formed for all pH ranges, from acidic to basic, and exhibit chromophoric peaks in the 300–360 nm range. It is believed that these oxidized products are N-oxides (possibly even nitrosamines). Owing to their high redox potentials during IEF, especially in alkaline pH ranges, they are able to capture two H + and two electrons and be reduced, while oxidizing in proteins SH group of Cys to —S—S— (possibly even to cysteic acid). This generates artefactual band heterogeneity, not present in the starting sample, which could never be eliminated as, on rerunning the zone, additional oxidation will take place. In such cases, the presentation of proof of sample homogeneity to agencies such as the FDA, for recombinant DNA products for human consumption, might be impossible. Such a cause of artefactual sample heterogeneity can be virtually eliminated by simply not exposing the carrier ampholytes to persulphate. In the proposed IEF protocol, “empty” gels are polymerized, washed, dried and reswollen in the desired pH interval.

Device for miniscale isoelectric focusing of proteins

A simple device is developed for mini-scale electrofocusing of proteins. The main apparatus consists of only two glass tubes joined by a small tubing. No special cooling system, stopcocks, stands, etc., are needed. Even the need for a peristaltic pump for fractionation is eliminated. The apparatus does not require very high voltages and the amount of Ampholines is drastically reduced. The model can be used for analytical as well as semi-quantitative purposes.

歯肉（溝）しん出液の分析 等電点電気泳動法によるタンパク分画と臨床評価の関連について

The purpose of this study was to determine the protein patterns in gingival crevicular fluid relation to the isoelectric focusing protein patterns of GCF and to clinical evaluations. GCF was collected with filter paper from 105 subjects. The probing depth, the gingival index (Löe & Silness) and the plaque index (Silness & Löe) as clinical evaluations The results follow: 1. The main isoelectric focusing protein patterns of GCF were between pH 5.5 and 7.5. In comparison, the GCF and the serum from the same patients showed patterns to similar serum albumin. 2. Between of GCF pH 5.5 and 7.5 the protein patterns that ranged over 60% was pI 5.65, 6.45, 6.55, 6.75 and 7.00. The frequencies of the ranges of protein patterns and clinical evaluation were compared by the X2 test. pI 5.65, 6.45, 6.55 and 6.75 and PD were significant different, as were pI 6.45, 6.55 and 6.75 and GI. But each pI and PIl. were not significantly different.

Prenatal diagnosis of alpha-1-antitrypsin deficiency by fetal blood sampling.

Fetal blood sampling for the diagnosis of alpha-1-antitrypsin deficiency using protein isoelectric focusing was carried out in the period 1980-1985. The results of 25 cases from 18 mothers are reported. All had a previous history of a PiZ child affected by liver disease. The method was found to be technically satisfactory and the fetal results were subsequently confirmed in all 18 cases where follow-up was possible. The fetus was found to be PiZ in nine cases and all these pregnancies were terminated. Of the remaining pregnancies three cases aborted or were delivered prematurely and 13 proceeded to term without complications.

Isoelectric focusing of proteins in rewetted ultrathin polyacrylamide gel layers

Ultrathin layers of polyacrylamide gel bound to glass can be washed, air-dried, and stored for at least 1 year before rewetting in ampholyte solutions for isoelectric focusing. Short-term drying affects neither fluorescent banding of the ampholytes (not evident in conventional gels) nor resolution of complex protein mixtures while prolonged storage seems to have no deleterious effects. Layers are fully functional after soaking for 10 min in solutions that may contain 8 m urea and 10% sorbitol. Rewetting allows the rapid survey of different ampholytes, gradient stabilizers, separator compounds, or protein reagents and is adaptable to concentration modification of the pH gradient (alone or with a gel overlay), to focusing in a transverse urea gradient, and to electrophoresis across a preformed pH gradient. The procedure avoids protein modification by residual polymerizing reagents while adding to the convenience and economy of using ultrathin layers in relatively small formats.

Pollen gene expression analysed by micro-isoelectric focusing of proteins from isolated pollen grains in Cucurbita pepo L.

Isoelectric focusing of proteins from single pollen grains of Cucurbita pepo L. has been developed for large scale study of pollen grain populations' heterogeneity. Forty to forty-five protein bands from one pollen grain are revealed after silver staining. Applications of this technique to pollen grain populations from different genotypes are described in this paper. Possible applications and limits of this technique are discussed with respect to plant breeding especially for the measure of gene frequencies in pollen grain populations.