Preparative Cell Electrophoresis

Abstract

A simple method is described for preparative cell electrophoresis. Solid glass beads (? 0.1 mm diameter) are used as packing material in a cylindrical glass column, the interstices of which contain a citric acid-phosphate buffer of pH 7.6 and 0.015 ionic strength. Cell mixtures containing ? 6 × 10⁹ erythrocytes in 0.2 ml are electrophoresed at 25 V/cm for 1–2 hours after which the column contents are extruded and the separated cell fractions recovered. Complete separation was achieved between mixtures of human and chicken erythrocytes and untreated and papain-treated human erythrocytes. The electrophoretic mobilities of cells subjected to this procedure are in good agreement with previously published mobilities, obtained by microelectrophoresis.     

Preparative Electrophoresis of Isotopically Labeled L-Cell Interferons

The preparative method of polyacrylamide gel electrophoresis was adapted for purification and characterization of isotopically labeled L-cell interferons. Re-covery of interferon activity was quantitative, and purification and resolution were comparable to those obtained by analytical polyacrylamide gel electrophoresis. Ultimate specific activities attainable ranged from 2 x 10(6) to 3 x 10(6) international units per mg of protein.     

A New Apparatus for Preparative Gel Electrophoresis

Syntheses of two carbonyl derivatives of hydantocidin 1, a potent, naturally occurring herbicide, and their herbicidal activity are described. Spiroimidazolidinone 2, the descarbonyl compound at C9, was prepared by employing reductive demethylsulfurization with tri-n-butyltin hydride as the key step. Another derivative, spiroimidazolinone 10, was obtained from α-azidoamide 8 and benzyl isocyanate via the aza-Wittig reaction. 2 had lost almost all herbicidal activity, whereas 10 retained herbicidal activity against such dicotyledonous weeds as ragweed and cocklebur, but lost activity against monocotyledonous weeds. These results imply the possibility that proper modification of the carbonyl group at C7 of the parent compound would afford hydantocidin analogues possessing crop selectivity.     

An Improved System for Preparative Starch Block Electrophoresis

A starch block electrophoresis system is described which includes (a) a single-unit Lucite electrophoresis chamber, (b) temperature control with a circulating water bath and casting resin-coated brass cooling plate, (c) continuous monitoring of block surface temperature and (d) an easily-assembled apparatus for rapid elution of protein from the starch segments.     

Isolation of cu -Antitrypsin in a New Preparative Electrophoresis Apparatus

The construction and operation of an apparatus is described which enables α-antitrypsin to be isolated from human serum by preparative electrophoresis in polyacrylamide gel. The initial pass through the chamber yields a fraction that is predominantly albumin and several α₁-proteins. After removal of albumin by affinity chromatography, a second pass through the chamber separates the individual α₁-proteins. A volume of 50 ml of serum may be accommodated by the chamber, and the recovery of activity in each step is greater than 60%. The entire procedure may be completed in 36 hrs.     

Preparative Electrophoresis of Human Lymphocytes I. Purification of Non-Immunoglobulin-Bearing Lymphocytes by Electrophoretic Levitation

When 10–30 × 10⁶ human peripheral lymphocytes are electrophoresed in an upward direction in a vertical column for 1 to 1–1/2 hour at 12 V/cm at pH 7.1, the fastest migrating fraction of 3–10 × 10 lymphocytes consists of 98–100%. non-immunoglobulin-bearing lymphocytes, as determined by immunofluorescence with anti-human immunoglobulin conjugate. The method can be applied to fresh human lymphocytes as well as to lymphocytes that have been frozen and thawed and, if glycerol is added to the buffer as a cryoprotectant, the fast “T” cell fraction can be frozen immediately, to be stored for later use. Similar separations can be obtained with lymphocytes from human tonsils.     

