Electrophoretic Separation Of Proteins Research Articles

State of preservation of polymorphic plasma proteins recovered from ancient human bones

Protected by the mineral matrix, bone proteins are capable of surviving inhumation periods of several hundreds or thousands of years in soil. While the preservation of the bone matrix protein, collagen I, is the prerequisite for a variety of archaeometric approaches, such as radiocarbon dating and the reconstruction of palaeodiet by stable isotope analysis, little is known about both the rate and state of preservation of non-collagenous proteins. We succeeded in the isolation, electrophoretic separation (SDS-PAGE, IEF) and immunological detection (radial immunodiffusion, IEF immunoblotting and ELISA) of plasma proteins preserved in archaeological human bones. However, sample preparation and electrophoretic methods had to be adapted to the specific demands of these aged proteins, since they are not only degraded and fragmented but also cross-linked to other organic components, either indigenous to the bone or to contaminants from the burial environment. Complex decomposition phenomena are responsible for the altered mode of migration of aged proteins through a gel. After isoelectric focusing, the ancient proteins mainly concentrate below pH 4.45 in the pH-gradient. Thus, highly negatively charged protein components have a better chance of preservation in bone after death. Isoelectric focusing with subsequent immunoblotting of ancient protein samples revealed protein patterns which showed marked charge-modifications in comparison with those of modern human plasma proteins due to protein degradation (e.g. α2-HS-glycoprotein and α1-antitrypsin). Nevertheless, in combination with different immunological analyses, previous results concerning the selective enrichment of α2-HS-glycoprotein in bone compared with other plasma glycoproteins could be confirmed. Copyright © 1999 John Wiley & Sons, Ltd.

Clinical analysis of human serum proteins using the Beckman automated Paragon 2000 CZE system.

Electrophoretic separation and quantitation of serum proteins, including paraproteins, remain essential for the diagnosis and management of a variety of disorders. We examined the Beckman automated Paragon 2000 capillary zone electrophoresis (CZE) system and compared its electropherograms to those obtained by cellulose acetate electrophoresis (CAE). Within-run CVs using seven different capillaries and between-day CVs for CZE were <2% for albumin; they were <6% for α1-globulin, α2-globulin, β-globulin, and γ-globulin. From 2.7 to 15.0g/dl of total protein concentrations, the same electrophoretic patterns could be observed. Comparisons of the five principal bands obtained by CZE and cellulose acetate electrophoresis (CAE) gave correlation coefficients from 0.8 to 0.9. On a quantitative basis in 55 specimens, albumin by CZE was on average 10% lower than CAE, and α1-globulin and β-globulin were 93% and 15% higher than by CAE, respectively (p<0.001). Based on our use of the Beckman Paragon software and our reference values (percent of total protein) for CZE of 56.3 to 68.9% for albumin, 3.9 to 6.3% for α1-globulin, 4.8 to 8.4% for α2-globulin, 7.9 to 11.9% for β-globulin, and 9.9 to 21.5% for γ-globulin, we found 75% to 100% agreement with CZE; i.e., both methods gave either normal or abnormal results of the time. Patients showing a gammopathy gave values that were higher by CZE 60% of the time. Furthermore, the CZE system can resolve prealbumin, α1-acid glycoprotein, α1-antitrypsin, hemopexin, transferrin, and complement besides to the usual five bands.

Cationic and anionic polymeric additives for wall deactivation and selectivity control in the capillary electrophoretic separation of proteins in food samples

Both cationic and anionic polymeric additives were used for the capillary electrophoretic separation of proteins in food samples. The cationic polyelectrolyte polydiallyldimethylammonium chloride was more effective in minimizing protein–wall interactions at pH 3 than at pH 7, presumably due to greater repulsion between the adsorbed polymer and proteins. Improved resolution was observed in the presence of the co-additive sodium octanesulphonate, presumably due to ion-pairing interactions with protein sample components. The anionic polymer dextran sulfate produced relatively high efficiencies, 120 000–180 000 theoretical plates, for protein separation, presumably because the polymer adsorbed to the capillary wall, rendering the surface more hydrophilic. In addition to reduced protein–wall interactions, improved resolution was observed, presumably due to analyte–polymer ion-exchange/ion-pairing interactions. When poly(vinyl sulphonic acid) was used instead of dextran sulfate, broader profiles were obtained and fewer components were resolved, presumably due to reduced wall deactivation that is related to the lower hydrophilicity of poly(vinyl sulphonic acid).

