Analytical Polyacrylamide Gel Isoelectric Focusing Research Articles

Endo-(1→4)-β- d-glucanases from sclerotium rolfsii. Purification, substrate specificity, and mode of action

Three purified endo-(1→4)-β- d-glucanases (EC 3.2.1.4), A, B, and C, from Sclerotium rolfsii culture filtrates showed homogeneity in disc-gel electrophoresis and in analytical isoelectric-focusing in polyacrylamide gel. The three endo- d-glucanases are glycoproteins, endo B and endo C being composed of a single polypeptide chain, and endo A of two dissimilar polypeptide chains that are covalently bound by a disulfide bridge. Endo B and endo C do not contain half-cystine residues. With carboxymethylcellulose as substrate, the liquifying activity of the three enzymes was inhibited by cellobiose but not by d-glucose. The specificity of the enzymes is restricted to β-(1→4) linkages, but they showed some differences in the mode of attack on cellodextrins, phosphoric acid-swollen cellulose, and lichenan to give cellobiose, cellotriose, and small proportions of d-glucose. Endo B in addition showed endo- d-xylanase activity.

Purification and characterization of bovine placental lactogen.

Bovine placental lactogen (bPL) has been purified to homogeneity from bovine placental tissue. A radioreceptor assay (RRA), using a pregnant rabbit liver membrane fraction and human GH (hGH) as standard and tracer, was employed to monitor hormonal activity during the purification. The apparent molecular weight of bPL is approximately 30,000-32,000 as determined by analytical sodium dodecyl sulfate polyacrylamide gel electrophoresis. Somewhat larger estimates were obtained upon Sephadex gel filtration columns. The isoelectric point of the purified hormone as determined by analytical polyacrylamide gel isoelectric focusing is 5.5. The binding and displacement curves of radioiodinated bPL and hGH in the RRA for GH are superimposable, whereas the displacement curve of bPL in the RRA using the particulate fraction of a pregnant rat liver homogenate is parallel to the ovine PRL standards. Purified bPL is capable of modulating hGH-specific receptor sites in cultured human IM-9 lymphocytes. Stimulation of the growth of quiescent NB2 node lymphoma cells in culture demonstrates that bPL and bovine PRL are equipotent in this bioassay.

Identification of Ungulate Hemoglobins for Law Enforcement

Identification of Ungulate Hemoglobins for Law Enforcement

Comparison of early embryonic and differentiating cell surfaces: Interaction of lectins with plasma membrane components

1. 1. Cells of the unincubated as well as those of primitive streak chick blastoderm, which are preparing for or are involved in morphogenetic movements, are agglutinated by wheat germ agglutinin, Ricinus communis agglutinin and concanavalin A, but not by fucose-binding protein. 2. 2. Agglutination of these cells with soybean agglutinin occurs only after neuraminidase treatment, while that induced by concanavalin A, wheat germ and Ricinus communis agglutinins is not affected. 3. 3. Trypsin treatment of blastoderm cells had no effect on lectin-mediated agglutination. 4. 4. In contrast, cells derived from 10- and 12-day differentiating chick liver were agglutinated by wheat germ agglutinin only after trypsinization. 5. 5. Mechanically dissociated embryonic liver cells, which are not agglutinated, more 3H-labelled wheat germ agglutinin per cell than trypsinized cells, suggesting that during differentiation there may be a spatial reorganization of wheat germ agglutinin receptors within the plasma membrane. 6. 6. Membranes isolated from the above cell types were examined by analytical polyacrylamide gel isoelectric focusing and, in combination with affinity chromatography using wheat germ agglutinin conjugated to agarose, membrane material in the differentiating liver membrane, which binds to this lectin, was identified.

Studies of hyperimmune restricted and partially restricted anti-pneumococcal polysaccharide antibodies from allotype-defined pedigreed rabbits. I. Preparative liquid isoelectric focusing of the antibodies and characterization of the isolated fractions by electrophoretic techniques.

AbstractFour heterozygous rabbits (a2 a2, b4 b5; a1 a2, b6 b9; a2 a2, b5 b9; a3 a3, b4 b5) and one homozygous rabbit (a2 a2, b6 b6) were hyperimmunized with either type 3 or 8 pneumococcal vaccines. Four antisera were partially restricted and one antiserum was highly restricted. The immunoglobulins (Ig) were obtained by salt precipitation, ion‐exchange chromatography, and affinity chromatography. The total Ig, obtained by salt precipitation, were fractionated by preparative liquid isoelectric focusing (IEF) at various pH ranges. From each antiserum a number of fractions were obtained with significantly different isoelectric points. Many major fractions had reduced electrophoretic heterogeneity when compared with the Ig isolated before IEF. These fractions were tested for isoelectric purity by analytical IEF in polyacrylamide gels and by preparative refocusing over a narrow pH range. After complete reduction and alkylation, the light and heavy polypeptide chains of the focused antibody fractions were examined both by disc electrophoresis in alkaline urea gels and by IEF in polyacrylamide gels containing urea. From each antiserum, a number of fractions were obtained which yielded single light chains often with different electrophoretic mobilities. This method provided a successful means of preparatively obtaining different antibody fractions from a single antiserum.