Electrophoretic Separation Of Proteins Research Articles

Improved two-dimensional electrophoretic separation of acidic proteins extracted from Norway spruce needles by using immobilized pH gradients.

AbstractAn improved method for two‐dimensional electrophoretic separation of acidic proteins extracted from Norway spruce needles is presented. With the use of immobilized pH gradients in the first dimension, this method gave stable separations, with the proteins evently distributed over the entire gel surface. The number of spots in the electrophoretograms have increased fourfold compared to our earlier separations.

5] Membrane proteins

Publisher Summary Depending on the type of interaction, membrane proteins are either embedded into a lipid bilayer or associated with the membrane structures, in the latter case, usually by ionic interaction or by hydrogen bonds. To isolate and analyze these proteins, they must be extracted from the membrane structures— that is, solubilized. Solubilization is mostly carried out with detergents. In this chapter, to illustrate the strategy for high-resolution separation of membrane proteins, the chromatographic and electrophoretic separation of proteins from the plasma membranes of the liver and Morris hepatomas is shown. Three groups of proteins are chosen, with different degrees of hydrophobicity. Membrane-associated annexins [calcium-binding protein (CBP) 65/67, CBP 35, and CBP 33] are less hydrophobic and can be solubilized, without using detergents. These proteins can be phosphorylated, but they are not glycosylated. Dipeptidyl-peptidase IV (DPP IV; EC 3.4.14.5) and the cell-cell adhesion protein cell-CAM (cell adhesion molecule or gp110) are intrinsic membrane proteins, and they are also glycosylated. Other membrane proteins, e.g., from eukaryotic cells or of bacterial and viral origin, can be assigned to one of the above-mentioned groups, in accordance with their behavior in chromatographic as well as electrophoretic separations.

Use of plate numbers achieved in capillary electrophoretic protein separations for characterization of capillary coatings

The number of theoretical plates achievable in capillary electrophoresis has been proposed for the characterization of the quality of capillary coatings. It is shown that, as predicted by theory, the efficiency is independent of capillary inner diameter and increases with increasing field strength as long as dispersive effects can be neglected. On the other hand, in contrast to chromatography, there is no linear range within which the plate numbers are independent of the amount or volume of sample injected (from aqueous solutions). By optimization of the injection technique (use of dilute aqueous protein solutions, electrokinetic injection techniques, short injection times), up to 3 · 10 6 theoretical plates can be achieved. The plates generated by injection of similar amounts of sample from buffer solutions are lower by a factor of 20.

Monoclonal antibodies reveal complex structure in the membrane skeleton of Tetrahymena.

Twelve monoclonal antibodies were raised that are specific for the membrane skeleton of Tetrahymena. Five were directed against T. pyriformis and seven were directed against T. thermophila. Some cross-reactivity between species was found. Each monoclonal antibody recognized one of the three major components of epiplasm, i.e. the bands A, B, and C identified in electrophoretic separations of epiplasmic proteins. It was found, using these antibodies, that the epiplasmic proteins A, B and C have overlapping but independent distributions within the cell.

Expression of monoclonal-antibody-defined antigens in fractions isolated from human breast carcinomas and patients' serum.

The aim of this study was to examine tissue from patients with breast carcinoma or benign breast disease for the presence of monoclonal-antibody-defined antigens, including the MUC1 mucin and carcinoembryonic antigen CEA. The tests were performed by sodium dodecyl sulphate/polyacrylamide gel electrophoretic separation of proteins, electrophoretic transfer to nitrocellulose membranes and immunostaining with the monoclonal antibodies. Some of the antigens identified are known to circulate at high levels in some but not necessarily all, breast carcinoma patients. Serum from a panel of ten breast cancer patients was subjected to a fractionation procedure designed to release antigen from immune complexes, and again these samples were analysed for the presence of monoclonal-antibody-defined antigens. A high frequency of positive reactions was detected by the anti-MUC1 monoclonal antibody C595 with both breast carcinoma subcellular membrane fractions as well as antigen fractions eluted from circulating immune complexes. No reactions were observed with equivalent materials from benign breast disease samples. The findings illustrate the variability in antigen expression between breast tumours. The data also indicate that a proportion of patients respond to their tumour by the production of antibodies that recognise the MUC1 antigen in their circulation.

Capillary zone electrophoretic separation of proteins using a column coated with epoxy polymer

Fused-silica capillaries used in capillary zone electrophoresis were covalently coated with epoxy polymer and then cross-linked with ethylenediamine to form a stable layer. This coating was of sufficient thickness and hydrophilicity to reduce both protein adsorption and electroosmotic flow. The electroosmotic flow-rate showed about a twofold decrease but was still sufficient to carry both positive and negative species to the detector. As a consequence, high separation efficiencies were obtained in the pH range 2–10. The coatings were stable after more than 350 injections in 2 months. Further, reproducible separations were achieved from run to run and day to day.

