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At least 37 records · Page 2

Computer simulation of two electrophoretic columns coupled for isoelectric focusing in simple buffers

Computer simulation is used to analyze a system of two electrophoretic columns coupled by mixing the anolyte of one with the catholyte of the other. A mathematical model is presented which is used to predict the pH gradients formed by monovalent buffers in this system, when the currents in the columns are unequal. In the column with the higher current a pH gradient is created which increases from anode to cathode and is potentially useful for isoelectric focusing. The breadth of this gradient is dependent upon the ratio of the currents. The function of the second column is the compensation of buffer migration which occurs in the first column, thereby maintaining constant electrolyte composition. The effects of buffer pKs and mobilities are evaluated.

Tsai, Amos↗

Isoelectric focusing of proteins and peptides

Egg-white solution was chosen as the reference solution in order to assess the effects of operational parameters (voltage, flow rate, ampholine pH range and concentration, and protein concentration) of the RIEF apparatus on protein resolution. Topics of discussion include: (1) comparison of RIEF apparatus to conventional IEF techniques (column and PAG) with respect to resolution and throughput; (2) peptide and protein separation (AHF, Thymosin - Fraction 5, vasoactive peptide, L-asparaginase and ACP); and (3) detection of peptides - dansyl derivatives of amino acids and peptides, post-focusing fluorescent labeling of amino acids, peptides and proteins, and ampholine extraction from focused gels.

Egen, N.↗

Isoelectric focusing of red blood cells in a density gradient stabilized column

The effects of Ficoll and cell application pH on red blood cell electrophoretic mobility and focusing pH were investigated by focusing cells in a density gradient stabilized column. Sample loading, cell dispersion, column conductivity, resolution of separation, and the effect of Ampholines were examined.

Smolka, A. J. K.↗

Isoelectric focusing of dansylated amino acids in immobilized pH gradients

The 21 free amino acids commonly encountered in proteins have been transformed into 'carrier ampholyte' species by reacting their primary amino groups with dansyl chloride. These derivatives can thus be focused in an immobilized pH gradient covering the pH interval 3.1 to 4.1, except for arginine, which still retains a pI of 8.8. Due to their inherent fluorescence, the dansyl derivatives are revealed in UV light, with a sensitivity of the order of 2-4 ng/sq mm. All nearest neighbors are separated except for the following couples: Asn-Gln, Gly-Thr, Val-Ile and Cys-Cys2, with a resolving power, in a Delta(pI) scale, of the order of 0.0018 pH units. Except for a few cases (notably the aromatic amino acids), the order of pI values is well correlated with the pK values of carboxyl groups, suggesting that the latter are not altered by dansylation. From the set of pK(COOH)-pI values of the different amino acids, the pK of the tertiary amino group in the dansyl label has been calculated to be 5.11 + or - 0.06. Knowing the pK of the amino-dansyl and the pI of the excess, free dansyl label (pI = 3.34), a pK of 1.57 is derived for its sulfonic acid group.

Bianchi-Bosisio, Adriana↗

Hormone purification by isoelectric focusing in space

The objective of the program was the definition and development of optimal methods for electrophoretic separations in microgravity. The approach is based on a triad consisting of ground based experiments, mathematical modeling and experiments in microgravity. Zone electrophoresis is a rate process, where separation is achieved in uniform buffers on the basis of differences in electrophoretic mobilities. Optimization and modeling of continuous flow electrophoresis mainly concern the hydrodynamics of the flow process, including gravity dependent fluid convection due to density gradients and gravity independent electroosmosis. Optimization of focusing requires a more complex model describing the molecular transport processes involved in electrophoresis of interacting systems. Three different focusing instruments were designed, embodying novel principles of fluid stabilization. Fluid stability was achieved by: (1) flow streamlining by means of membrane elements in combination with rapid fluid recycling; (2) apparatus rotation in combination with said membrane elements; and (3) shear stress induced by rapid recycling through a narrow gap channel.

