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.     

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.     

Purification of cAMP-specific phosphodiesterase from rat heart by affinity chromatography on immobilized rolipram

Affinity chromatography on a cAMP-specific phosphodiesterase inhibitor related to Rolipram, immobilized to AH Sepharose allowed to perform an efficient purification of the cAMP-specific phosphodiesterase isoenzyme from rat heart cytosol (102-fold purification with a 35% yield in a single step). This affinity chromatography involved a biospecific interaction since a 2 mM cAMP elution step at 30 degrees C was necessary for releasing the cAMP specific form tightly bound on the affinity gel. The cAMP eluate fraction exhibited a high specificity towards cAMP (cAMP/cGMP hydrolysis ratio 5-10), a marked sensitivity to Rolipram inhibition and could be resolved in two cAMP-specific, highly Rolipram-sensitive peaks of pI 6.7 and 4.8 by IEF on polyacrylamide gel plates. Protein stain of the IEF gel revealed a single band at pI 6.7.     

Molecular characterization by high resolution isoelectric focusing of the products encoded by the class II region loci of the MHC in humans. II. DP alpha and DP beta gene variants

To characterize the molecular polymorphism of the DP alpha and DP beta gene products, the HLA-DP molecules expressed by more than 200 cell lines were individually immunoprecipitated by using the mAb B7/21 and their neuraminidase-treated DP alpha and DP beta chains analyzed in IEF gels. These cell lines, most of them from members of 32 families, allowed the definition, by segregation analysis, of the IEF patterns of the DP polypeptide chains encoded by 129 distinct haplotypes. Both DP alpha and DP beta chains display polymorphic IEF-banding patterns. Two DP alpha (A and B) and seven DP beta (A, B, C, D, E, F, and G) IEF variants were characterized. The DP alpha B variant was found in linkage disequilibrium with both DP beta B and DP beta D. Linkage disequilibrium was also encountered with alleles at the DR and DQ loci. Finally, the correlations between the IEF DP alpha and DP beta variants and the primed lymphocyte test-defined HLA-DP specificities were determined by using a panel of 24 primed lymphocyte test-typed cell lines.     

Apolipoproteins as the basis for heterogeneity in high-density lipoprotein2 and high-density lipoprotein3. Studies by isoelectric focusing on agarose films

A method is described for the isoelectric focusing (IEF) of lipoproteins on thin films of agarose. Within a pH gradient of 4.60-5.30 both high-density lipoproteins 2 and 3 (HDL2 and HDL3) are resolved into more than 10 fractions which could be stained either for protein or for lipids. The isoelectric focusing patterns for HDL2 and HDL3 are similar although HDL2 appears richer in the more alkaline bands. Narrow film strips from the IEF separation of HDL2 and HDL3 were interfaced with various agarose plates containing antisera against apolipoproteins apoAI, apoAII and apoCIII either alone or in combination, to provide two-dimensional IEF immunoelectrophoresis patterns. This technique demonstrated that apoAI and apoAII were present throughout the IEF gel for both subclasses of HDL. It also provided evidence for the existence of lipoproteins containing both apoAI and apoAII and other lipoproteins present in the alkaline region of the gel which contained apoAI but no apoAII. ApoCIII was found mostly in acidic lipoproteins and was not distributed identically in HDL2 and HDL3. The lipoproteins separated by IEF on agarose were also analysed by two-dimensional IEF-SDS electrophoresis and the individual apolipoproteins were identified by reaction with antibodies to apolipoproteins AI, AII, CI, CII, CIII, D, and E. This technique confirmed that in IEF of HDL, apoAI extended throughout the spectrum of lipoproteins whereas apoE was only present in alkaline lipoproteins and apoD was only present in acidic lipoproteins. IEF on agarose of either HDL2 or HDL3 allowed us to collect eight different fractions, which have the same pI in either lipoprotein class. The apolipoprotein composition of each isolated band was analysed by electroimmuno-assays for apolipoproteins AI, AII, CI, CII, CIII, D, and E and the results expressed as the ratio of the measured apolipoprotein to measured apoAI. In both HDL2 and HDL3, acidic lipoprotein fractions were enriched in apoAII, apoCIII and apoD. ApoCII and apoCII were not similarly distributed in HDL2 and HDL3 subfractions whereas the apoCI distribution was similar in both classes. Noteworthy in all experiments was the difference in the distributions of apoCI, apoCII, and apoCIII in HDL2 and HDL3, which indicated that the existence of a lipoprotein containing simultaneously CI, CII and CIII can only account for a small fraction of these apolipoproteins. Therefore these experiments substantiate the theory of the protein basis of HDL heterogeneity and suggest that the majority of apolipoproteins are present in complexes which upon IEF result in lipoprotein fractions of identical pI for both HDL2 or HDL3.(ABSTRACT TRUNCATED AT 400 WORDS)     