Capillary electrophoretic separations of biotechnology-derived proteins in E. coli fermentation broth.

A capillary electrophoresis (CE) method was developed for the analysis of a recombinant DNA protein in a fermentation broth matrix. A polyacrylamide-coated capillary was employed to eliminate electroosmotic flow, facilitating relatively rapid run times. Selectivity and efficiency were enhanced significantly through the incorporation of magnesium sulfate and acetonitrile, an organic modifier, into the separation buffer. Both of these additives appeared to influence the separation by diminishing the interaction of the protein analyte with the charged micelles present in the buffer. The quantitation capabilities of the CE method were validated and found to be comparable to the standards set by chromatographic techniques previously developed for similar applications.

Electrophoretic size separation of proteins treated with sodium dodecyl sulfate in 1% agarose gels.

Separation of proteins treated with sodium dodecyl sulfate (SDS) according to molecular size was achieved by discontinuous electrophoresis in vertical low-concentration agarose gels. A linear relationship was found between the migration distance and the square root of the molecular weight. This holds for proteins in the range of 7-200 kDa separated in 1.25% w/v agarose gel slabs (7 x 7 x 0.15 cm) with 0.1% w/v SDS and sulfate as leading ion. The linear regression coefficient was 0.998. The molecular weight and charge of coions influenced the separation. Small ions with low pKa values were found suitable as coions. The migration distance of proteins treated with SDS varied linearly with the agarose concentration of the gel. The agarose type and quality affected the resolution of the SDS-protein bands. We conclude that agarose gels can substitute polyacrylamide gels for the separation of proteins treated with SDS. A homogeneous agarose gel at a concentration of about 1% is a nonsieving support for electrophoresis. Therefore, the separation described here cannot be explained by the pore size of the gel. The results suggest that the separation is mainly due to the relative migration velocities of the coion and the proteins treated with SDS.

N,N,N',N'-tetramethyl-1,3-butanediamine as effective running electrolyte additive for efficient electrophoretic separation of basic proteins in bare fused-silica capillaries.

The effect of N,N,N',N'-tetramethyl-1,3-butanediamine (TMBD) in the running electrolyte on the electroosmotic flow and the migration behavior of four standard basic proteins in bare fused-silica capillaries was examined at pH 4.0, 5.5, and 6.5. Depending on the electrolyte pH and additive concentration the electroosmotic flow was either cathodic or anodic. A similar Langmuirian-type dependence of the electroosmotic flow on the concentration of TMBD in the running electrolyte was found at the three experimented pH values, which may be indicative of the specific adsorption of the additive in the immobilized region of the electric double layer at the interface between the capillary wall and the electrolyte solution. Electrophoretic separations of the four standard basic proteins performed at the three above pH values, showed well-resolved, efficient and symmetric peaks, demonstrating the utility of this additive for protein electrophoresis in bare fused-silica capillaries. The variations in separation efficiency, peak capacity, resolution and reproducibility of migration times as a function of the additive concentration at pH 6.5 were also examined.

High resolution one-dimensional electrophoretic separation and partial characterisation of human head hair proteins.

A reproducible, rapid procedure for the extraction, labelling and separation of human hair proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) has been developed. Hair proteins were extracted in 8 M urea, containing 0.2 M mercaptoethanol, followed by sonication. Extracts were neutralised with Tris and incubated with either labelled (14C) or unlabelled iodoacetic acid to S-carboxymethylate cysteine groups. Proteins were separated on 12.5% SDS-polyacrylamide gels and gels stained with Coomassie Brilliant Blue and/or silver nitrate to reveal major protein bands. Gels were then treated with a fluorographic agent, dried and autoradiographed to reveal major sites of S-carboxymethylation. A given gel was scanned by laser densitometry after Coomassie and/or silver stain to quantitate the protein content of each major protein zone. An autoradiogram of the same gel was scanned to estimate the cysteine content of each major zone. In this way it was possible to partially characterise rapidly and reproducibly many different protein zones in different individual samples on one gel at the same time. By calculating the ratio of autoradiograph absorbance to Coomassie Blue absorbance, protein zones could be assigned to four different categories, viz: very high cysteine (VHC) proteins, high cysteine (HC) proteins, low cysteine (LC) proteins and very low cysteine (VLC) proteins. The method described is reproducible, rapid and inexpensive enough to be suitable for mass screening. Overall the results were more informative than previously reported one-dimensional separations and indeed this technique may well be more suited to forensic and/or population investigations than the much more laborious and time-consuming two-dimensional techniques.

High-performance capillary electrophoretic separation of proteins and peptides using a bonded hydrophilic phase capillary.