Capillary zone electrophoretic separation of basic proteins and drugs using guaran as a buffer modifier

Guaran, a neutral polysaccharide, has been used as a buffer modifier to improve the separation of basic proteins and drugs. Migration reproductibility, peak shape and efficiency were improved when 0.1% guaran was added to the buffer. The concentration of guaran, ionic strength, and pH of buffer solution were optimized to obtain the optimum separation of proteins. Possible separation efficiencies of 700,000 plates per meter were obtained for test proteins. The relative standard deviation (% RSD) of the migration time of all test proteins was less than 0.5%. Improved separation of β-blockers was also observed when guaran was added to the buffer.

Capillary electrophoresis of peptides and proteins in fused-silica capillaries coated with derivatized polystyrene nanoparticles.

High-resolution capillary electrophoretic separation of proteins and peptides was achieved by coating the inner wall of 75 microm ID fused-silica capillaries with 40-140 nm polystyrene particles which have been derivatized with alpha-omega-diamines such as ethylenediamine or 1,10-diaminodecane. A stable and irreversibly adsorbed coating was obtained upon deprotonation of the capillary surface with aqueous sodium hydroxide and subsequent flushing with a suspension of the positively charged particles. At pH 3.1, the detrimental adsorption of proteins to the capillary inner wall was suppressed efficiently because of electrostatic repulsion of the positively charged proteins from the positively charged coating which enabled protein separations with maximum efficiencies of 400000 plates per meter. A substantial improvement of separation efficiency in particle-coated capillaries was observed after in-column derivatization of amino functionalities with 2,3-epoxy-l-propanol, resulting in a more hydrophilic coating. Five basic and four acidic proteins could be separated in less than 7 min with efficiencies up to 1900000 theoretical plates per meter. Finally, coated capillaries were applied to the high-resolution analysis of protein glycoforms and bioactive peptides.

NAD+ Kinase Activities in Euglena Gracilis and Phaseolus Vulgaris

After an electrophoretic separation of proteins from Euglena gracilis and dry seeds of Phaseolus vulgaris in native conditions in polyacrylamide gels, gels were incubated in mixtures containing NAD+, Mg-ATP2-, glucose 6-phosphate, G6P dehydrogenase, and either phenazine ethosulfate and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (PES/MTT) or phenazine methosulfate and nitro blue tetrazolium (PMS/NBT) as coupled redox system for NAD+ kinase activity detection. In the presence of PES/MTT, 4 bands were revealed for E. gracilis, among which two corresponded to NAD+ kinase activity, the other corresponding to a NAD+ reductase activity due to alcohol dehydrogenase (ADH). In the presence of PMS/NBT, only the bands of NAD+ kinase activity were revealed. With Phaseolus vulgaris, 3 bands of ADH were always revealed in both mixtures, and only the use of PMS/NBT allowed the detection of NAD+ kinase as a fourth band. With both materials, NAD+ reductase staining in gels was intensifed in the presence of GTP or ATP and even further with ADP or GDP. The results demonstrate that: 1) the NAD+ kinase and NAD+ reductase are two distinct enzymes; 2) the NAD+ reductase corresponds to ADH.

Two-dimensional electrophoretic separation of yeast proteins using a non-linear wide range (pH 3-10) immobilized pH gradient in the first dimension; reproducibility and evidence for isoelectric focusing of alkaline (pI > 7) proteins.

