Immobilized pH gradients (IPG)
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Hydrogen-Ion Concentration↗
Biomedical subjects
E Gianazza
Publications and source records attributed to E Gianazza.
Monitoring by two-dimensional electrophoresis somatic embryogenesis in leaf and petiole explants from Vitis.
A two-dimensional electrophoretic analysis of the total proteins was carried out in Vitis rupestris as model system in order to characterize the different developmental stages--from callus to plantlets--of somatic embryogenesis events in the grapevine. The patterns of callus, embryogenetic callus, somatic embryos and plantlets derived from leaf and petiole explants were compared. Each differentiation step was characterized by specific peptide spots.
Cells, Cultured↗
A homozygous missense arginine to histidine substitution at position 482 of the beta-galactosidase in an Italian infantile GM1-gangliosidosis patient.
We have studied, by the polymerase chain reaction, the beta-galactosidase cDNA from several Italian patients with infantile GM1-gangliosidosis. One homozygote for a previously undiscovered G > A mutation at position 1479, causing an arginine to histidine change, was detected. The same mutation, in heterozygosis, was identified in 6 unrelated patients, but not in 100 normal chromosomes.
Arginine↗
Biomedical relevance of two-dimensional protein mapping.
State-of-the-art and future perspectives are discussed for the application of two-dimensional protein maps to basic medical research and routine clinical chemistry problems. Despite the technical advances that allow effective processing of a large number of samples and the refinement of devices and procedures for image analysis, at present two-dimensional maps are mostly confined to research purposes, i.e. to the inventory of normal constituents of body fluids and tissues on the one hand, and to qualitative-quantitative alterations of some protein spots in a number of instances (genetic, degenerative, infectious or xenobiotic diseases) on the other. It is hoped that in some instances a single primarily affected component will be able to be identified and then specifically tested (for instance by immunological means) as a diagnostic marker, but complex pathological patterns would still require the analysis of a large number of peptides at the resolution level only afforded by two dimensions. Further simplification of the protocols, for example with ready-made gels, and data reduction systems might then allow the application of the technique to be extended to general clinical laboratories.
Genetic Diseases, Inborn↗
Isoelectric focusing in immobilized pH gradients: applications in clinical chemistry and forensic analysis.
The applications of isoelectric focusing in immobilized pH gradients in clinical chemistry and forensic analysis are reviewed. Strong emphasis is given to the separation of serum proteins, in particular alpha 1-acidic glycoprotein, acid phosphatase, alkaline phosphatase, alpha 1-antitrypsin, apolipoproteins, complement component, factor B, factor XIIIB, group-specific component, lecithin:cholesterol acyltransferase, phosphoglucomutase, prealbumin, protein C and transferrin. The analysis of human parotid salivary proteins is discussed and an assessment is given of the state of the art in thalassaemia screening.
Blood Proteins↗
Sodium dodecyl sulphate electrophoresis of urinary proteins.
The analysis of urinary proteins and their identification are discussed, particularly in regard to the technique of sodium dodecyl sulphate electrophoresis in polyacrylamide gradient gels. Urine collection, storage and preparation are evaluated, especially in regard to problems connected with concentration and dialysis of such samples. The instrumental approach to sodium dodecyl sulphate polyacrylamide gel electrophoresis represented by the Phast System appears to be particularly valuable in routine clinical analysis of urine specimens, since no sample pretreatment is required. The following types of proteinurias are evaluated: (a) orthostatic proteinurias; (b) post-renal proteinurias; (c) Bence-Jones proteinuria; (d) lower and upper urinary tract infection (cystitis and pyelonephritis) and (e) diabetes mellitus proteinurias.
Diabetes Mellitus↗
On the computational approach to immobilized pH gradients.