Bier, M.↗

The onset of electrohydrodynamic instability in isoelectric focusing

The onset of electrohydrodynamic motion associated with the imposition of an electric field across a thin layer of liquid has been investigated for the case in which the electrical conductivity varies linearly over the depth of the layer. The variation of the conductivity is due to concentration gradients in the charge-carrying solutes and its spatio-temporal evolution is represented by a convective-diffusion equation. When the viscous relaxation time is long compared to the time for charge relaxation, the analysis reveals that the neutral stability curves for the layer can be characterized by three dimensionless parameters: Ra(sub e) is equivalent to d(epsilon)E(sup 2, sub 0) Delta sigma/mu K(sub eff) sigma(sub 0), an electrical Rayleigh number; delta sigma/sigma(sub 0), the relative conductivity increment; and alpha, the transverse wave number of the disturbance. Here d is the thickness, epsilon is the dielectric constant, and mu is the viscosity of layer, E(sub 0) is the applied field strength at the lower conductivity boundary, and K(sub eff) is an effective diffusivity associated with the Brownian motion of the charge-carrying solutes. With viscous-stress-free boundaries, at which the electrical conductivity and the normal component of the electric field are prescribed, the critical Ra(sub e) is 1.504 x 10(exp 4) at a critical transverse wave number of 1.97 when Delta sigma/sigma(sub 0) is 10. As Delta sigma/sigma(sub 0) increases, the critical Ra(sub e) increases and shifts to shorter wavelength disturbances; the critical imposed field strength, however, passes through a minimum because the lower-conductivity boundary exerts a considerable stabilizing influence in the presence of steep conductivity gradients. Similar trends were obtained for liquid layers with rigid boundaries.

Baygents, James C.↗

Isoelectric focusing-affinity immunoblot analysis of mouse monoclonal antibodies to the four human IgG subclasses

Isoelectric focusing (IEF)/affinity immunoblotting and enzyme-linked immunosorbent assay (ELISA) were used for parallel analysis of murine monoclonal antihuman IgG-subclass antisera (MoAbs). Coomassie Blue-stained protein bands in the pH region 5.5-8.0 were shown to be murine IgG by direct blotting onto nitrocellulose followed by detection with conjugated antimouse IgG. Use of IgG myeloma antigen-coated nitrocellulose in the IEF-affinity immunoblot allowed detection of the charge microheterogeneity of MoAbs. The MoAb group contained one to five major dense bands flanked by up to four minor fainter bands, all with pIs ranging from 6.1 to 7.8. Semiquantitative estimates of binding specificity in the IEF-affinity blot compared well with cross-reactivity data obtained from a quantitative ELISA.

Hamilton, Robert G.↗

Rapid Protein Separations in Microfluidic Devices

This paper describes fabrication of glass and plastic microfluidic devices for protein separations. Although the long-term goal is to develop a microfluidic device for two-dimensional gel electrophoresis, this paper focuses on the first dimension-isoelectric focusing (IEF). A laser-induced fluorescence (LIF) imaging system has been built for imaging an entire channel in an IEF device. The whole-channel imaging eliminates the need to migrate focused protein bands, which is required if a single-point detector is used. Using the devices and the imaging system, we are able to perform IEF separations of proteins within minutes rather than hours in traditional bench-top instruments.

MICROFLUIDICS↗

Recent advances in preparative electrophoresis

Various approaches for preparative electrophoresis, and three new instruments for preparative electrophoresis are discussed. Consideration is given to isoelectric focusing, isotachophoresis, and zone electrophoresis, three gel-based electrophoresis methods. The design, functions, and performance of the Elphor VaP 21 device of Hannig (1982), the shear-stabilized BIOSTREAM separator of Thompson (1983), and the recycling isoelectric focusing device are described.

Mosher, Richard A.↗

Isoelectric leukocyte focusing

Modified electrophoretic separation procedure yields 70 to 80 percent visible cells for small-scale immunological and clinical profiling. All reagents and preparations used in separation and characterization procedures are commercially available.

