Electrophoretic cell separation by means of microspheres

The electrophoretic mobility of fixed human erythrocytes immunologically labeled with poly(vinylpyridine) or poly(glutaraldehyde) microspheres was reduced by approximately 40%. This observation was utilized in preparative scale electrophoretic separations of fixed human and turkey erythrocytes, the mobilities of which under normal physiological conditions do not differ sufficiently to allow their separation by continuous flow electrophoresis. We suggest that resolution in the electrophoretic separation of cell subpopulations, currently limited by finite and often overlapping mobility distribution, may be significantly enhanced by immunospecific labeling of target populations using microspheres.     

Electrophoretic separation of the three Rhizobium meliloti replicons.

The megaplasmids and the chromosome from the bacterium Rhizobium meliloti 1021 were separated in preparative quantities by using transverse alternating-field gel electrophoresis. The genetic content of each electrophoretically separated band was determined by Southern hybridization with replicon-specific probes and by comparison with Agrobacterium tumefaciens transconjugants harboring either pSym-a or pSym-b megaplasmids. Pulsed-field gel electrophoresis analyses of PacI (5'-TTAATTAA-3') and SwaI (5'-ATTTAAAT-3') digests of the whole genome and of the separated replicons were used to calculate genome sizes in two R. meliloti strains. In these strains, PacI digestion yielded only four fragments for the entire genome. The sizes of the PacI fragments from R. meliloti 1021 in megabase pairs (Mb) were 3.32 +/- 0.30, 1.42 +/- 0.13, 1.21 +/- 0.10, and 0.55 +/- 0.08, for a total genome size of 6.50 +/- 0.61 Mb. Southern hybridization with replicon-specific probes assigned one PacI fragment to the chromosome of R. meliloti 1021, one to pRme1021a, and two to pRme1021b. PacI digestion of A. tumefaciens pTi-cured, pSym transconjugants confirmed these assignments. In agreement with PacI data, the addition of the six SwaI fragments from R. meliloti 1021 gave a genome size of 6.54 +/- 0.43 Mb. pRme1021a was calculated to be 1.42 +/- 0.13 Mb, 1.34 +/- 0.09 Mb, and 1.38 +/- 0.12 Mb on the basis of PacI digestion, SwaI digestion, and the migration of uncut pRme1021a, respectively. pRme1021b was calculated to be 1.76 +/- 0.18 Mb, 1.65 +/- 0.10 Mb, and 1.74 +/- 0.13 Mb on the basis of PacI digestion, SwaI digestion, and the migration of uncut pRme1021B, respectively. The R. meliloti 1021 chromosome was calculated to be 3.32 +/- 0.30 Mb, 3.55 +/- 0.24 Mb, and 3.26 +/- 0.46 Mb on the basis of PacI data, SwaI data, and the migration of uncut chromosome, respectively.     

Electrophoretic separation of cells in a density gradient.

A bulk electrophoretic method is described for the separation of mammalian cells in Ficoll-sucrose density gradients. The fractionation of cells is performed in a simple commercially available apparatus (Buchler Poly-Prep) which should facilitate the application of the technique in various laboratories. Details of the experimental procedure are presented along with typical model separations of erythrocytes from various species and mouse lymphocytes.     

Electrophoretic separation and characterization of pea protoplasts

A carrier-free electrophoresis system was developed to separate protoplasts according to their zeta-potential and to study their behaviour in subsequent cell culture. Apex protoplasts from young cotyledon-free pea embryos ( Pisum sativum L. cv. Belman), including primordia, were analysed with respect to their electrophoretic mobility, viability, cell size, cell division capacity, chlorophyll content, and expression of a 50 kDa protein involved in early somatic embryogenesis. Protoplasts fractionated on the basis of their electrophoretic mobility were viable and able to divide and form microcallus in cell culture. Cell culture procedures were modified in order to handle low numbers of protoplasts (50–2000 per fraction). Electrophoretic separation revealed distinct classes of protoplasts, differing in all parameters tested except cell size. Therefore, a connection between zeta potential and other physiological parameters can be proposed.     

