A new method for the preparation of solid-phase immunoadsorbents

A solid-phase immunoadsorbent was prepared by insolubilization of antibody during precipitation with Na₂SO₄. The polyaldehyde macromolecule created by periodate oxidation of dextran (macrofixative) served as the insolubilizing, crosslinking agent. After appropriate fixation, the precipitate was stable to removal of the precipitating salt, to washing, and, to a large extent, to heating in the denaturing detergent, sodium dodecyl sulfate. Under proper conditions, the precipitate retained satisfactory antibody activity, although very high molecular weight antigens were apparently excluded from the internal active sites of the solid-phase matrix. This method provides the advantage of insolubilization of the primary antibody in a small volume; for analytical work, the entire precipitate, with bound antigen, may be quantitatively applied to a polyacrylamide gel (tube or slab) and electrophoresed without overloading by the binding antibody. This method might also be extended for use in immunoisolation procedures employing standard eluting agents, as well as insolubilization of proteins other than immunoglobulin.     

Comparative studies of the preparation of alpha-gliadin]

alpha-gliadin was prepared from wheat flour by two different methods. The products were compared electrophoretically and by double radial immuno-diffusion. The alpha-gliadin fraction proved to be identical in the immunological test. Only the alpha-gliadin preparation received by ion exchange chromatography is suitable for further purification by multiple gel filtrations.     

Spin-labelling studies of the agarose gelling-system

Rates of tumbling and exchange of free nitroxides in aqueous solution are unaffected by the presence of quite high concentrations of agarose gel. Low-level, covalent attachment of labels to the polysaccharide by means of stable, acetamido ether linkages, however, causes considerable diminution in their rate of reorientation. Dissolution of this material to form a gel and melting and setting of the gel cause further changes in the e.s.r. spectrum. Experimental correlation-times for reorientation of label may be decomposed into contributions from rotation about bonds in the linking group and from polysaccharide motions. This allows information to be obtained about the microscopic characteristics of the gel state, which is found to vary greatly depending on its history.     

Nonimmunospecific protein-protein interactions of IgG: studies of the binding of IgG to IgG immunoadsorbents.

Nonimmunospecific interactions of IgG and IgG-agarose columns were systematically studied under varying conditions. Nonimmunospecific binding to the columns was primarily due to protein-protein interactions. These nonimmunospecific protein-protein interactions of IgG were enhanced with heat-induced or chemical aggregation of IgG, low pH, low ionic strength (at pH above 4), or low temperature. Conversely, this binding was decreased with proteolytic fragmentation of IgG, high ionic strength (at pH above 4), or temperatures above 4 degrees C. Chemical modification of IgG by acetylation, formalinization, carbamylation, or reaction with 1,2-cyclohexanedione significantly decreased these interactions. These observations suggest that above pH 4, ionic interactions caused the protein-protein binding. Below pH 4, hydrophobic interactions presumably play a major role. These results permit the development of rational methodology for avoiding nonimmunospecific protein-protein interactions in immunologic procedures for detection, isolation, or quantification of rheumatoid factors and other antibodies to IgG.     

Comparative studies of four protein preparation methods for proteomic study of the dinoflagellate Alexandrium sp. using two-dimensional electrophoresis

Alexandrium is a wide-spread genus of dinoflagellate causing harmful algal blooms and paralytic shellfish poisoning around the world. Proteomics has been introduced to the study of Alexandrium, but the protein preparation method is still unsatisfactory with respect to protein spot number, separation and resolution, and this has limited the application of a proteomic approach to the study of dinoflagellates. In this study we compared four protein preparation methods for the two-dimensional electrophoresis (2DE) analysis of A. tamarense: (1) urea/Triton X-100 buffer extraction with trichloroacetic acid (TCA)/acetone precipitation; (2) direct precipitation with TCA/acetone; (3) 40 mM Tris (hydroxymethyl) aminomethane (Tris) buffer extraction; and (4) 50 mM Tris/5% glycerol buffer extraction. The results showed that, among the four protein preparation methods, the method combining the urea/Triton X-100 buffer extraction and TCA/acetone precipitation allowed detection of the highest number and quality of protein spots with a clear background. Although the direct TCA/acetone precipitation method also detected a high number of protein spots with a clear background, the spot number, separation and intensity were not as good as those obtained from the urea/Triton X-100 buffer extraction with TCA/acetone precipitation method. The 40 mM Tris buffer and 50 mM Tris/5% glycerol buffer methods allowed the detection of fewer protein spots and a pH range only from 4 to 7. Subsequently, the urea/Triton X-100 buffer extraction with TCA/acetone precipitation method was successfully applied to profiling protein expression in A. catenella under light stress conditions and the differential expression proteins were identified using MALDI TOF–TOF mass spectrometry. The method developed here appears to be promising for further proteomic studies of this organism and related species.     