Current trends in capillary isoelectric focusing of proteins

Isoelectric focusing (IEF) in thin capillaries is reviewed here. After an introduction on the genesis and chemistry of the carrier ampholyte buffers, different approaches to IEF are discussed and evaluated. The classical approach consists on IEF under conditions of suppressed electroosmotic (EOF) flow, usually obtained by covalently bonding hydrophilic polymers to the inner capillary wall. The other approach consists of IEF in dynamically (and partially) coated capillaries, so as to allow a reduced EOF flow to coexist with the IEF process, so that focusing and transport of the train of stacked bands occurs simultaneously. The various experimental parameters: focusing, elution and detection steps, pI measurements, as well as typical drawbacks, such as isoelectric precipitation are evaluated. The review ends with some examples of analytical separations, at the moment mostlyl limited to focusing of native hemoglobins (normal and point mutants). These separations are compared with those obtained by slab-gel IEF and in immobilized pH gradients.     

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 mitochondrial proteome of the model legume Medicago truncatula

Legumes carry out special biochemical functions, e.g. the fixation of molecular nitrogen based on a symbiosis with proteobacteria. At the cellular level, this symbiosis has to be implemented into the energy metabolism of the host cell. To provide a basis for future analyses, we have characterized the protein complement of mitochondria of the model legume Medicago truncatula using two-dimensional isoelectric focussing (IEF) and blue-native (BN)-SDS-PAGE. While the IEF reference map resulted mainly in resolution of those proteins associated with the mitochondrial matrix, the BN proteomic map allowed separation of protein subunits from the respiratory chain protein complexes, which are located in the organelle's inner membrane. The M. truncatula mitochondrial BN reference map revealed some striking similarities to the one from Arabidopsis thaliana but at the same time exhibited also some special features: complex II is of increased abundance and additionally represented by a low molecular mass form not reported for Arabidopsis. Furthermore three highly abundant forms of prohibitin complexes are present in the mitochondrial proteome of M. truncatula. Special features with respect to mitochondrial protein complexes might reflect adaptations of legumes to elevated cellular energy requirements enabling them to develop symbiotic interactions with rhizobial bacteria.     

A qualitative investigation of major urinary proteins in relation to the onset of aggressive behavior and dispersive motivation in male wild house mice (<Emphasis Type="Italic">Mus musculus domesticus</Emphasis>)

The physiological basis for population differentiation of dispersal timing during individual development in male wild house mice is still unknown. As major urinary proteins (MUPs) are known to convey information about competitive ability in male mice, we examined individual MUP profiles defined by isoelectric-focusing (IEF) patterns in relation to developmental timing of dispersive motivation. As an experimental paradigm marking the development of the dispersal propensity, we used agonistic onset between litter mate brothers when kept in pairs under laboratory conditions. Agonistic onset is known to reflect the initiation of dispersive motivation. Hence, we compared individual MUP IEF patterns between fraternal pairs that did or did not develop agonistic relationships before the age of 2 months. Urine was collected on the day of weaning and at the beginning of adulthood. We investigated whether there was a significant co-occurrence of particular MUP IEF patterns with the agonistic onset in male mice. We assumed that, based on this co-occurrence, particular MUP IEF patterns and/or a particular dynamic of MUP IEF expression from weaning to adulthood may be considered a physiological predictor of a specific behavioral strategy in male mice (i.e. submissive-philopatric or agonistic-dispersive strategy). We found that agonistic males expressed more MUP IEF bands than amicable ones at weaning, but these differences disappeared later on. The presence of two particular IEF bands at weaning was significantly associated with early agonistic onset. Our study suggests that MUPs could have a predictive value for the onset of aggressive behavior and dispersal tendency in male wild house mice.     

