Two-dimensional protein analysis by isoelectric focusing in a multi-cell plate system

A practical and low cost system for isoelectric protein focusing (IEF) was developed. The system uses a multi-cell glass plate compatible with a common vertical one-dimensional electrophoresis chamber, dispensing specific IEF apparatus. After focusing, the IEF gels are easily recovered. The resulting two-dimensional (2-D) gels have provided efficient protein separation for different concentrations and extracts.     

Preparative isoelectric focusing of human serum very low-density and low-density lipoproteins.

Ten percent glycerol prevented the usual precipitation of human serum very low-density lipoproteins (VLDL) and low-density lipoproteins (LDL) at their isoelectric points during their preparative isoelectric focusing (IEF), IEF separated VLDL and LDL into two major fractions. The observed optical density peaks are not artifacts caused by binding of Ampholines to VLDL or LDL since no radioactivity accumulated in the fractions containing VLDL or LDL during IEF in the presence of [¹⁴C]Ampholine, and gel filtration completely separated the lipoproteins from [¹⁴C]Ampholine. These results suggest that IEF may separate subspecies of VLDL and LDL under suitable experimental conditions.     

Isolation of three creatine kinases MM from porcine skeletal muscle

The purified creatine kinase MM of porcine skeletal muscle [Takasawa, T. & Shiokawa, H. (1981) J. Biochem. 90, 195-204] was separated into three distinct fractions by isoelectric focusing (IEF) in a sucrose gradient column, and the three active fractions were isolated by repeated IEF. There were one major fraction with isoelectric point (pI) 6.57 and two minor fractions with pI 6.74 and pI 6.34, respectively. No differences were observed in the IEF pattern of the enzyme in the presence and absence of dithiothreitol throughout the column. There was no interconversion from one form to another during IEF. The distribution of the three forms on IEF was not affected by adding protease inhibitor to the extraction medium. Of the three fractions, the major fraction had the highest specific activity. The three fractions differed from one another in their amino acid compositions. Not only porcine muscle but also rabbit muscle creatine kinase displayed this type of heterogeneity. Such microheterogeneities may occur widely in muscle creatine kinases.     

Quantitative variation in cystic fibrosis-associated proteins in cystic fibrosis patients,carriers, and controls

Summary Serum samples from patients with cystic fibrosis (CF), obligate heterozygotes, and normal controls have been examined by isoelectric focusing (IEF). Our results suggest that cystic fibrosis protein (CFP) is a normal serum protein exhibiting quantitative variation primarily dependent on possession of the CF allele. It is concluded that detection of CFP by IEF is an inappropriate screening test for the CF gene due to lack of specificity.     

Characterization and preparation of chymosin from calf rennet samples by means of isoelectric focusing

Chymosin has been identified in rennet samples from stomaches of living calfs by means of it's isoelectric points (IP), molecular weight (Mol. Wt.) and enzymatic activity. The values obtained by isoelectric focusing (IEF) and a two-dimensional electrophoretic technique, amount 4,5…4,7 (IP) and 36 300 (Mol. Wt.) respectively. Furthermore the fractionation of calf rennet by IEF has been performed in a preparative scale. From one gramme of calf rennet we obtained 53 mg of purified chymosin, which shall be used for preparing antisera against chymosin.     

On the possibility of applying noncovalent dyes for protein labeling in isoelectric focusing

Noncovalent fluorescent dyes are widely used for protein quantification and postcolumn detection in electrophoretic separations and recently some attempts to separate the precolumn labeled proteins using isoelectric focusing (IEF) have been made. In the present study, the possibility of applying the technique of protein labeling with noncovalent dyes for IEF is investigated. We found that fluorescent signal emitted by NanoOrange dye increases essentially in presence of carrier ampholyte (CA) components, which makes problematic a reliable protein detection in CA environment. Since in an isoelectric focusing mode the CA species are present in much greater concentration than the concentrations of fractionated proteins, the method of protein labeling with NanoOrange is not suitable for precolumn labeling and cannot be used for CA-IEF, at least without more detailed study of the dye-protein interaction mechanism.     