Steady-state and pulsed NMR studies of gelation in aqueous agarose

Steady-state and pulsed NMR techniques have been used to investigate molecular motion in sols and gels of agarose. In passing through the sol–gel transition, the molecular mobility of water molecules is reduced only by a small amount, whereas motion of the polymer chains is greatly attenuated. The results are discused in terms of the network theory of gelation, with references to the role of water in the process and the nature of the “junction zones” between polymer chains. T₂ and line-width measurements are dominated by exchange broadening. The effects of exchange rate and differences in relaxation time between the exchanging sites are discussed. The temperature hysteresis behavior of agarose gels has been investigated and the effects of “ageing” correlated with changes in nuclear relaxation times. The synergistic increase in gel strength obtained on adding locust bean gum (LBG) to agarose has been investigated. The results indicate that LBG does not form double-helix junctions and may decrease rates of gelation by steric effects. At high agarose concentration, the LBG remains mainly in solution in interstitial water, but at low agarose concentration, it is suggested that the LBG can link gel aggregates together into a self-supporting structure, producing a synergistic increase in gel strength. Comparisons have been made between the nature of the agarose–LBG interaction and agarose–cellulose interactions in biological systems.     

Use of the intact mouse skeletal-muscle preparation for metabolic studies. Evaluation of the model.

1. We examined the isolated mouse skeletal-muscle model in vitro, commonly used by many investigators, for its suitability for metabolic studies. 2. Despite the fact that pH, O2 saturation, osmolality and the release of the enzyme creatine kinase remained stable, histochemical studies showed large cores devoid of glycogen, suggesting that the incubated muscle had lost its viability. 3. This study indicates that caution should be exercised when interpreting the results of studies with intact isolated mouse muscles.     

Comparative studies of purinergic nerves.

Purinergic nerves supply the gastrointestinal tract of vertebrates, including fish, amphibians, reptiles and birds, as well as mammals. Their cell bodies are located in Auerbach's plexus and their axons extend in an anal direction before innervating mainly the circular muscle coat. In the stomach they are controlled by preganglionic cholinergic fibres of parasympathetic origin. They are involved in "receptive relaxation" of the stomach, "descending inhibition" in peristalsis and reflex relaxation of oesophageal and internal anal sphincters. The terminal varicosities of purinergic nerves are characterised by a predominance of "large opaque vesicles," which can be distinguished from the "large granular vesicles" found in small numbers in both adrenergic and cholinergic nerves. Stimulation of purinergic nerves with single pulses produces hyperpolarisations of up to 25 mV (inhibitory junction potentials) in smooth muscle cells. These potentials are unaffected by atropine, adrenergic neuron blocking agents or sympathetic denervation, but are abolished by tetrodotoxin. The "rebound contraction" which characteristically follows cessation of purinergic nerve stimulation is probably due to prostaglandin. Evidence that ATP is the transmitter released from purinergic nerves includes: (1) synthesis and storage of ATP in nerves; (2) release of ATP from the nerves when they are stimulated; (3) exogenously applied ATP mimicking the action of nerve-released transmitter, both producing a specific increase in K+ conductance; (4) the presence of Mg-activated ATPase, 5'-nucleotidase and adenosine deaminase, enzymes which inactivate ATP; (5) drugs (including quinidine, some 2-substituted imidazolines, 2-2'pyridylisatogen and dipyridamole) which produce similar blocking or potentiating effects on the response to exogenously applied ATP and nerve stimulation. Speculations are made about the evolution and development of the nervous system, including the possibility that purinergic nerves are a primitive nerve type.     

Successful preparation and characterization of biotechnological grade agarose from indigenous Gelidium amansii of Taiwan

This paper reports the first successful preparation of biotechnological grade agarose from Gelidium amansii found in Taiwan. The scale-efficiency preparation was achieved by shortening EDTA treatment time through dispersing G. amansii agar in water in the presence of heat and EDTA, removing agaropectin impurity with a heat-compatible and strong-anion exchange resin, and precipitating agarose with a cost-effective isopropanol method. The yield of agarose from prepared G. amansii agar was 11.3%. The acquired agarose has a gel strength of 853 g cm^?2, a sulfate content of 0.14%, a pyruvate content of 1.03%, a degree of electroendosmosis of 0.16 and very limited binding affinity to DNA. The excellent properties of agarose from G. amansii of Taiwan confirm its potential diverse biotechnological applications. This innovative agarose preparation method with the significantly improved scale-efficiency can be modified for large-scale preparation of agarose for use in biotechnological industry and biochemical research.     

Ultrastructure of beaded agarose.

Beaded agarose was dehydrated and embedded in Epon by a procedure that preserves the size, shape and, most likely, the native ultrastructure of the beads. Thin sections of the embedded beads reveal under the electron microscope a network sponge-like structure, uniform throughout the bead. The matrix skeleton is fairly rigid, though it occupies only a small percentage of the bead volume. This skeleton is composed of thin filaments (~20 Å in diameter) bundled in a side-by-side assembly. The pores or channels between the filament bundles vary in shape and diameter (up to 0.3 μm). This structure accounts for some of the known physicochemical properties of beaded agarose.     

A method for the recovery of DNA from agarose gels.

We describe a quick and versatile method for the isolation of DNA from agarose gels. The DNA is electrophoresed into a trough containing hydroxyapatite, where it is bound. The hydroxyapatite is taken out and the DNA eluted with phosphate buffer. By putting the hydroxyapatite on a small column of Sephadex G50, elution and subsequent removal of phosphate can be performed in one step. The DNA recovered can be used equally well in enzymatic incubations as DNA not purified through agarose gel electrophoresis. Several applications of this technique are described.