Preparative Electrophoresis of Living Mammalian Cells in a Stationary Ficoll Gradient

An apparatus was designed for the vertical density-gradient electrophoresis of viable mammalian cells. Cultured Chinese hamster cells (M3-1F3) were grown is suspension and layered on top of a 0–10% ficoll gradient which was also an inverse 6.8-5.1% sucrose gradient in phosphate buffer, pB 7.2 and 1Z glucose. A 60-ml vertical gradient of this composition covered the density range 1.04–1.05 g/cm³ over a distance of 15 cm in a 2.3 cm diameter glass column. An electric field of approximately 2.3 V/cm was applied for 5 hr. The cells migrated 4.7 cm during this period. The migration rate was consistent with the cells having an electrophoretic mobility of -1.15 ± 0.20 μm/sec per volt/cm, equal to that determined by microelectrophoresis. The gradient was harvested in 50 fractions after electrophoresis, and the viability of the cells was 75% on the basis of colony formation.     

连续自由流电泳的研制和应用于分离蛋白质和细胞

连续自由流电泳(Continuous free-flow dectrophoresis,CFE)自1980年起有了相当大的发展,其主要特点是将只能分析少量物质(1μg)的分析型电泳转变成能分离lg左右物质的制备型电泳,以达到分离和纯化各种生物产品目的^[1]。近年来CFE广泛应用于蛋白质和细胞分离,是一种很有前途的制备级电泳方法^[2,3]。在连续自由流电泳中,一种恒定pH称为载体缓冲液的液体．沿垂直方向缓慢流过一矩形的电泳薄腔,电泳腔的两侧是电极室,能被横向地加上直流电场,欲分离的蛋白质混合物通过一根细管从样品入口处连续注入腔体,随缓冲液措径向流动,同时又向与液流垂直的方向迁移．每种蛋白质的侧移距离与它的泳动率、电场强度及它在分离腔中的保留时间成正比。在分离腔出口处,混合物中各种蛋白质根据它们的泳动率被分配在各股液流中并由腔体出口处的一组收集管收集。但是有许多次级现象能损害连续自由流电泳的分离结果,诸如沉降效应、电渗流、焦尔热和自然对流。本文报道了仪器的研和我们对模型蛋白分离条件进行了优化,并成功地将人和兔的红细胞分开。     

Preparative Gel Electrophoresis at High Sample Load. The Effect of Some Experimental Variables on Separation Performance

The separation of model protein pairs (hemoglobin/ albumin, trypsin/chymotrypsin, hemoglobin A/hemoglobin F) was studied in an apparatus for preparative gel electrophoresis at loads up to 40 mg/cm ²of the cross-sectional area of the gel bed. Separation was favored by higher ionic strength and by longer migration path. Under the conditions used and within the load range studied, increasing total protein load had no adverse effect but increased voltage gradient, temperature, or gel strength were all unfavorable.     

Isolation of Protein uH2A Using a one Step Preparative Gel Electrophoresis

Abstract

A one step electrophoretic procedure for the isolation of protein uH2A has been devised which may improve the overall yield. The improvement involves elimination of intermediate steps which might result in the decrease of the yield. The method may serve as an alternate to the conventional methods and can also be used successfully for the isolation of several different proteins.     

Preparative Polyacrylamide-Agarose Gel Electrophoresis of Proteins and RNAS by the Use of New Device

Quantitatively reproducible results were obtained by using a new device for preparative gel electrophoresis combined with polyacrylamide-agarose composite gel. When an adequate gel-buffer system was selected according to the procedure described in this paper, proteins and RNA's were well separated and recovered. The new device for preparative gel electrophoresis and the method for preparation of polyacrylamide-agarose composite gel are presented together with the elution profiles of the recovered substances.     

Preparative Electrophoresis with On-column Optical Fiber Monitoring and Direct Elution into a Minimized Volume

A “column-format” preparative electrophoresis device which obviates the need for gel extraction or secondary electro-elution steps is described. Separated biomolecules are continuously detected and eluted directly into a minimal volume of free solution for subsequent use. An optical fiber allows the species of interest to be detected just prior to elution from the gel column, and a small collection volume is created by addition of an ion-exchange membrane near the end of the column.     