A procedure for the immunoblotting of proteins separated on isoelectric focusing gels

A method has been devised for performing Western blot assays on proteins resolved by isoelectric focusing. Electrophoretic transfer of proteins directly from isoelectric focusing (IEF) tube gels to nitrocellulose sheets allowed their immunoassay without conventional second dimension SDS gel electrophoresis. The same method can also be used for IEF slab gels. For the immunostaining of nonmuscle actin isoforms in extracts of cultured cells, the resolution of this technique was much improved over that of Western blots of two-dimensional gels.     

Multiple zeins from maize endosperms characterized by reversed-phase high performance liquid chromatography

The major storage proteins of maize (Zea mays L.) endosperm are located in protein bodies, and may be separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) into two major classes and four minor classes of polypeptides. The two major classes (commonly known as zeins) have been separated previously into a large number of components by isoelectric focusing (IEF). Reversed-phase high performance liquid chromatography (HPLC) further separated the major classes into additional components, and gave distinctive peaks for each minor zein class. Some IEF bands produced two or more HPLC fractions, while some HPLC fractions produced two or more IEF bands. Apparently identical IEF bands from different inbreds may appear in different fractions after HPLC. Thus the total number of zeins revealed by separations based on apparent size (SDS-PAGE), net charge (IEF), and hydrophobicity (HPLC) is very large. Different laboratories have developed diverse nomenclatures which cause much confusion. A key is presented to provide a flexible and expandable nomenclature for this complex group of proteins.     

Detection of 10 variants of biliverdin reductase in rat liver by two-dimensional gel electrophoresis

We have identified and characterized multiple forms of biliverdin reductase (BVR) in control rat liver cytosol. Two-dimensional electrophoresis of the purified BVR resolved a minimum of 10 discrete protein zones. All 10 proteins were BVR as judged by immunological cross-reactivity toward rabbit anti-rat BVR. Based on the isoelectric focusing pattern of separation, the BVR variants could be organized into five net-charge groups designated as BVR-IEF1 to BVR-IEF5 and three molecular mass groups designated as BVR-MW1-BVR-MW3, respectively. The pI values of the net-charge groups were: BVR-IEF1, 6.23; IEF2, 5.91; IEF3, 5.76; IEF4, 5.61; IEF5, 5.48. The Mr values of the molecular mass groups were: BVR-MW1, 30,400; MW2, 30,700; MW3, 31,400. Single dimension slab gel isoelectric focusing offered greater resolution of the net charge variants, and BVR-IEF3 was further resolved into two variants, IEF3a and IEF3b, with pIs of 5.77 and 5.75, respectively. The six net-charge variants also resolved on a preparative chromatofocusing column and were designated as BVR-CF1-BVR-CF6. The pH values of the peak fractions were: BVR-CF1, 6.91; CF2, 6.33; CF3, 6.03; CF4, 5.82; CF5, 5.45; CF6, 5.27. Correspondence between the isoelectric focusing net-charge variants and the chromatofocusing net-charge variants was established. The Mr and net-charge variants did not represent partially degraded forms of biliverdin reductase produced during purification since the pattern of resolution of variants on slab gel isoelectric focusing or two-dimensional electrophoresis did not change by purifying the proteins in the presence of protease inhibitors and 5 mM EDTA. BVR-CF2 and BVR-CF4 were purified and examined for pH-dependent cofactor requirements for activity. Both net-charge variants and two pH optima that were cofactor-dependent; maximum activity with NADPH, however, was at pH 8.5 and with NADH at pH 6.7. With both variants, however, a higher catalytic rate was observed with NADH than with NADPH at their respective pH optima. Furthermore, BVR-CF2 exhibited a higher catalytic rate than did BVR-CF4 with either cofactor throughout the pH range of 5-9.     

