Colored pI standards and gel isoelectric focusing in strongly acidic pH

Colored, low molecular weight pI markers have been developed for isoelectric focusing (IEF) in acidic pH range. Their isoelectric points (pIs) were determined by direct measurement of the pH of the focused bands after completion of IEF on polyacrylamide gels. The practicable suitability of the proposed pI markers as pI standards for IEF was tested by applying gel IEF. The acidic pH gradient was created either by commercial synthetic carrier ampholytes or by mixture of simple buffers consisting of acids (non-ampholytes) and ampholytic buffers. By applying simple acids, it was possible to extend the acidic pH range beyond those achievable with commercial synthetic carrier ampholytes. By using an experimental arrangement without electrode electrolyte reservoirs with electrodes creating the fixed end of the gel, the strongly acidic pH gradient was stable even for prolonged focusing time.     

The human keratinocyte two-dimensional gel protein database (update 1992): towards an integrated approach to the study of cell proliferation, differentiation and skin diseases.

The master two-dimensional gel database of human keratinocytes currently lists 2980 cellular proteins (2098 isoelectric focusing, IEF; and 882 nonequilibrium pH gradient electrophoresis, NEPHGE) many of which correspond to posttranslational modifications. About 20% of all recorded proteins have been identified (protein name, organelle components, etc.) and they are listed in alphabetical order together with their M(r), pI, cellular localization and credit to the investigator(s) that aided in the identification. Also, we have listed 145 microsequenced proteins that are recorded in this database. As an aid in localizing the polypeptides we have included blow-ups of the master images (IEF, NEPHGE) displaying all the protein numbers. In the long run, the master keratinocyte database is expected to link protein and DNA sequencing and mapping information (Human Genome Program) and to provide an integrated picture of the expression levels and properties of the thousands of proteins that orchestrate various keratinocyte functions both in health and disease.     

Conductivity properties of carrier ampholyte pH gradients in isoelectric focusing

The conductivity properties of natural pH gradient created by carrier ampholytes were studied during the process of isoelectric focusing (IEF). IEF was performed in capillaries (10-30 mm long) or in microchips with the same channel length. A 10-30x reduction of the conductivity of the separation medium was observed during the establishment of pH gradient. Results obtained using different IEF voltages indicate that there is a nonlinear relationship between the conductivity of an established pH gradient and the applied electric field. Our theoretical analysis using a simplified model generated values that reasonably agree with the experimental data. In addition, we found that above a certain electric field ( approximately 300 V/cm), resolution does not increase with the applied voltage as predicated; we observed band-broadening and gel breakdown. The approach presented in this work can be used for optimization of the IEF separation and judicious selection of IEF conditions.     

Isoelectric focusing in immobilized pH gradients with carrier ampholytes added for high-resolution phenotyping of bovine beta-lactoglobulins: characterization of a new genetic variant

The genetic variants of bovine beta-lactoglobulin (beta-lg) from the "Murnau-Werdenfelser" breed were analyzed in three different isoelectric focusing (IEF) systems. While carrier ampholyte IEF with a pH gradient of 0.2 pH/cm did not resolve the new variant W from the B variant and IEF in immobobilized pH gradients (IPG) with 0.1 pH/cm only partially resolved it, adequate separation was achieved with IPG-IEF in a pH 5.25-pH 5.7 gradient, in presence of 0.8 % w/v carrier ampholytes, both over a 10 and 17 cm separation distance. Apparent isoelectric points (pI's) and genetic frequencies (f) were as follows: beta-lg A, pI = 5.370, f = 0.364; beta-lg B, pI = 5.485, f = 0.480; beta-lg W, pI = 5.492, f = 0.076; and beta-lg D, pI = 5.610, f = 0.080. The small difference of delta pI = 0.007 between beta-lg B and beta-lg W respectively, seems to originate from a "silent" substitution of neutral amino acid residues as compared to the larger delta pI's of the other genetic variants of beta-lg, which result from substitution of charged amino acids.     