Preparative parallel protein purification (P4)

In state of the art drug discovery, it is essential to gain structural information of pharmacologically relevant proteins. Increasing the output of novel protein structures requires improved preparative methods for high throughput (HT) protein purification. Currently, most HT platforms are limited to small-scale and available technology for increasing throughput at larger scales is scarce. We have adapted a 10-channel parallel flash chromatography system for protein purification applications. The system enables us to perform 10 different purifications in parallel with individual gradients and UV monitoring. Typical protein purification applications were set up. Methods for ion exchange chromatography were developed for different sample proteins and columns. Affinity chromatography was optimized for His-tagged proteins using metal chelating media and buffer exchange by gel filtration was also tested. The results from the present system were comparable, with respect to resolution and reproducibility, with those from control experiments on an AKTA purifier system. Finally, lysates from 10 E. coli cultures expressing different His-tagged proteins were subjected to a three-step parallel purification procedure, combining the above-mentioned procedures. Nine proteins were successfully purified whereas one failed probably due to lack of expression.     

Fractionation of Subunits in Xylanases from Trichoderma viride with a New Simple Preparative Polyacrylamide Gel Electrophoresis Apparatus

Formic acid was identified as the acidic component of antibiotic K-52B by gas-liquid chromatography, whereas amino sugar I hydrochloride was isolated from the acid hydrolysate as the basic sugar component. On the basis of infrared analyses of constituent oligosaccharides, formic acid was assumed to be linked to the hydroxyl group of galactose in oligosaccharide III. From the results of physicochemical properties and ninhydrin degradation, periodate oxidation and peracetylation studies of amino sugar I hydrochloride, C₈H₁₈N₂O₅. 2HC1 was considered to be a new diaminohexose with a substituent group.     

Preparative scale isolation of galactosylhydroxylysine.

A method is described for the isolation and purification of gram quantities of the hydroxylysine-monosaccharide from commercially available marine sponge. The procedure utilized alkaline hydrolysis followed by purification by ionexchange chromatography and gel filtration. Compositional analysis indicated that the final product contained only galactose, hydroxylysine, and HCl which were present in equimolar quantities and comprised 94% of the dry weight. This preparation has been utilized as a substrate for the assay of UDP-glucose:collagen glucosyltransferase (EC 2.4.1.66) of human platelets.     

Immunoabsorption of membrane-specific antibodies for determination of exposed and hidden proteins in human erythrocyte membranes.

Human erythrocyte membrane proteins solubilized with the non-ionic detergent Berol EMU-043 have been characterized by crossed immunoelectrophoresis with rabbit antibodies raised against the membrane material. Three out of sixteen membrane-specific immunoprecipitates disappeared when the antisera were first absorbed with intact erythrocytes. This finding indicates that three antigens are exposed on the outside of the erythrocyte membrane. One of these antigens showed acetylcholinesterase activity, and another was the major glycoprotein (glycophorin) as shown by crossed-line immunoelectrophoresis. No antigenic determinants of the latter protein were detected within the membrane or on its inner surface. In crossed immunoelectrophoresis with antisera after absorption with washed, non-sealed membranes only one precipitate remained. This precipitate corresponded to albumin. Accordingly, several proteins seem to have antigenic determinants exposed on the inside of the membrane.     

Editorial board page for “Preparative Biochemistry”, Volume 4, Number 4

This is a scanned image of the original Editorial Board page(s) for this issue.     

Preparative purification of dipeptidyl peptidase IV.

Dipeptidyl-Peptidase IV was purified from pig kidney by ammonium sulfate fractionation, gel filtration, QAE-cellulose chromatography and affinity columns with Gly-Pro- and Concanavalin A-Sepharose. The specific activity of the purified enzyme is 41.8 units/mg. Polyacrylamide gel electrophoresis and silver staining show a single band. The enzyme preparation is free of aminopeptidase and dipeptidase activity, proved fluorimetrically and by gas chromatography/mass spectrometry. The most important procedure for removal of contaminating enzyme activities is a stepwise NaCl-gradient on a QAE-ZetaPrep ion exchange disk.