The proteome of the yeast Saccharomyces cerevisiae was analysed by two-dimensional (2D) polyacrylamide gel electrophoresis utilizing a non-linear immobilized pH gradient (3-10) in the first-dimensional separation. Cells were labelled by [35S]methionine incorporation in the respiro-fermentative phase during exponential growth on glucose. Gels were run, visualized with phosphoimager technology and all resolved proteins automatically quantified. Proteins were well resolved over the whole pH interval, and evidence for isoelectric focusing on the basic side of the pattern was generated by sequencing of some spots, revealing the 2D positions of Tef1p, Pgk1p, Gpm1p, Tdh1p and Shm2p. Roughly 25% of the spots were resolved at the alkaline side of the pattern (pI > 7). The position reproducibility was high and in the range 1-2 mm in the x- and y-dimension, respectively. No quantitative variation was linked to a certain size or charge class of resolved proteins, and the average quantitative standard deviation was 17 +/- 11%. The obtained immobilized pH gradient based pattern could easily be compared to the old ampholine-based 2D pattern, and the previously reported identifications could thus be transferred. Our yeast pattern currently contains 43 known proteins, all identified by protein sequencing. Utilizing these identified proteins, relevant pI and Mr scales in the pattern were constructed. Normalization of the expression of identified spots by compensating for the number of methionine residues a protein contains allowed stoichiometric comparisons. The most dominant proteins under these growth conditions were Tdh3p, Fba1p, Eno2p and Tef1p/Tef2p, all being expressed at more than 500,000 copies per cell. The differential carbon source response during exponential growth on either glucose, galactose or ethanol was examined for the alkaline proteins identified by micro-sequencing in this study.

Further characterization of Renibacterium salmoninarum extracellular products.

Renibacterium salmoninarum, the agent of bacterial kidney disease in salmonids, releases high concentrations of extracellular protein in tissues of infected fish. The extracellular protein consists almost entirely of a 57-kDa protein and derivatives of degradation and aggregation of the same molecule. The 57-kDa protein and its derivatives were fractionated into defined ranges of molecular mass. Separated fractions continued to produce degradation and aggregation products. One-dimensional electrophoretic separation of extracellular protein revealed a number of proteolytically active bands from > 100 to approximately 18 kDa associated with various 57-kDa protein derivatives in the different molecular mass fractions. Two-dimensional separation of extracellular protein showed that continued degradation and aggregation, similar both in location and behavior to some of the 57-kDa protein derivatives, was also displayed by the proteolytically active bands after their separation. Effects of reducing agents and sulfhydryl group proteinase inhibitors indicated a common mechanism for the proteolytically active polypeptides characteristic of a thiol proteinase. The results suggested that the 57-kDa protein and some of its derivatives undergo autolytic cleavage, releasing a proteolytically active polypeptide(s) of at least 18 kDa. Soluble polysaccharide-like material also was detected in extracellular products and tissue from infected fish. Antiserum to the polysaccharide-like material cross-reacted with O-polysaccharide of the fish pathogen Aeromonas salmonicida, suggesting some structural similarity between these polysaccharides. The polysaccharide and the proteolytic activity associated with the 57-kDa protein derivatives should be investigated with respect to the pathogenesis of R. salmoninarum infections.

Developmentally regulated masking of an intracellular epitope of the 180 kDa isoform of the neural cell adhesion molecule NCAM