Electrophoretic separation and quantitation of creatine kinase isozymes

A method is described for separating and quantitating the isozymes of creatine kinase in various tissue extracts. We have tested the sensitivity and precision of the method over a wide range of mixtures of isozymes. The quantitation is linear in the range of 0–5 mIU of CK analyzed, and the lower limit of quantitation of an isozyme in a mixture is 10 mIU/ml. For certain tissues the presence of dithiothreitol is necessary to prevent inactivation of the brain-type isozyme.     

Electrophoretic separation of plasma lipoproteins in agarose gel

A method has been developed for the separation of serum or plasma lipoproteins by electrophoresis in an agarose-agar gel mixture. The gel is applied to the surface of a thin polyester photographic film strip. With minor alterations in technique either single samples on individual strips or many samples on one large sheet may be processed. After fixation and dehydration the transparent film is stained with Sudan Black B and washed with water. The finished electrophoretogram can be obtained in 5 hr and consists of widely separated bands of lipoprotein fractions on a colorless transparent background, ideally suited for scanning with a densitometer. Plasma samples from different subjects show pre-beta lipoproteins of different mobilities. An effect of gel concentration on the extent of lipoprotein migration is demonstrated. The clearcut separation of lipoproteins by this method will facilitate the classification of hyperlipoproteinemias and improve quantitative estimates of lipoprotein distribution.     

Light microscope identification of murine B and T cells by means of functional polymeric microspheres.

Covalent bonding of purified antibodies to polymeric microspheres of 0.4 to 0.8 μm diameter yields conjugates which can be used to label lymphocytes in the light microscope. Nonadherent microspheres can be separated by means of a discontinuous density gradient and quantitative measurements of adherent microsphere distributions can be made through examination of Wright's stained dry mounts or through fluorescent microscopic examination of cells in suspension.In general the distributions of adherent microspheres on mouse splenic and thymic lymphocytes in direct or indirect labelling assays show good agreement with results obtained from fluorescent antibody techniques. In comparison to fluorescent antibody the use of these antibody-microsphere conjugates has the advantage of allowing direct correlations between the surface antigens of cells and their histologie morphology.     

Rapid separation of proteins and their higher-molecular fragments by means of Spheron ion-exchanges.

Ion-exchange derivatives are described. of a hydrophilic rigid macroporous glycolmethacrylate gel called Spheron, suitable for rapid high-performance liquid chromatography (HPLC) of proteins and their fragments. Their flow parameters are compared with those of ion exchange derivatives of cellulose and polydextran. The conditions for work with them are described (regeneration, cycling, equilibration, column packing) as well as the construction of a simple apparatus for medium-pressure ion exchange chromatography of proteins. The efficiency of these ion exchangers for the separation of proteins is illustrated with examples of chromatography of an artificial mixture of serum albumin, chymotrypsinogen and lysozyme. Chromatography of cyanogen bromide fragments of serum albumin and the A and B chains of oxidized insulin showed that the method can be applied in chromatography on higher molecular protein fragments. A review of all proteins, including technical enzymes, which have already been chromatographed on Spheron ion exchangers is also given. The prospects of Spheron ion exchangers for HPLC of proteins and their fragments are briefly discussed.     

Electrophoretic separation of high-molecular protein components of low-density lipoproteins

Apolipoprotein B (apoB) and 40 different protein standards have been studied at electrophoresis under various conditions of separation. The system was selected that is capable of fractionating well the apoB and its high-molecular weight species formed after the treatment of serum lipoproteins with malondialdehyde. The separation of proteins is based on the usage of complex gels combining in one unit the regions with constant (T = 3 and 10%) or continuously changing (gradient gel with T = 3-6%) concentration of acrylamide. The technique allows to create the optimum conditions for simultaneous separation of both high and low-molecular weight polypeptides. The method was applied to determine the relative molecular mass (Mr) of apoB. When apoB was isolated in strictly controlled environment its Mr has been found to be about 500-540 kilodaltons. The result is in a good accordance with the data on the amino acid sequence of mature apoB.