Several small GTP-binding proteins are strongly down-regulated in simian virus 40 (SV40) transformed human keratinocytes and may be required for the maintenance of the normal phenotype

High resolution two-dimensional (2-D) gel electrophoresis in combination with the blot overlay nucleotide binding assay was used to reveal low molecular weight GTP-binding proteins expressed by primary cultured, normal human keratinocytes. Forty one small GTP-binding proteins (30 isoelectric focusing, IEF; and 11 nonequilibrium pH gradient electrophoresis, NEPHGE) ranging in molecular weights from 18000 to 30000 and isoelectric points from 4.4 to 8.0 were detected and mapped in the master human keratinocyte database. Four GTP-binding proteins were identified by 2-D gel immunoblotting and these correspond to rap 1 and 2 and two forms of rab6. ras Proteins are most likely present in the [α³²P]GTP 2-D gel blots but their levels may be too low to be detected by immunoblotting. Quantitative changes in the relative expression levels of [α³²P]GTP-binding proteins in normal proliferating and simian virus 40 (SV40) transformed human keratinocytes (K 14) were determined by scintillation counting of the radioactive spots excised from the nitrocellulose blots. The results showed that thirteen of these proteins were not expressed in transformed K14 keratinocytes, implying that they may play a role in the maintenance of the normal cell phenotype.     

Two-dimensional gel isoelectric focusing

Two-dimensional gel isoelectric focusing (2-D gel IEF) is presented as the combination of the same separation method used consecutively in two directions of the same gel. In this new method, after completion of IEF process in the first dimension the gel was cut into the separate strips, each containing selected analytes together with the appropriate part of the original broad pH gradient, and the strips were rotated by 90 degrees (with regard to the first IEF) and left to diffuse overnight. After diffusion the strips were subjected to the second IEF. During the second IEF, the corresponding narrow part of pH gradient in each strip was restored again, however, now along the strip. The progress of the separation process can be monitored visually by using colored low-molecular-weight isoelectric point (pI) markers loaded into the gel simultaneously with proteins. The unique properties of IEF, focusing and resolution power were enhanced by using the same technique twice. Two forms of beta-lactoglobulin (pI values 5.14 and 5.31, respectively) non-separated in the first IEF were successfully separated in the second dimension at relatively low voltage (330 V) with the resolution power comparable to the high-resolution gels requiring the high voltage during the run and long separation time. Glucose oxidase loaded as diluted solution into ten positions across the gel was finally focused into a single band during 2-D gel IEF. Since the first and second IEF are carried out on the same gel, no losses and contamination of analyte occur. The suggested method can be used for separation/fractionation of complex biological mixtures, similarly as other multidimensional separation techniques applied in proteomics, and can be followed by further processing, e.g., mass spectrometry analysis. The focusing properties of IEF could be useful especially in separation of mixtures, where components are at low concentration levels.     

New azo dyes as colored isoelectric point markers for isoelectric focusing in acidic pH region

Newly prepared azo compounds and several commercially available indicators were investigated for their applicability as colored isoelectric point (pI) markers for isoelectric focusing (IEF) in the acidic range below pH 5. The majority of compounds described here can serve as primary standards since their pI values were determined by UV-VIS spectrophotometry independently IEF and direct measurement with a pH electrode. Subjected to gel IEF they show narrow and well-observable zones of different colors. Finally, our work resulted in suggestion of a color ladder composed of pI markers covering the pH range from 1.5 to 4.7.     

Development of non-denaturing off-gel isoelectric focusing for the separation of uranium–protein complexes in fish

An off-gel non-denaturing isoelectric focusing (IEF) method was developed to separate uranium–biomolecule complexes from biological samples as a first step in a multidimensional metalloproteomic approach. Analysis of a synthetic uranium–bovine serum albumin complex demonstrated the focusing ability of the liquid-phase IEF method and the preservation of most of the uranium–protein interactions. The developed method was applied to gill cytosol prepared from zebrafish (Danio rerio) exposed to depleted uranium. The results were compared in terms of resolution, recovery, and protein identities with those obtained by in-gel IEF using an immobilized pH gradient gel strip.     