The neural cell adhesion molecule NCAM is a cell surface glycoprotein that occurs in several isoforms. It was previously shown that the largest 180-kDa isoform of NCAM (NCAM 180) accumulates at sites of cell contact and in postsynaptic densities and may be responsible for the stabilization of cell contacts by its interaction with the membrane-cytoskeleton linker protein brain spectrin. In immunohistochemical studies on the expression of the NCAM 180, we noticed that two NCAM 180 specific monoclonal antibodies, termed 481 and D3, showed different patterns of immunoreactivity in sections of fresh-frozen adult mouse brain. Here we show that the D3-specific, but not the 481-specific epitope becomes inaccessible to the antibody during development of the hippocampal formation, coincident with the establishment of stable cell-cell contacts. In contrast, in the olfactory bulb with its continually regenerating olfactory nerve fibers, both NCAM 180 antibodies remain fully immunoreactive throughout development and adulthood. We also show that the D3-specific epitope becomes inaccessible in primary cerebellar neuron cultures with time in culture. Electrophoretic separation of hippocampal membrane proteins under nondenaturating conditions showed NCAM to be present in protein complexes of different molecular weights at different developmental stages. We propose that NCAM is involved in the formation of developmentally regulated, noncovalent complexes with as yet unknown partner molecules that could be responsible for the masking of the D3-specific epitope.

Free Solution Capillary Electrophoretic Separation of Basic Proteins and Drugs Using Cationic Polymer Coated Capillaries

A simple capillary coating which exhibits reversed (anodic) electroosmotic flow (EOF) has been developed for the separation of cationic compounds by free solution capillary electrophoresis. Poly(2-aminoethyl methacrylate hydrochloride) (PAEM) was chemically bound onto the fused-silica capillary inner wall. Capillaries modified by this method exhibit an essentially pH-independent EOF in the pH range from 3 to 5.5, and have been applied to the improved separation of basic proteins and drugs (e.g., beta-adrenergic blocking drugs). High efficiency and precise migration (relative standard deviation was less than 1%) were observed.

Relationships between health and weight loss during lactation and between health and ability to return to oestrus after weaning in primiparous sows.

The study comprises observations in 301 Swedish Yorkshire primiparous sows belonging to a research herd. During the 6 weeks of lactation, they were fed according to a conventional feeding regime based on litter size. The sows were weighed at farrowing and at weaning. After weaning the sows were checked daily for standing reflex and blood samples for determination of plasma progesterone were drawn regularly. Diseases occurring during lactation were recorded in 223 sows. A clinical health examination of these sows was also performed on the day of weaning, including body temperature, condition scoring, appetite, locomotor disorders, remarks on legs and mammary glands. A blood sample was drawn 1 week before weaning in 155 sows for analysis of blood haemoglobin concentration, packed cell volume, total white blood cell count, serum concentration of urea, creatinine, triglycerides, free fatty acids, total protein and electrophoretic separation of proteins. Sows with large weight loss had a higher total incidence of diseases during lactation, mastitis being the most common disease, than sows with small weight loss. Sows with large weight loss seemed to be more catabolic during late lactation than sows with small weight loss, according to the blood analyses. The blood analyses did not show any signs of subclinical infection being more common among sows with large weight loss than among sows with small weight loss. There were neither any significant differences in the total incidence of diseases during lactation nor in the health at weaning between sows returning to oestrus within 10 days of weaning and sows not returning within this time.

Electrophoretic Separation and High-Sensitivity Detection of Dyestuff-Labeled Proteins Using an Untreated Fused-Silica Capillary and Sodium Dodecyl Sulfate-Containing Buffer for Migration and Labeling.

Proteins labeled with the dye Rhodamine B isothiocyanate were separated by capillary zone electrophoresis using an untreated fused-silica capillary and buffer containing sodium dodecyl sulfate. The sodium dodecyl sulfate in the migration buffer eliminated or minimized the protein adsorption to the inner wall of the capillary and rendered the proteins negatively charged through hydrophobic interactions. Furthermore, an appropriate use of SDS during the labeling procedure was found to increase the solubility of the dyestuff, which in turn results in sensitive detection of the protein. The labeled proteins were separated successfully in an untreated capillary using a buffer containing sodium dodecyl sulfate and detected spectrophotometrically and fluorometrically with high sensitivity. Bovine serum albumin reacted with the dyestuff for 4, 12 and 24h at 20°C was detected by spectrophotometry at 556nm with approximately 2.5, 4.2 and 6.0 times, respectively, higher sensitivity than unlabeled protein. Fluorometric detection(λex=563nm, λem= 577nm) further increased the detection sensitivity to approximately twice that of spectrophotometric detection at 556nm for the labeled protein.