The unified treatment for computing the pH of complex mixtures of mono- and polyprotic buffers, including ampholytes, as utilized in the gradient simulation program PGS, is presented. Its ability to compute pH, buffering power and ionic strength is shown by discussing a few simulations. The problems arising in the automatic formulation of optimal mixtures are presented, as well as the merits and limits of several target functions utilized in such optimizations. It is shown that no universal target function exists and that a proper optimization method should account for the fact that more than one formulation is possible for a given pH range.
Buffers↗
Amidosulfobetaines, a family of detergents with improved solubilization properties: application for isoelectric focusing under denaturing conditions.
The synthesis of a family of new detergents and their use for isoelectric focusing are described. These detergents differ from the conventional sulfobetaines by the presence of an amido group bridging the hydrophobic linear tail and the polar head, which increases their water solubility and urea tolerance considerably. Four different linear alkyl hydrophobic tails were tested, together with three different polar heads. The solubilization of red blood cell ghosts by the 12 resulting detergent performances was evaluated both in solution and in isoelectric focusing runs. Six chemicals gave performances equal or better than those of conventional detergents and could replace them for general use in focusing experiments. In addition, the synthesis of a whole range of compounds allowed valuable insights in the empirical understanding of protein-detergent interactions, which are extensively discussed.
Amides↗
LAK1: a novel leucocyte differentiation antigen shared by lymphoid and endothelial cells.
Lymphokine activated killer (LAK) cells have been utilized as a useful tool in cancer adoptive immunotherapy. The lineage origin of this population has always been controversial since it shares phenotypic markers with both myelomonocytic cells and T lymphocytes. Recently we described a new monoclonal antibody (MoAb), termed LAK1, which recognizes a 120 Kd surface molecule expressed on human large granular lymphocytes (LGL) and LAK precursors and effectors. LAK1 MoAb defines two different populations of positive cells amongst peripheral lymphocytes: the first subset (20%), represented by brightly stained cells, belongs to the non T-LGL population, whereas the second subset (30%) displays low fluorescence intensity and was partially composed of T lymphocytes. More interestingly, LAK1 is shared by some other cell types, such as monocytes and vascular endothelial cells. Immunohistochemical staining performed on muscle, endometrium and lymphoid or other non lymphoid tissues shows that LAK1 antigen is selectively expressed by the reticuloendothelial system.
Antibodies, Monoclonal↗
Formulations for immobilized pH gradients including pH extremes.
Formulations are given both for narrow (less than 2 pH units) and for wide range (up to 8 pH units) immobilized pH gradients, spanning between pH 2.5 and pH 11. The contribution from water to the buffering power (beta) at these pH extremes requires the recipes to be optimized (in terms of gradient linearity) for each desired level of beta av.
Buffers↗
Identification of a new surface molecule expressed by human LGL and LAK cells production of a specific monoclonal antibody and comparison with other NK/LAK markers.
Recently we described a new monoclonal antibody, termed LAK1, which recognizes a 120-kDa surface antigen that is expressed on virtually all LGL and LAK precursors and effectors. In the present study we describe a second mAb, termed LAK2, which was derived against cloned LAK cells. The LAK2 mAb, similar to the LAK1 mAb reacts with a subset of peripheral blood lymphocytes which includes the precursors of LAK cells. In addition, among IL2-activated peripheral lymphocytes, this antibody defines cells displaying LAK activity. The expression of the LAK2 molecule on PBMC was analyzed by two-color cytofluorometric analysis in comparison with the expression of both T cell and LGL markers. We show that most resting LAK2+ cells lack surface expression of CD3, whereas nearly 60% express CD2 antigen. Moreover, all CD16+ and CD56 (NKH1)+ lymphocytes coexpressed both LAK2 and LAK1 antigens. Morphological analysis of LAK2+ lymphocytes indicated that the majority of these cells was represented by LGL. Thus the expression of the LAK2 molecule on LGL-enriched populations was compared by two-color cytofluorometric analysis to that of other known LGL markers such as CD16, CD57 (HNK1), and LAK1. Most LGL coexpressed LAK1, LAK2, CD16 and CD57 antigens Finally, the surface molecule recognized by LAK2 mAb is composed of two chains with apparent molecular masses of approximately 110 and 140 kDa.