Electrophoretic phenotyping of erythrocyte enzymes.

Erythrocyte acid phosphatase (EAP), esterase D (ESD) and phosphoglucomutase (PGM) phenotypes among the erythrocyte enzyme types of blood groups are surveyed and a modified cellulose acetate membrane isoelectric focusing (CAM-IEF) method for their exploration is described. The phenotyping procedures are usually classified as either equilibrium or non-equilibrium IEF. Equilibrium IEF, which is based on differences in pI values, includes three methods: (i) a narrow pH range of carrier ampholytes, (ii) a relatively narrow pH range of carrier ampholytes containing chemical separators and (iii) immobilized pH gradient gels. Among the three methods, immobilized pH gradients provides a better resolution of isozymes. Conversely, the disadvantages of immobilized pH gradients include longer focusing times and complex gel preparations. Moreover, immobilized pH gradients are unsuitable for stain analysis because of the insensitivity of PGM1 detection. A hybrid IEF system and a commercial immobilized pH gradient dry plate have overcome these problems. However, EAP typing is extremely expensive and ESD typing is not well distinguished by hybrid IEF. As each method has both merits and demerits, the most suitable technique should be selected based on the kind of erythrocyte enzyme types and sample conditions. On the other hand, non-equilibrium IEF is a rapid method because isozymes are detected on the basis of their charge differences under non-equilibrium conditions. Moreover, the appropriate addition separators increases the charge difference and provides a good resolution within a shorter time. Addition of more separators produces a narrow pH range in the gel and takes a substantially longer time to reach the optimum pH range for charge difference.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isoelectric focusing in long immobilized pH gradient gels to improve protein separation in proteomic analysis.

The number of protein spots detected on two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) gels increases as the gel size increases. The largest commercially available systems resolve a few thousand spots, being only a fraction of the total proteome. We have developed an extremely long isoelectric focusing (IEF) system aimed at more complete protein profiling. The system is especially well suited to sensitive detection methods, such as radioactive detection. The major constraint preventing progress in this area has been the inability to create an even density gradient during the immobilized pH gradient (IPG) casting process. We demonstrate for the first time that this constraint can be effectively overcome, to enable greatly increased IEF separating power with all the advantages of IPG technology,     

The transitional isoelectric focusing process

The transitional isoelectric focusing (IEF) process (the course of pH gradient formation by carrier ampholytes (CAs) and the correlation of the focusing time with CA concentration) were investigated using a whole-column detection capillary isoelectric focusing (CIEF) system. The transitional double-peak phenomenon in IEF was explained as a result of migration of protons from the anodic end and hydroxyl ions from the cathodic end into the separation channel and the higher electric field at both acidic and basic sides of the separation channel. It was observed that focusing times increase logarithmically with CA concentration under a constant applied voltage. The correlation of focusing time with CA concentration was explained by the dependence of the charge-transfer rate on the amount of charged CAs within the separation channel during focusing.     

Separation of protein mixtures using pH-gradient cation-exchange chromatography

Historically, separation of a protein mixture after adsorption to a cation-exchange column is effected by alteration in ionic strength. An alternative separation method using pH induced gradient in the range of 4–7.5 was studied. A cation-exchange column with large particle beads containing excessive carboxyls was employed. A pH gradient across the column was generated by a step change at the column entrance using a non-retained buffer system. Consistency and accuracy of pH values in timed intervals were demonstrated in three different batches. In development of the application, we found a correlation coefficient of >0.9 between the elution pH values of six acidic proteins and their isoelectric points. One case study showed the resolution between a monoclonal antibody and non-retained protein species from a protein A column. Another case study showed the feasibility of separating polyethylene glycol conjugated protein from native protein.     