Comparison of isoelectric focusing in Sephadex vs immobiline flatbeds for the recovery of follicle regulatory protein from porcine follicular fluid

We describe and compare the use of isoelectric focusing (IEF) in a granulated Sephadex matrix and in polyacrylamide immobilized pH gradients to separate an aromatase inhibitor (follicle regulatory protein: FRP) in preparative amounts from porcine follicular fluid (PFF). The starting material for IEF was derived from pFF after passage through agarose immobilized textile dye Orange A (0.5 KC1 eluent). Before IEF, some Orange A bound (OAB) material was further purified on a FPLC employing a Mono-Q anion exchange column. Previous use of chromatofocusing indicated that aromatase inhibitory activity is largely concentrated in OAB fractions with a pI in the ranges of pH approximately 4.5 and approximately 6.5. The current study revises these findings to provide a more precise measure of the isoelectric points in question to pH 4.73 +/- 0.05 and pH 6.41 +/- 0.06. The use of Sephadex was limited by gradient instability and the selection of pH ranges available. IEF using immobilized pH gradients had several advantages over Sephadex: 1) broader selection of gradients from 0.1 to 7.0 pH units; greater resolving power, and enhanced stability. The principal disadvantage of the immobiline system was the recovery of focused material from the gel matrix. The use of isoelectric focusing with immobilized pH gradients on a preparative scale to purify FRP from OAB resulted in a greater than 50% recovery with a substantial increase in specific activity (from ID50 approximately 300 micrograms/ml to 20 ng/ml).     

Charge heterogeneity of cholera toxin and its subunits

Abstract Analytical isoelectric focusing (IEF) in thin layers of polyacrylamide gels resolved cholera toxin into 3 isomeric forms differing in charge (isoelectric points 6.80, 6.65 and 6.55). All these forms had identical molecular weights, and were also antigenically similar, as demonstrated by their reactivity to antisera to cholera toxin. Both the B and A subunits possessed charge heterogeneity. The B subunit was detected in a free form when a solution of cholera toxin was aged for a few days. Antisera to cholera toxin, irrespective of mode of immunisation, contained antibodies to both the intact cholera toxin and the free B subunit as demonstrated by the immunoblotting technique based on IEF.     

Isoelectric focusing of Gc (vitamin D binding globulin) in parentage testing

Summary Gc subtyping with isoelectric focusing (IEF) in paternity cases allowed us to increase the exclusion probability (P) of Gc in Whites to 31% from a (P) of 16% using agarose electrophoresis. Both IEF and agarose are necessary to identify certain rare variants, therefore it is recommended that both techniques be performed in cases of disputed parentage and population studies.     

Transferrin polymorphisms in Japanese populations: north-south cline in the distribution of the TF*C2 allele

Allele frequencies for human transferrin (TF) subtypes were determined using serum samples from Japanese subjects living in Fukui prefecture, Japan, and compared with other Japanese populations using isoelectric focusing (IEF) and immunoblotting. The application of IEF revealed considerable heterogeneity in the TF system, enhancing its potential value for anthropologic and genetic studies. So far, TF subtypes of about 27,000 Japanese individuals from 35 population groups have been analyzed to evaluate the degree of genetic variation at the TF locus. Possible geographic and biologic factors are discussed.     

Quantitative assay of deoxyribonuclease activity after isoelectric focusing in polyacrylamide gels: pH control and effects of enzyme diffusion

A method for the quantitative assay of nuclease activity in crude cell lysates after isoelectric focusing (IEF) in polyacrylamide slab gels is described. After IEF, an agarose overlay gel containing DNA is placed on the IEF gel and the nuclease activity quantified by the loss of ethidium bromide fluorescence of the DNA. With this method a linear response was obtained for 1 to 10 ng of DNase I. Various methods of pH equilibration after IEF were also evaluated. The use of a high buffer concentration in the overlay gel is recommended to control the pH during the enzyme reaction. An analytical solution for the diffusion of enzymes from the IEF gel to the overlay gel is also presented and an equation that may be used to choose optimum times for transfer of the enzyme from the IEF gel to the overlay gel is given.     

The novel peptide composition of the seeds of Trichosanthes dioica Roxb

The seeds of Trichosanthes dioica contain a large amount of peptides in the range of 2-8 kD. These peptides can be resolved in a discontinuous sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) system using Tricine as the trailing ion. The seed proteins contain a number of charge species as determined by isoelectric focusing (IEF) in polyacrylamide gels. The peptides were focused in the basic region as determined by two-dimensional electrophoresis involving IEF and SDS-PAGE. The seed peptides have the unique property of being resistant to the action of silver nitrate, a sensitive reagent commonly used to stain proteins. The seed contains haemagglutinating activity which is inhibited by galactose.     

