Curtain Electrophoresis Research Articles

Theory of Simultaneous Multiple Streak Collimation in Continuous-Flow Electrophoresis by Superposition of Electro-Osmosis and Thermal Convection

Abstract The resolving power in continuous flow streak deflection electrophoresis (the “free flow” curtain electrophoresis as well as in “endless fluid belt” electrophoresis) is limited by the broadening of the streaks during the process of electro-migration. This effect becomes more pronounced as the diameter of the injector is increased to augment the throughput. A gain in the latter is thus achieved at a sacrifice in resolution. It has been possible until now to avoid this limitation for only one fraction of an electrophoretic mixture, namely that component whose electrophoretic velocity is equal and opposite to the velocity of the electro-osmotic flow adjacent to the wall of the electrophoretic curtain (6,7). This paper elucidates a way to achieve streak collimation (i.e. elimination of streak broadening) simultaneously for all components of an electrophoretic mixture and to do it in an optimal velocity configuration where the throughput increases with the square of the injector diameter without an es...

Isolation and purification of human serum alpha-1 acid glycoprotein.

Abstract Alpha-1 acid glycoprotein (or orosmucoid) was obtained in a pure state from normal human serum by ion exchange chromatography followed by curtain electrophoresis and a final ion exchange chromatography step. Pure α1 acid glycoprotein (α1A) has a sedimentation coefficient of 3.1 S and a diffusion coefficient of 5.2 × 10−7cm2 sec−1, which yields a molecular weight of 44,680 Daltons and an asymmetry factor of 14.6. The αA prepared in the manner here described appears less denatured than the same protein isolated by the Cohn fractionation method.1,2 ' Alpha-1 A acts as a depressant of phagocytosis3 and is one of the constituents of Mowbray's serum fraction,″which induces a prolongation of skin homografts.

Isolation of 2 macroglobulin subunits from human serum.

Abstract Subunits of α2 macroglobulin (α2M) with sedimentation rates lower than 10 S were found in ultrafiltrates (through macroglobulin-stopping membranes) of normal human sera. Five to six percent of the total α2M in human sera exists in subunit form. After isolation by means of ammonium sulfate precipitation, curtain electrophoresis, gel filtration and ultrafiltration these subunits were shown to have sedimentation rates of 5–10 S.

Purification and characterization of a thyroglobulin reactive phytoprecipitin from Pisum sativum.

Abstract By means of paper curtain electrophoresis and polyacrylaaide gel filtration, it has been possible to isolate the thyroglobulin-reactive phytoprecipitin from pea (Pisum sativum; American pea; Russian pea) extract. An of 4.0 Svedbergs and a of 5.7×10−7 cm2/sec were found from analytical ultracentrifuge experiments and a specific volume of 0.61 was determined by pycnometry. These data yield a molecular weight of 43,000 for the phytoprecipitin and, postulating an oblong ellipsoid shape, an asymmetry factor of 12. Its electro-phoretic mobility corresponds with that of globulin. It could be shown that the phytoprecipitin attaches to the carbohydrate part of the thyroglobulin.

Purification and some properties of two boticins.

Two bacteriocins (boticins) were elaborated without induction by strain S5, a nontoxigenic variant of Clostridium botulinum type E. After separation of the two active entities by gel filtration on Sephadex G-50, a large particle with boticin activity was isolated by density gradient ultracentrifugation, and a small soluble boticin was purified by continuous curtain electrophoresis and chromatography on sulfoethyl-Sephadex. Large and small boticins were purified 200- and 3,000-fold, respectively, with yields of 50% or more. The small boticin, a basic substance with molecular weight under 30,000, was the predominant species; the large boticin, a negatively charged particle with particle weight greater than 40 x 10(6), represented less than 20% of the total activity. Both purified boticins were resistant to heat and were attacked by proteolytic enzymes, but the large boticin was less thermostable and less sensitive to proteolytic enzymes than was the smaller variety. The activity of the large boticin was not reduced by treatment with urea or deoxyribonuclease. Both boticins exhibited sporostatic and bactericidal activities for C. botulinum type E, strain 070. A suspension of type E strain 070 vegetative cells was rendered nonviable within 9 min by the small boticin. The lethal action of this bacteriocin was not reversed by trypsin.