Evolution of the theoretical description of the isoelectric focusing experiment: II. An open system isoelectric focusing experiment is a transient,bidirectional isotachophoretic experiment

The carrier ampholytes-based (CA-based) isoelectric focusing (IEF) experiment evolved from Svensson's closed system IEF (constant spatial current density, absence of convective mixing, counter-balancing electrophoretic and diffusive fluxes yielding a steady state pH gradient) to the contemporary open system IEF (absence of convective mixing, large cross-sectional area electrode vessels, lack of counter-balancing electrophoretic- and diffusive fluxes leading to transient pH gradients). Open system IEF currently is described by a two-stage model: In the first stage, a rapid IEF process forms the pH gradient which, in the second stage, is slowly degraded by isotachophoretic processes that move the most acidic and most basic CAs into the electrode vessels. An analysis of the effective mobilities and the effective mobility to conductivity ratios of the anolyte, catholyte, and the CAs indicates that in open system IEF experiments a single process, transient bidirectional isotachophoresis (tbdITP) operates from the moment current is turned on until it is turned off. In tbdITP, the anolyte and catholyte provide the leading ions and the pI 7 CA or the reactive boundary of the counter-migrating H₃O⁺ and OH⁻ ions serves as the shared terminator. The outcome of the tbdITP process is determined by the ionic mobilities, pK_a values, and loaded amounts of all ionic and ionizable components: It is constrained by both the transmitted amount of charge and the migration space available for the leading ions. tbdITP and the resulting pH gradient can never reach steady state with respect to the spatial coordinate of the separation channel.     

Efficient separation and analysis of peroxisomal membrane proteins using free-flow isoelectric focusing

We have elaborated a protocol for the fractionation of both hydrophilic and hydrophobic proteins using as a model the matrix and membrane compartments of highly purified rat liver peroxisomes because of their distinct proteomes and characteristic composition with a high quota of basic proteins. To keep highly hydrophobic proteins in solution, an urea/thiourea/detergent mixture, as used in traditional gel-based isoelectric focusing (IEF), was added to the electrophoresis buffer. Electrophoresis was conducted in the ProTeam free-flow electrophoresis (FFE) apparatus of TECAN separating proteins into 96 fractions on a pH 3-12 gradient. Consecutive sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis demonstrated that both matrix and the integral membrane proteins of peroxisomes could be successfully fractionated and then identified by mass spectrometry. This is documented by the detection of PMP22, which is the most hydrophobic and basic protein of the peroxisomal membrane with a pI > 10. The identification of 96 prominent spots corresponding to polypeptides with different physical and chemical properties, e.g., the most abundant integral membrane polypeptides of peroxisomes and specific ones of the mitochondrial and microsomal membrane, reflects the fractionation potential of free-flow (FF)-IEF, accentuating its value in proteomic research as an alternative perhaps superior to gel-based IEF.     

Immobilized pH gradient isoelectric focusing and immunoblotting for investigations of anti-Borrelia burgdorferi IgG antibodies.

Anti-Borrelia burgdorferi immunoglobulin G (IgG) responses in cerebrospinal fluid, serum, and joint fluid from Lyme disease patients were investigated by immobilized pH gradient (IPG) isoelectric focusing (IEF) in pH 4-10 and pH 4-7 gels. After focusing, the anti-B.-burgdorferi antibodies were blotted by affinity-driven transfer to antigen-coated polyvinylidene difluoride membranes (immunoblot) and the IgG antibodies were immunoenzymatically stained. IPG-IEF gels gave an excellent resolution of IgG and the immunoblot proved advantageous for the detection of anti-B. burgdorferi IgG antibodies. These antibodies, as judged from the electromigration characteristics, were found to contain oligoclonal as well as polyclonal subpopulations. This latter group included IgG antibodies that were inadequately resolved when separated by conventional carrier ampholyte IEF.     