Animals↗
Ion retention by immobilized pH matrices.
The effect of the interaction between polyacrylamide matrices containing covalently bound acidic and basic residues and free ions was indirectly evaluated from the width of the residual salt fronts after electrophoresis under standard conditions. Around neutrality the front width is larger than at alkaline, and much larger than at acidic pH, and in all instances much higher for anions than for cations. The width decreases faster with the buffering power of the matrix than with its ionic strength. After about 5000 V x h, the migration of the salt front is very slow, and becomes negligible when diffusion is prevented. When inadequately buffered by the matrix, H+ and OH(-)--comigrating with anions and cations--increase the conductivity within the salt front and drastically lower the electrical field strength.
Acrylamides↗
Properties of thin-rod immobilized pH gradients.
Immobilized pH gradient gel rods, 1.5 mm in diameter, were cast with a manifold connected to high-precision burettes. The reproducibility of gel length was ca. 1.7 mm. The average standard deviation sigma x for spot position was 2 mm after one-dimensional and 5.8 mm after two-dimensional runs. In order to bring to completion the elution of the salt fronts into the electrode compartments, carrier ampholytes had to be included in the gel formulation at concentrations of at least 0.5-1%, depending on the pH range. The presence of carrier ampholytes, however, was troublesome in two respects: the gel tended to shrink and the cathodic bands drifted with time. Ionic components in the sample were tolerated up to the following concentrations: NaCl 8 mumoles, sodium dodecyl sulfate 10 micrograms per tube. In presence of non-ionic detergents, the gels moved as a whole towards the cathode.
Ampholyte Mixtures↗
The use of dye-ligand affinity chromatography for the purification of non-enzymatic human plasma proteins.
Literature data are analysed in this review on the use of immobilized triazine dyes for the characterization, isolation and purification of non-enzymatic human plasma proteins in both conventional and high-pressure liquid chromatography systems. Attention is focused on the mode of interaction between the dyes and these proteins, as well as on the advantages over previously reported techniques. Future developments are discussed.
Blood Proteins↗
Immobilized pH gradients.
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Hydrogen-Ion Concentration↗
Isoelectric focusing in immobilized pH gradients: theory and newer methodology.
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Hydrogen-Ion Concentration↗
Additives for immobilized pH gradient two-dimensional separation of particulate material: comparison between commercial and new synthetic detergents.
We describe the synthesis of two detergents, L and A15, whose performances as solubilizing agents and as additives in the first-dimension step of a two-dimensional separation are compared with those of some commercial compounds, i.e., Nonidet P-40, 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate(Chaps), and sulfobetaine, on three membrane protein preparations: rat RBC ghosts, beef kidney microvilli, and spinach thylakoids. L is 3-]3-dodecylamidoprophylcbdimethylammonio propane-1-sulfonate; owing to the substitution of a dodecylamido for the dodecyl residue of SB 3-12, the concentration of urea compatible with 2% detergent increases from 4.5 M for the parent molecule up to 7 M. With all three biological samples on which the panel of different detergents has been tested in parallel, L + urea scores as the most effective solubilization medium. On red blood cells a notable qualitative difference is observed with the selective extraction by L as well as by N-dodecyl-N,N-dimethylammonio-3-propanesulfonate of a major protein (pI = ca. 5.5, Mr = ca. 100,000). A15 is derived from a tertiary amine, with one alkylic substituent (either C11 or C13) and two poly(ethylene oxide) tails (totaling 15 ethoxy residues), which is reacted with propane sultone. Approximately 30% of the product corresponds to the N-adduct and is a truly zwitterionic detergent, while 60% is an O-derivative and still contains a titratable amino group (with a pK of 7.2). A15 can thus be used for isoelectric focusing on immobilized pH gradients, as in this work, but would not be compatible with carrier ampholyte isoelectric focusing.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