Capillary electrophoretic separation of acidic and basic proteins in the presence of cationic and anionic fluorosurfactants

We report the use of mixture of cationic and anionic fluorosurfactants as addtives for free-flow capillary electrophoresis of proteins. Effective deactivation of the capillary wall is obtained, which allows the use of raw fused-silica capillaries. The magnitude and direction of the electroosmotic flow is strongly affected by the composition of the surfactant mixture, and a suggested model for this behaviour in terms of micellation and formation of admicellar surfactant layers is described. By utilizing mixtures of the oppositely charged surfactants, it is possible to separate both positively and negatively charged proteins in the same run. Due to charge interactions of the fluorosurfactants with the proteins, it is possible to tune the separation selectivity, without having to change the buffer strength or pH. This was demonstrated in particular for the model substances myoglobin and ribonuclease, where the order of elution could be reversed, compared to their elution order in a normal buffer system. Another advantage of the fluorosurfactants additives is their effectiveness in low concentrations (<100 μg/ml) even when buffers of low ion strength are employed. Thus, rapid separations at a high field strength can be accomplished, without suffering from excessive Joule heating. This is demonstrated with an example, where a mixture of positively and negatively charged proteins is separated in less then 2 min in a 10 m M phosphate buffer at pH 7.

Effect of zwitterionic surfactants on the separation of proteins by capillary electrophoresis

The effect of zwitterionic surfactants on capillary electrophoretic separation of proteins was investigated using an uncoated silica capillary column and different buffers containing zwitterionic surfactants. The effects of N-alkyl-N,N-dimethyl ammonio-1-propane-sulfonates on the electrophoretic mobility of three different proteins, namely albumin, lysozyme and myoglobin, were examined. The addition of N-alkyl-N,N-dimethylammonio-1-propane sulfonate in phosphate saline buffer is important in minimizing protein-capillary wall interactions, and facilitated an efficient electrophoretic mobility of myoglobin and lysozyme. The separation efficiency of proteins also depends on the injection pressure for the control of migration time and the peak sharpness. The electrophoretic conditions were applied to evaluate the separation of lysozyme, myoglobin and albumin. The zwitterion surfactants can form a dynamic coating on the capillary surface, thereby reducing the number of adsorption sites to which proteins may adsorb.

Argon-laser-induced fluorescence detection in sodium dodecyl sulfate-capillary gel electrophoretic separations of proteins

The use of argon-laser-induced fluorescence detection in sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE) separations of proteins is described. A simple, rapid (10 min) precolumn labeling reaction for a series of standard proteins (molecular masses of 20100–77000) was developed which utilizes 4-fluoro-7-nitrobenzofurazan and 4-chloro-7-nitrobenzofurazan as fluorogenic labeling reagents. Detection limits of 25 ng/ml for labeled bovine serum albumin were obtained. These detection limits compare favorably with alternate detection techniques such as Coomassie blue staining (2.5 μg/ml) and UV-Vis absorption detection in SDS-CGE (0.5 μg/ml). Conditions were identified for the labeling reactions which produce no measurable zone broadening effects for labeled proteins relative to unlabeled species. By using relative migration data from labeled proteins and molecular mass calibration plots obtained exclusively from unlabeled proteins, molecular mass estimates of labeled proteins typically accurate to within 4.5% of literature values can be obtained. The labeling reaction thus maintains the molecular mass information inherent in the separation.