Optimized two‐dimensional gel electrophoresis in an alkaline pH range improves the identification of intracellular CFDA‐cisplatin‐protein adducts in ovarian cancer cells

Intracellular binding of cisplatin to proteins has been associated with acquired resistance to chemotherapy. In our previous study we established an analytical method for the identification of intracellular cisplatin‐binding proteins. The method used a fluorescent carboxyfluorescein‐diacetate‐labeled cisplatin analogue (CFDA‐cisplatin), two‐dimensional gel electrophoresis (2DE) and mass spectrometry, which allows detecting and identifying intracellular CFDA‐cisplatin‐containing protein adducts in the acidic pH range (pH 4–7). Based on this analytical method we extended the identification of intracellular cisplatin‐protein adducts to the alkaline pH range (pH 6–10) giving chance to discover new important binding partners. 2DE analysis of alkaline proteins is challenging due to the difficult separation of basic proteins during the isoelectric focusing (IEF). The establishment of an optimized IEF protocol for basic proteins enabled us to identify several intracellular CFDA‐cisplatin‐binding proteins including enzymes of the glucose and serine metabolism like alpha enolase and D‐3‐phosphoglycerate 1‐dehydrogenase.     

Two-dimensional gel electrophoresis of platelet polypeptides with immobilized pH gradients in capillary tubes

Two-dimensional electrophoresis (2-DE) with immobilized pH gradient (IPG) gels in capillary tubes was used in the first-dimensional isoelectric focusing (IEF) for the separation of human platelet polypeptides. Two types of IPG tube gels, pH ranges 4-8 and 7-10, containing 8 M urea, 1% Nonidet P-40 and 0.1% pH 3.5-10 Ampholine carrier ampholytes (CA) were prepared by a simple method not requiring special equipment. The addition of CA to both gel and sample solutions was essential in the tube gel IPG system. Proteins were visualized by a modification of Wray's silver-staining technique. The degree of resolution and the number of spots observed on an IPG 2-DE gel with pH 4-8 were comparable with those obtained with O'Farrell's high-resolution 2-DE. Approximately 200 basic polypeptides, which are difficult to separate by conventional CA-based IEF 2-DE or the non-equilibrium pH gradient system, were well resolved by 2-DE with a pH 7-10 IPG tube gel in the first-dimension. The gel patterns with either pH gradient 4-8 or 7-10 were highly reproducible among gels prepared and run simultaneously. These results demonstrated the potential and usefulness of the 2-DE system with IPG gels in capillary tubes.     

Isoelectric focusing of proteins in capillary electrophoresis with pressure-driven mobilization

Chromatographia - An isoelectric focusing (IEF) method in the capillary format with wide linear pH range (pH 3–10) and high resolution has been developed for separations of proteins. The...     

Analysis of human apolipoproteins C by isoelectric focusing in immobilized pH gradients

Apolipoproteins C are involved in many ways in the metabolism of plasma lipoproteins. Apolipoproteins C from the delipidated VLDL of 35 controls and 165 normo- and hyperlipoproteinemic patients were analyzed by isoelectric focusing on an immobilized pH gradient, pH 4.0-5.0, with 7 M urea, which raised the apparent pH range to 4.8-5.7. This method is an improvement over conventional isoelectric focusing with carrier ampholytes with regard to both resolution and reproducibility. Due to the high resolution (0.1 pH units per cm) additional apolipoprotein C-III bands: C-III0 A1, C-III0 A2, C-III1 C and C-III2 C (the designations A, anodic, and C, cathodic, refer to direction of migration on IEF in relation to the main band) are described for the first time. The possible artifactual nature of these protein bands could be excluded. Cleavage with neuraminidase and peptidases, immunological detection and/or two-dimensional electrophoresis were used to obtain more information. The additional bands seem, in part, to be hydrolysis products of carboxypeptidase A (C-III1 C, C-III2 C). The appearance of C-III1 C and C-III2C was dependent upon the serum triglyceride concentration. The percent distribution of C apolipoproteins in very low density lipoproteins (VLDL) from control serum agreed with previously published data. Apolipoproteins C can also be focused in immobilized pH gradients from VLDL and serum without delipidation.