Isoelectric focusing management: an investigation for salt interference and an algorithm for optimization

Two-dimensional electrophoresis (2-DE) has evolved into a robust separation technique in proteomic research. However, one of the major challenges in 2-DE experiments, the reproducibility of the first dimensional electrophoresis (IEF), has remained unsolved. It is well-known that the quality of IEF experiments is significantly affected by the salt interference. Nevertheless, the interference mechanisms of salts in IEF have never been systematically investigated. In this study, we comprehensively investigated the interference effects in IEF due to various kinds of simple and buffer salts in protein samples. Two interference schemes were proposed accordingly to elucidate the interference mechanisms of salts in IEF. Furthermore, to increase the reproducibility of IEF, we proposed that conductivity measurement is a feasible method to assess the salt content of 2-DE samples and developed an algorithm to predict the optimal total volt-hours (Vh) required for protein focusing in IEF. The developed algorithm had been evaluated under various IEF conditions for a variety of 2-DE samples and proven to be a reliable guide. In sum, information disclosed in this study should be of use for increasing the reproducibility and thus the applicability of 2-DE in current proteomics.     

Diagnosis of congenital disorders of glycosylation type-I using protein chip technology

A method for the diagnosis of the congenital disorders of glycosylation type I (CDG-I) by SELDI-TOF-MS of serum transferrin immunocaptured on protein chip arrays is described. The underglycosylation of glycoproteins in CDG-I produces glycoforms of transferrin with masses lower than that of the normal fully glycosylated transferrin. Immobilisation of antitransferrin antibodies on reactive-surface protein chip arrays (RS100) selectively enriched transferrin by at least 100-fold and allowed the detection of patterns of transferrin glycoforms by SELDI-TOF-MS using approximately 0.3 microL of serum/plasma. Abnormal patterns of immunocaptured transferrin were detected in patients with known defects in glycosylation (CDG-Ia, CDG-Ib, CDG-Ic, CDG-If and CDG-Ih) and in patients in whom the basic defect has not yet been identified (CDG-Ix). The correction of the N-glycosylation defect in a patient with CDG-Ib after mannose therapy was readily detected. A patient who had an abnormal transferrin profile by IEF but a normal profile by SELDI-TOF-MS analysis was shown to have an amino acid polymorphism by sequencing transferrin by quadrupole-TOF MS. Complete agreement was obtained between analysis of immunocaptured transferrin by SELDI-TOF-MS and the IEF profile of transferrin, the clinical severity of the disease and the levels of aspartylglucosaminidase activity (a surrogate marker for the diagnosis of CDG-I). SELDI-TOF-MS of transferrin immunocaptured on protein chip arrays is a highly sensitive diagnostic method for CDG-I, which could be fully automated using microtitre plates and robotics.     

蛋白质组学研究中的对角线电泳

经典的蛋白质组学研究方法包括IEF/SDS-PAGE双向电泳和质谱技术的联用,但由于IEF的一些不足,限制了其应用范围。对角线电泳是蛋白质组学研究中的一项特殊分离技术,由于其原理与IEF/SDS-PAGE不同,正逐渐成为蛋白质组学中电泳分离技术的重要补充,特别是在膜蛋白和蛋白质相互关系的研究中将起到重要作用。本文综述了对角线双向电泳技术的特点、发展和在蛋白质组学研究中的最新进展,比较了双向电泳和对角线电泳的优缺点,展望了对角线电泳在蛋白质组学研究中的应用前景。     

Identification of Two Human Rho GDP Dissociation Inhibitor Proteins Whose Overexpression Leads to Disruption of the Actin Cytoskeleton

Proteins (IEF's 1120, 8118, 8120) sharing similarity to the bovine Rho GDP dissociation inhibitor (GDI) have been identified in the human two-dimentional-gel database of keratinocyte proteins. Molecular cloning of the corresponding cDNAs showed that IEF 8118 is the human homolog of bovine GDI while IEF 8120 is a distinct although related protein. All available information indicates the IEF 1120 is a derivative of IEF 8120. The cDNAs coding for IEF's 8118 and 8120 were recombined into vaccinia virus and expressed in differentiated human keratinocytes and their effect on the actin cytoskeleton was assessed by immunofluorescence using TRITC-phalloidin. The results showed that overexpression of both GDI proteins leads to rounding up of the cells and loss of stress fibers and focal contact sites. In addition, the cell to cell adhesion belts gradually disappeared, an effect that was particularly pronounced in infected cells overexpressing IEF 8120. Taken together, the results imply that Rho GDI's play a role in modulating the activity of the Rho proteins as their overexpression mimics phenotypic changes associated with the inactivation of these proteins.     