The hemolymph proteins of the blue crab, callinectes sapidus—I. Hemocyanins and certain other major protein constituents

1. 1. Starch-gel electrophoresis revealed considerable qualitative and quantitative variation in the proteins of hemolymph samples from individual adult specimens of Callinectes sapidus. 2. 2. Partial separation of the hemolymoh proteins was achieved by continuous-flow paper curtain electrophoresis. 3. 3. Two hemocyanins, fast and slow, are not interconvertible. 4. 4. Removal of copper from isolated hemocyanins by dialysis against cyanide-containing buffers produced apohemocyanins with electrophoretic mobilities identical to those of the intact hemocyanins. 5. 5. Fast hemocyanin has a copper-protein ratio of 0·2:100 and dissociates at alkaline pH. 6. 6. Slow hemocyanin is found almost exclusively in hemolymph from females. 7. 7. Another female-limited protein is found only in those specimens with maturing oocytes; this yellow, high-density lipoprotein is apparently associated with vitellogenesis. 8. 8. A “complex” protein, extremely variable in quantity, binds 59Fe and possesses amylase activity. 9. 9. A clotting protein is more abundant in serum than in plasma from the same animal. 10. 10. Species specificity of the electrophoretic patterns is confined to the characteristic mobility and dissociation of fast hemocyanin, the only protein found to be present in all samples of blue crab hemolymph.

Studies on the relation between plasma antithrombin and heparin-cofactor.

A method for the preparation of purified plasma antithrombin and heparin-cofactor is described. The method involves adsorption by aluminium hydroxide, separation on a DEAE-cellulose column by means of a graded salt concentration, and vertical curtain electrophoresis. A 100-fold increase in the specific activity of antithrombin and a 30-fold increase in the specific activity of heparin-cofactor have been achieved. In spite of the increased purification, no separation of the two activities was achieved. When a highly purified fraction was subjected to starch-gel electrophoresis for 16–18 h and then eluted from the gel, there was significant loss of heparin-cofactor activity but not of antithrombin activity. The electrophoretic patterns of the recovered proteins were not altered.

Breakdown of Royal Jelly Protein in the Midgut of the Larval Honeybee

SummaryHomogenates of the midgut of honeybee larvae 3–5 days old hydrolysed all five protein fractions isolated from royal jelly by paper curtain electrophoresis. The amino acids lysine and threonine were identified by thin-layer chromatography in the hydrolysate of one fraction. The end-products of digestion of the other fractions were not identified but appeared to be peptides. Gel electrophoretograms of royal jelly protein before and after digestion by isolated midguts showed an increase in the number of protein bands to seven. After digestion four of the original bands were missing. Apparently royal jelly protein passes through the midgut as a mixture of polypeptides and/or peptides and a minor proportion of amino acids. Comparison of the hydrolysing characteristics of midgut homogenate and crystalline beef trypsin showed major differences. The homogenate had no effect on a synthetic substrate T.A.M.E. (p-toluene sulphonyl-L-arginine methyl ester HCl), which is readily hydrolysed by trypsin. No antige...

Studies on thyroid proteins: II. Purification of human thyroid proteins by gel filtration and the isolation of 19-S, 7-S, and 4-S components

A procedure is described by which the major soluble proteins of normal human thyroid tissue can be separated. Gel filtration on Sephadex G-200 yields three fractions. The first eluted fraction (A) contains samples of well-purified thyroglobulin. Each of these samples also contains varying proportions of the 34-S, 27-S and 12-S thyroid proteins. Some of the samples from Fraction A contain only the 27-S and heavier proteins. The second fraction (B) contains nearly pure 7-S thyroid protein, as judged by ultracentrifugal analysis. By this same criterion, the third fraction (C) has essentially pure 4-S material, designated thyralbumin. The components in Fraction A cannot be further separated by paper curtain electrophoresis. The 7-S protein in Fraction B is, however, separated into nearly equal amounts of two subfractions by curtain electrophoresis. Likewise, the 4-S protein in Fraction C is separated into two nearly equal amounts. These procedures have separated eight major protein fractions in human thyroid extract. Furthermore, one of the 7-S protein subfractions and both of the 4-S subfractions have been shown to contain thyroid-specific antigenic activity.