High performance capillary electrophoresis (HPCE) was applied to the separation of protein and peptide mixtures with molecular masses ranging from 1300 to 96000 Da using a new bonded hydrophilic phase capillary, CElect-P150. This coated capillary reduces the interaction between proteins and silanol groups in capillary walls, allowing a complete recovery of the proteins and peptides of interest. HPCE was also used for the analysis of a complex mixture of tryptic fragments and to monitor the process of enzymatic digestion. Moreover, using a CElect-P150 capillary, highly reproducible analysis was possible without preconditioning the capillary with acid or basic solutions before each new analysis.

Evaluation of a neutral hydrophilic coated capillary for capillary zone electrophoretic separation of proteins

A neutral, hydrophilic coated capillary with negligible electroosmotic flow was characterized as to migration time reproducibility and separation efficiency for protein separation by capillary zone electrophoresis. Consecutive runs (over 200 runs) of the basic proteins at pH 6.0 yielded excellent migration time reproducibility (< 2% R.S.D.) and high separation efficiency (ca. 3–5 · 10 5 plates m ). The acidic proteins were separated at pH 8.0 under the reversed polarity (cathode at the injection end), and excellent migration time reproducibility of less than 3% was achieved. Separation of egg white proteins at pH 3.0 showed migration time reproducibility of less than 0.5% R.S.D, ( n = 36) for lysozyme, conalbumin and ovalbumin.

Protein (western) blotting.

The different steps involved in protein (Western) blotting and subsequent analysis of the proteins are reviewed. Electrophoretic separation of proteins, procedures of transfer to membranes, immunological and nonimmunological protein detection systems, and characterization of protein-nucleic acid and protein-protein interactions are described. Emphasis is on the sensitivity of the methods described and on possible variations that allow the individual steps of Western blotting to be adapted to specific questions.

Chitinase isoforms in roots of various pea genotypes infected with arbuscular mycorrhizal fungi

Chitinase activity has been investigated in mycorrhiza-resistant (myc −), non-nodulating (nod −) isogenic pea ( Pisum sativum L.) mutants in an attempt to understand the reasons for such resistance to symbiotic fungi. Activities from control and Glomus mossease-inoculated roots of myc − mutants were compared to the corresponding mycorrhizal (myc +), nodulating (nod +) wildtype pea genotype cv. Frisson. A pea mutant only deficient for the nod − character [myc +, nod −] was also studied. Two acidic and two basic chitinase isoforms were detected in control roots of all peas tested after native polyacrylamide gel electrophoretic (PAGE) separation of proteins at pH 8.9 or pH 4.3. No difference in basic chitinase isoforms patterns occurred in the various pea genotypes in the presence of G. mosseae. However, as soon as 1 week after infection, an additional acidic chitinase isoform was observed in extracts of G. mosseae-colonized roots from the wildtype pea genotype and the [myc +, nod −] mutant. The isozyme had a molecular mass of approximately 27 kDa, estimated by sodium dodecyl sulfate (SDS)-PAGE under non-reducing conditions, and exhibited a faint lysozyme activity by lysis of Micrococcus luteus cells. It was not detected in the [myc −, nod −] pea mutant, unless plants were grown under conditions which increased the number of appressoria. The host origin of the 27 kDa chitinase isoform was indicated by its presence after infection with another Glomus species, and from the fact that it was not detected in a mixture of germinated spores and mycelia of G. mosseae.

Improved capillary zone electrophoretic separation of basic proteins in uncoated fused-silica capillary by using ethylene diamine as a buffer additive

Ethylene diamine was used as a buffer additive to improve capillary zone electrophoretic separation of basic proteins in an uncoated fused-silica capillary and theoretical plate numbers of the order of a hundred thousand were obtained for all five standard proteins at pH 6.5, 8.0 and 9.5. The critical factors that affected the separation efficiency were the ethylene diamine concentration and the applied voltage at each pH value. Adsorption of ethylene diamine ions to the capillary inner wall significantly reduced the charge density of the wall, and therefore reduced the adsorption of basic proteins. Increasing the concentration of ethylene diamine increased this reduction, and caused an increase in the theoretical plate numbers of all proteins. Increasing the applied voltage caused an increase in the theoretical plate numbers of all proteins in the low-voltage region and a decrease in high-voltage region, which maybe attributed to excessive Joule heat.

Identification of Distinct Internal and External Isozymes of Carbonic Anhydrase in Chlorella saccharophila.