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.     

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.     

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.     

Separation of bacterial cells by isoelectric focusing, a new method for analysis of complex microbial communities.

A simple isoelectric focusing (IEF) method for whole bacterial cells was developed. In a pH gradient of 2 to 10 and an electric field of 11.5 V cm-1, mixtures of cells from the three different bacterial strains Chlorobium limicola 6230, Pseudomonas stutzeri DSM 50227, and Micrococcus luteus DSM 20030 could be separated. A density gradient of Ficoll prevented convective currents in the system. The method was tested with a concentrated mixture of bacteria from a shallow eutrophic lake and yielded up to 10 different bands. Species composition in each IEF band was analyzed by PCR plus denaturing gradient gel electrophoresis (DGGE). Each IEF band exhibited a different species composition. After the separation of cells by IEF three times more 16S ribosomal DNA signals could be detected by DGGE than in the unfractionated natural bacterial community. It is concluded that the resolution of these molecular biological methods is significantly enhanced if cells are first separated by IEF. At the same time, the IEF fractions are enriched for certain species, which can be used in subsequent cultivation experiments.     

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.     

DETECTION AND DEMONSTRATION OF INHIBITORY ACTIVITIES OF BACTERIOCINS BY ISOELECTRIC FOCUSING

A technique utilizing isoelectric focusing (IEF) was developed and compared with a modified Bhunia's sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) method for directly detecting antimicrobial activities of inhibitory peptides or proteins (bacteriocins). In IEF, the gel containing separated peptide or protein bands was directly overlaid with indicator bacteria without any fixation or rinsing as done in the SDS-PAGE method. The IEF gave clear zones of inhibition surrounding inhibitory bands. Bhunia's method was modified to reduce the time for fixation and rinsing, and a 15-min fixation followed by 2-h rinsing with dd H₂O was determined to be necessary for a detection similar to the IEF. In addition, results of this study showed that denaturation of bacteriocins and, subsequently, failure to detect the presence of a bacteriocin could occur in the SDS-PAGE analysis while little denaturation was observed in the IEF assay. Therefore, the IEF assay developed in this study was more rapid and less destructive as compared with the SDS-PAGE method.     

Evaluation of strong cation exchange versus isoelectric focusing of peptides for multidimensional liquid chromatography-tandem mass spectrometry

Shotgun proteome analysis platforms based on multidimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS) provide a powerful means to discover biomarker candidates in tissue specimens. Analysis platforms must balance sensitivity for peptide detection, reproducibility of detected peptide inventories and analytical throughput for protein amounts commonly present in tissue biospecimens (< 100 microg), such that platform stability is sufficient to detect modest changes in complex proteomes. We compared shotgun proteomics platforms by analyzing tryptic digests of whole cell and tissue proteomes using strong cation exchange (SCX) and isoelectric focusing (IEF) separations of peptides prior to LC-MS/MS analysis on a LTQ-Orbitrap hybrid instrument. IEF separations provided superior reproducibility and resolution for peptide fractionation from samples corresponding to both large (100 microg) and small (10 microg) protein inputs. SCX generated more peptide and protein identifications than did IEF with small (10 microg) samples, whereas the two platforms yielded similar numbers of identifications with large (100 microg) samples. In nine replicate analyses of tryptic peptides from 50 microg colon adenocarcinoma protein, overlap in protein detection by the two platforms was 77% of all proteins detected by both methods combined. IEF more quickly approached maximal detection, with 90% of IEF-detectable medium abundance proteins (those detected with a total of 3-4 peptides) detected within three replicate analyses. In contrast, the SCX platform required six replicates to detect 90% of SCX-detectable medium abundance proteins. High reproducibility and efficient resolution of IEF peptide separations make the IEF platform superior to the SCX platform for biomarker discovery via shotgun proteomic analyses of tissue specimens.