Heterogeneity of cellulase complexes from Trichoderma reesei: A preparative isoelectric focusing study of some extracellular hydrolases

Abstract Homogeneity of extracellular proteins of Trichoderma reesei found after isoelectric focusing (IEF) has been shown to reflect purity of a (multi)enzyme complex rather than purity of one enzyme isolated. The intention of the present study was to demonstrate to what extent distribution of (multi)enzyme complexes reflects a general concept of extracellular hydrolases in the culture fluid of T. reesei . The complete culture fluid studied by preparative IEF on the distribution of cellulases, β-glucosidase, xylanase and xylosidase confirms this view. (Macro)heterogeneity found after IEF is discussed on the basis of different charges of complexes.     

Human transferrin polymorphism

The application of isoelectric focussing (IEF) has revealed a large amount of heterogeneity in the human transferrin (TF) system and has enhanced its potential value in anthropological and genetic studies. The average heterozygosity has been elevated from 0.05, observed by conventional methods of electrophoresis, to 0.29 detected by IEF. So far approximately 30,000 individuals from 122 population groups have been analyzed for TF subtypes to evaluate the magnitude of genetic variation at the TF locus. Possible environmental and biological factors, which may be operating to maintain the TF polymorphism, are discussed.     

The effect of ampholytes on ferritin isoelectric focussing patterns

Ferritin was subjected to isoelectric focussing (IEF) on agarose gels containing different commercial carrier ampholytes. In two gels protein staining revealed banded patterns which differed from one another, while a third gel yielded zones rather than discrete bands, indicating that the bands may be artefacts.The differences between banded patterns were studied by isolating bands from an IEF gel and refocussing these on gels containing either the original ampholyte or a different ampholyte preparation. Striking differences were noted.Chromatofocussing of ferritin resulted in the elution of broad peaks between the same pH limits as indicated by IEF patterns.     

Biochemical systematics of the genus Lipara using isoenzymes

ln the course of a systematic and population genetic study of the genus Lipara, a new approach was tested, involving the examination of isoenzymes using PAGE and isoelectric focusing (IEF). The enzymes from whole individual specimens of both larvae or adult flies were analysed. Twenty-five enzyme systems have been investigated with PAGE, 18 of which gave interpretable results. General proteins were analysed with both PAGE and IEF. By means of these techniques, the intra- and interspecific variation of structural proteins and different enzyme systems were examined. Possible changes in general proteins and enzymes during the development from larvae to adults were also investigated.     

Detection of protein-protein interactions and a group of immunoglobulin G-associated minor proteins in human plasma by nondenaturing and denaturing two-dimensional gel electrophoresis

The dissociation of noncovalently associated protein-protein complexes in human plasma was examined by comparing two-dimensional gel electrophoresis (2-DE) patterns obtained in two different electrophoretic conditions. A type I 2-DE pattern was obtained running nondenaturing isoelectric focusing (IEF) followed by nondenaturing gel electrophoresis and a type II 2-DE pattern was nondenaturing IEF followed by sodium dodecyl sulfate gel electrophoresis. Micro-sized gels (internal diameter(id) 1.3 x 35 mm polyacrylamide IEF gels and 38 x 38 x 1 mm polyacryamide slab gels) were used to follow the dissociation processes of major plasma proteins. Larger gel sizes (id 3.4 x 160 mm agarose IEF gels and 160 x 120 x 2.8 mm polyacrylamide slab gels) were used to detect minor plasma proteins dissociated from major proteins. About 110 spots, which have not been detected on type I (nondenaturing) 2-D gels, newly appeared on type II large-sized 2-D gels at molecular masses smaller than 67 kDa. Some of these spots had been analyzed and identified, but about 70 minor spots (isoelectric point 5.5-7.5 and relative molecular mass 8-45 kDa) were detected for the first time by applying large volumes of human plasma samples to the large type II 2-D gels. These minor spots could be concentrated on type II 2-D gels by enriching the immunoglobulin G (IgG) fraction under nondenaturing conditions, and they disappeared when IgG was removed from the fraction. These results strongly suggest that many of the minor spots newly detected were bound to IgG in physiological conditions.