Studies on the structure of collagen: I. The sequence analysis of peptides released by pronase

Peptides released during the solubilization of native insoluble collagen by the action of pronase were separated by successive curtain electrophoresis and paper chromatography and analyzed. Of the 47 peptides and 8 free amino acids liberated, the sequences of 40 peptides have been determined. From this data the sequences of 12 larger peptides have been deduced and their relation to the structure of collagen α chains is discussed. These peptides are mainly from the C-terminal end of the constituent chains and have a composition dissimilar to collagen, i.e. glycine occurs in every second position. The presence of three ketoacids, α-ketoisovaleric, α-ketoglutaric and pyruvic acid, in the tropocollagen molecule is reported.

Composition et structure de L'α 2-Macroglobuline isolée du serum de porc

α 2-Macroglobulin has been isolated from porcine serum by a combination of curtain electrophoresis, ultracentrifugation and gel filtration on Sephadex G-200. Analytical ultracentrifugation, acrylamide-gel electrophoresis and immunoelectrophoresis were used as criteria of purity and for identification of the α 2-macroglobulin. Amino acid composition and physicochemical constants (isoionic point, sedimentation coefficient, molecular weight) were found to be very close to those observed by Schultze and co-workers for human α 2-macroglobulin. The variation of viscosity as a function of concentration indicates that the molecule is highly expanded in very dilute solution. Measurements of fluorescence depolarization showed that the structure is rigid, in contrast to that of γ-macroglobulin. Values of optical rotatory parameters have been determined for the range 546 to 313 mμ; they suggest a high β structure content in the molecule.

Discontinuous Potential in Planar Electrophoresis

Abstract Direct experimental evidence has been obtained to show that in planar electrophoresis, as opposed to curtain electrophoresis, the electric and centrifugal forces act together in a synergistic fashion to yield an enhanced shift of the electromigration path of a migrant. For a mixture, this means an enhanced separation of its components, under otherwise similar experimental conditions. At the same time, it is evident that a discontinuous potential can also be utilized in planar electrophoresis, thus reducing heat production in the wet paper sheet and therefore obviating the many complicating factors that are involved.

Fractionation of poly(ethylenimine) by curtain electrophoresis

Journal of Polymer Science Part A: General PapersVolume 3, Issue 8 p. 3031-3037 Note Fractionation of poly(ethylenimine) by curtain electrophoresis T. D. Perrine, T. D. Perrine National Institute of Arthritis and Metabolic Diseases, National Institute of Health, Bethesda, MarylandSearch for more papers by this authorPatrick F. Goolsby, Patrick F. Goolsby National Institute of Arthritis and Metabolic Diseases, National Institute of Health, Bethesda, MarylandSearch for more papers by this author T. D. Perrine, T. D. Perrine National Institute of Arthritis and Metabolic Diseases, National Institute of Health, Bethesda, MarylandSearch for more papers by this authorPatrick F. Goolsby, Patrick F. Goolsby National Institute of Arthritis and Metabolic Diseases, National Institute of Health, Bethesda, MarylandSearch for more papers by this author First published: August 1965 https://doi.org/10.1002/pol.1965.100030831Citations: 1AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Citing Literature Volume3, Issue8August 1965Pages 3031-3037 RelatedInformation

ANTIHEPARIN ACTIVITY OF SOME HUMAN BLOOD PROTEIN FRACTIONS AND THEIR POSSIBLE RELATIONSHIP TO THROMBOSIS.

In view of the possible relationship to thrombosis the ;anti-heparin' activity of blood protein fractions was studied. Serum and plasma were separated by continuous paper curtain electrophoresis and two different groups of fractions with anti-heparin activity found. One group was associated with the fast gamma globulins and the other with the alpha globulins. The fast gamma activity appeared to be identical with the contact activation product (activated factors XI and XII). The alpha globulin activity is different from any of the known serum clotting factors. This activity may be due to a previously unrecognized clotting factor or may be a coagulant property of certain blood proteins which act by binding heparin. The clinical implications of these findings are discussed. It is suggested that the fast gamma globulin anti-heparin fraction may be identical with Wessler's serum thrombotic accelerator and the alpha globulin activity is a separate entity.

THE IDENTIFICATION AND ISOLATION OF TWO MOUSE-TOXIC PROTEIN COMPONENTS IN EXTRACTS FROM PASTEURELLA PESTIS.