External carbonic anhydrase (CA) was detected in whole cells of alkaline-grown Chlorella saccharophila but was suppressed by growth at acid pH or growth on elevated levels of CO2. Internal CA activity was measured potentiometrically as an increase in activity in cell extracts over that of intact cells. Cells grown under all conditions had equal levels of internal CA activity. Two isozymes were identified after electrophoretic separation of soluble proteins on cellulose acetate plates. The fast isozyme was found in cells grown under all conditions, whereas the slow isozyme was found only in cells grown at alkaline pH. Western blot analysis following sodium dodecyl sulfate-polyacrylamide gel electrophoresis using antibodies produced against the periplasmic form of CA from Chlamydomonas reinhardtii revealed a single band at 39 kD, which did not change in intensity between growth conditions and was associated only with proteins eluted from the fast band. The slow isozyme was inactivated by incubation of cell extract at 30[deg]C and by incubation in 10 mM dithiothreitol, whereas the internal form was unaffected. These results indicate that external and internal forms of CA differ in structure and their activities respond differently to environmental conditions.

High-efficiency capillary electrophoretic separation of basic proteins using coated capillaries and cationic buffer additives: Evaluation of protein—capillary wall interactions

The joint use of basic cationic additives (morpholine and several tetraazamacrocycles) in the buffer and chemically bonded cross-linked polyacrylamide-coated capillaries was evaluated as a method for decreasing the adsorption of basic proteins on the fused-silica capillary wall. The superiority of the tetraazamacrocycle Cyclen (1,4,7,10-tetraazacyclododecane) over morpholine and other tetraazamacrocycles is demonstrated. Using 20 m M phosphate-60 m M Cyclen buffer (pH 5.5) and cross-linked polyacrylamide-coated capillaries, separation efficiencies in the range of 10 6 plates/m were obtained for basic proteins. A simplified model that allows the quantification of the interactions between proteins and the capillary wall was developed. The model was assessed using the different buffers and capillaries evaluated in the first part. As the model predicts, a straight line for the plot of the inverse of the migration time versus the electric field strength with an intercept different from zero was observed. The value of the intercept correlates with the separation efficiency observed for the basic proteins studied and, therefore, with the interaction strength between proteins and the capillary wall.

Characterization of proteolytic activities stimulated by SDS or urea and two-dimensional gel electrophoresis of proteins from Brachionus plicatilis (Rotifera)

1. 1. Proteinases occurring in the 27,000 g supernatant of homogenates from Brachionus plicatilis have been characterized by electrophoretic techniques. 2. 2. Several individual proteases were detected which are active in the presence of either SDS or urea. 3. 3. By applying this knowledge about the properties of proteases occurring in Brachionus, two-dimensional electrophoretic separations of proteins from Brachionus plicatilis are made feasible without proteolytic artifacts.

Characterization and localization of an ATP-diphosphohydrolase on the external surface of the tegument of Schistosoma mansoni

An ATP-diphosphohydrolase (EC 3.6.1.5) was identified in the tegumental fraction isolated from Schistosoma mansoni worms. Both ATP and ADP were hydrolyzed to AMP at similar rates by the enzyme. Other nucleotides were also degraded by the tegument enzyme, revealing a broad substrate specificity. Electrophoretic separation of tegumental proteins under non-denaturing conditions followed by addition of ATP or ADP as substrate revealed a single band of activity with similar mobility. In addition, similar heat-inactivation profiles were obtained for ATPase or ADPase activities, indicating that a single enzyme is responsible for degrading both nucleotides. The enzyme was not inhibited by vanadate, levamisole, tetramisole, ouabain or sodium azide. The ADPase activity was not affected by adenosine (5′)-pentaphospho(5′)-adenosine (Ap 5A) or by an excess of glucose and hexokinase used as an ATP-trapping system, thus excluding the presence of any significant adenylate kinase activity. The ATP-diphosphohydrolase displayed micromolar affinities for both Mg 2+ and Ca 2+, and the calcium-activated enzyme was inhibited by millimolar Mg 2+. In intact live worms a calcium phosphate precipitate was formed on the outer tegumental surface upon incubation of the worms with either ATP or ADP, indicating the ecto-localization of this enzyme. In addition, ultrastructural hystochemical localization of the enzyme was obtained. A distinct deposition of lead phosphate granules on the outer surface of the tegument was observed by electron microscopy, in the presence of either ATP or ADP as substrate. It is suggested that the ATP-diphosphohydrolase could regulate the concentration of purine nucleotides around the parasites and hence enable them to escape the host hemostasis by preventing ADP-induced platelet activation.

Capillary electrophoretic protein separations in polyacrylamide-coated silica capillaries and buffers containing ionic surfactants

Capillary electrophoretic protein separations of high efficiency and resolution were obtained using polyacrylamide-coated silica capillaries and buffers containing ionic surfactants. The presence of micellar concentrations of sodium dodecyl sulfate or cetyltrimethyl- ammonium chloride minimized protein-capillary wall interactions, and facilitated concurrent separations of a mixture of both acidic and basic proteins, while the polyacrylamide coating provided increased resolution and migration time reproducibility via a reduction in electroosmotic flow. Attempts to obtain size-based protein separations via sieving through buffers containing the hydrophilic polymers methylcellulose and polyethylene glycol were unsuccessful.