Protein synthesis in developing ovaries of mealworm under in vivo and in vitro conditions: Effects of diflubenzuron

The effect of diflubenzuron (DFB) on protein synthesis in the ovaries of Tenebrio molitor was evaluated during oocyte maturation using in vivo and in vitro assays. When incorporated into the diet (5 and 10 mg g −1), DFB was found to affect both the weight, the protein levels and the incorporation of tritiated leucine into proteins of ovaries. In addition, electrophoretic separation of ovarian proteins by sodium dodecyl sulfate-polyacrylamide slab gels (SDS-PAGE) showed that DFB applied in vivo did not have a significant effect on the number of protein bands. When added to the culture medium (5 and 10 μg ml −1), DFB resulted in a slight but significant decrease in the rate of incorporation of tritiated leucine into proteins of ovaries cultured for 5 d compared to controls. These in vitro effects of DFB on protein synthesis were less marked than those observed in orally treated females.

Iron and copper nutrition-dependent changes in protein expression in a tomato wild type and the nicotianamine-free mutant chloronerva.

The nicotianamine-deficient mutant chloronerva resembles phenotypically an Fe-deficient plant despite the high accumulation of Fe in the leaves, whereas if suffers from Cu deficiency in the shoot. Two-dimensional electrophoretic separation of proteins from root tips and leaves of wild-type Lycopersicon esculentum Mill. cv Bonner Beste and the mutant grown with and without Fe showed a number of consistent differences. In root tips of the Fe-deficient wild type and the Fe-sufficient as well as the Fe-deficient mutant, the expression of glyceraldehyde-3-phosphate dehydrogenase, formate dehydrogenase, and ascorbate peroxidase was increased. In leaves of the Fe-sufficient and -deficient mutant, Cu-containing chloroplastic and cytosolic superoxide dismutase (Cu-Zn) and plastocyanin (Cu) were nearly absent. This low plastocyanin content could be restored by supplying Cu via the xylem, but the superoxide dismutase levels could not be increased by this treatment. The differences in the protein patterns between wild type and mutant indicate that the apparent Fe deficiency of mutant plants led to an increase in enzymes involved in anaerobic metabolism as well as enzymes involved in stress defense. The biosynthesis of plastocyanin was diminished in mutant leaves, but it was differentially induced by increased Cu content.

Immunoblot Detection

Immunoblotting (often referred to as western blotting) is used to identify specific antigens recognized by polyclonal or monoclonal antibodies. Following electrophoretic separation of proteins and transfer from the gel to an appropriate membrane, the immobilized proteins are probed with specific antibodies to identify and quantitate any antigens present. After being probed with primary antibody, the membrane is washed and the antibody-antigen complexes are identified using horseradish peroxidase (HRPO) or alkaline phosphatase (AP) enzymes coupled to the secondary anti-immunoglobulin-G (anti-IgG) antibody (e.g., goat anti-rabbit IgG). As described in this unit, the detection enzymes are attached directly or via an avidin-biotin bridge to the secondary antibody. Chromogenic or luminescent substrates are also described for visualizing the activity.

Selectivity change in the separation of proteins and peptides by capillary electrophoresis using high-molecular-mass polyethyleneimine

A study of the capillary electrophoretic separations of proteins and peptides using high-molecular-mass polyethyleneimine (PEI) is presented. Experiments were performed in the PEI-coated capillaries together with the use of this polymer as a buffer additive under different separation conditions. The effects of pH and the concentration of PEI in the buffer on the electroosmotic flow and the migration orders of biopolymers were investigated. The use of the cationic polymer offers an alternative for the modification of the separation selectivity and resolution of biopolymers.

Epoxy resin coatings for capillary zone electrophoretic separation of basic proteins

Epoxy resins, combining with a cross-linker, were coated on organosilanes modified fused-silica capillaries. After cross-linking, a tough three-dimensional network was attached to capillary surface via SiOSiC bonds. The most effective coating consisted of epoxy resins cross-linked with diaminodiphenylmethane, which was stable over the pH range 2–12 and suppressed protein adsorption effectively. The electroosmotic flow was also greatly reduced and maintained constant over the pH range 6–10. High resolutions and separation efficiencies for basic proteins were obtained in the pH range 3–10 and reproducible separations were achievedafrom run to run and day and day.