The toxin activity of Pasteurella pestis cells, strain "Tjiwidej," was found to be associated with two proteins. Using a disc electrophoresis technique in conjunction with mouse lethality, two toxic proteins were isolated exhibiting intraperitoneal LD(50)'s of less than 1.0 to 1.5 microg protein. Each produced a single characteristic precipitin band on agar gel diffusion plates. The slower migrating toxin in gel diffusion or disc electrophoresis was designated as toxin A. It was shown to be sensitive to deoxycholate and digitonin, did not accumulate in 5-fluorotryptophan treated cells, and was associated with the membrane fraction of the cell. The faster migrating toxin B, apparently is resistant to surface-active agents, and is not affected by treatment of cells with 5-fluorotryptophan. Toxin B is associated with the soluble or cytoplasmic fraction of the cell. This evidence suggested that each toxin represented a distinctly different molecular species. The possibility is discussed that toxin B is synonymous with the murine toxin previously isolated by paper curtain electrophoresis which revealed only one antigen band in the Oudin precipitin reaction.

Isolation and composition of staphylococcal alpha toxin.

SUMMARY: A method is described for the preparation, on a laboratory scale, of staphylococcal alpha toxin having a purity of about 70% and in a yield of 40%. The method entails ammonium sulphate fractionation followed by curtain electrophoresis. The toxin so obtained contains two impurities, one of which can be removed electrophoretically, and the other ultracentrifugally. The toxin itself is a protein of molecular weight 44,000. It contains most of the usual amino acids which vary in amount from 4 residues for histidine to 44 for aspartic acid. Although electrophoretically heterogeneous its electrophoretic components produce the biological effects heterogeneous associated with alpha toxin. As by-products of purification, two other proteins were isolated in crystalline form. Their significance for pathogenicity, if any, remains to be determined.

Immunologic studies of caddis fly: II. Isolation of the allergenic fractions of caddis fly extract

1. A component of Caddis fly extract has been isolated which appears to contain the major allergenic activity. This is fraction 2 of a zone electrophoresis procedure, that is, the fastest migrating major component. This fraction does not have all the allergenic components found in the initial extract. 2. Direct skin testing appears to give results which are comparable with and much easier to obtain than passive transfer skin tests. 3. Continuous flow paper curtain electrophoresis can be used as an alternative to the simpler starch block method, in order to give larger quantities of material, and in a purer state.

Curtain electrophoretic separation of phenyl acetate hydrolysing esterases of human serum. Demonstration of an EDTA-dependent enzyme.

SummaryBy repeated continuous flow curtain electrophoresis of human serum, 2 distinct phenyl acetate hydrolyzing esterases were purified 120 and 850 times. One esterase was heat-labile and inactivated by EDTA, while the other was heat-stable and required EDTA for activity. EDTA-inactivation could be prevented by manganese. The methods for purification and assay are described and the results discussed.

Bitterness in Celerya,b

SUMMARYThe bitter principle of celery was isolated by successive solvent extraction, paper chromatography and curtain electrophoresis. It is a colorless crystalline non‐flavonoid D‐glucoside with mp of 159°C. It is cationic at pH greater than 7, soluble in polar solvents, and fluorescent under ultraviolet light. Electrophoresis is introduced as a new technique in the isolation of a bitter principle from a vegetable. A new conception of bitterness in plants is introduced.

SERUM FRACTIONATION AND THE EFFECTS OF BOVINE SERUM FRACTIONS ON HUMAN CELLS GROWN IN A CHEMICALLY DEFINED MEDIUM

Serum has been fractionated by curtain electrophoresis using carboxymethyl cellulose dissolved in sodium bicarbonate electrolyte. Various fractions were produced from bovine serum and added to replicate cultures of Chang's endoepithelial cells and HeLa cells grown in a chemically defined medium. The effects of each of the various fractions on the appearance of the cultures and on cell multiplication were studied. Three different fractions were obtained and two were subjected to further purification. One fraction associated with albumin promoted survival, attachment, and flattening as well as cell multiplication. A second fraction associated with the alpha globulins promoted survival and multiplication of some cells. A third fraction caused cells to aggregate and form free floating clumps. An adequate chemically defined medium for continuous growth of human cells was used throughout the study. The response of cells to alterations in their environment which simulated some of the effects produced by serum fractions is described.