Leise, E. M.↗

Phosphorylation of the budgerigar fledgling disease virus major capsid protein VP1

The structural proteins of the budgerigar fledgling disease virus, the first known nonmammalian polyomavirus, were analyzed by isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The major capsid protein VP1 was found to be composed of at least five distinct species having isoelectric points ranging from pH 6.45 to 5.85. By analogy with the murine polyomavirus, these species apparently result from different modifications of an initial translation product. Primary chicken embryo cells were infected in the presence of 32Pi to determine whether the virus structural proteins were modified by phosphorylation. SDS-PAGE of the purified virus structural proteins demonstrated that VP1 (along with both minor capsid proteins) was phosphorylated. Two-dimensional analysis of the radiolabeled virus showed phosphorylation of only the two most acidic isoelectric species of VP1, indicating that this posttranslational modification contributes to VP1 species heterogeneity. Phosphoamino acid analysis of 32P-labeled VP1 revealed that phosphoserine is the only phosphoamino acid present in the VP1 protein.

Non-NASA Center↗

Comparison of bioseparation methods for microgravity experiments

The efficiency of the 1-g version of the continuous-flow electrophoresis (CFE) system flown on Space Shuttle missions is compared with the efficiency of a commercial CFE for separating living cells (human kidney, liver, and pituitary-gland cells and T-lymphocytes). In addition, the CFE system and a reciprocal isoelectric focusing (RIEF) system are compared with respect to protein pyrification efficiency. Correlations were made among electrophoretic mobilities (EPMs), secretory functions of cells, and input sample concentrations. A significant reduction in mean and range EPM was observed when input sample concentrations exceeded a low threshohold. This effect was not observed in microgravity experiments conducted at sample concentrations three times greater than the threshold for the controls. Comparison of CFE and RIEF methods showed that there are apparent advantages for each method depending on the product. For example, RIEF purification of urokinase removed more protein impurities, but focused the enzyme at a pH different than the enzyme's known isoelectric point.

Morrison, Dennis R.↗

Characterization of synthetic carrier ampholytes by repetitive scanning of the electric field during focusing

This paper reports the utilization of a potential gradient array detector for monitoring the dynamics of the electric field during isoelectric focusing. Transient and steady state electric field profiles are presented for synthetic carrier ampholyte mixtures with a wide (about 3-10) pH range. Two available commercial products (Ampholine and Pharmalyte) and a laboratory synthesized mixture (PEHA ampholytes) are compared. The formation of conductivity gaps and their migration toward the cathode in extended experiments (cathodic drift) can be visualized with this system.

Thormann, W.↗

Partial purification and characterization of a Ca(2+)-dependent protein kinase from the green alga, Dunaliella salina

A calcium-dependent protein kinase was partially purified and characterized from the green alga Dunaliella salina. The enzyme was activated at free Ca2+ concentrations above 10(-7) molar. and half-maximal activation was at about 3 x 10(-7) molar. The optimum pH for its Ca(2+)-dependent activity was 7.5. The addition of various phospholipids and diolein had no effects on enzyme activity and did not alter the sensitivity of the enzyme toward Ca2+. The enzyme was inhibited by calmodulin antagonists, N-(6-aminohexyl)-1-naphthalene sulfonamide and N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide in a dose-dependent manner while the protein kinase C inhibitor, sphingosine, had little effect on enzyme activity up to 800 micromolar. Immunoassay showed some calmodulin was present in the kinase preparations. However, it is unlikely the kinase was calmodulin regulated, since it still showed stimulation by Ca2+ in gel assays after being electrophoretically separated from calmodulin by two different methods. This gel method of detection of the enzyme indicated that a protein band with an apparent molecular weight of 40,000 showed protein kinase activity at each one of the several steps in the purification procedure. Gel assay analysis also showed that after native gel isoelectric focusing the partially purified kinase preparations had two bands with calcium-dependent activity, at isoelectric points 6.7 and 7.1. By molecular weight, by isoelectric point, and by a comparative immunoassay, the Dunaliella kinase appears to differ from at least some of the calcium-dependent, but calmodulin and phospholipid independent kinases described from higher plants.

Non-NASA Center↗

Electrophoresis in space at zero gravity

Early planning for manufacturing operations in space include the use of electrophoresis for purification and separation of biological materials. Greatly simplified electrophoresis apparatus have been flown in the Apollo 14 and 16 missions to test the possibility of stable liquid systems in orbit. Additionally, isoelectric focusing and isotachophoresis are of particular interest as they offer very high resolution and have self-sharpening boundaries. The value of possible space electrophoresis is substantial. For example, present technology permits large fractionation of only a few of blood proteins many fractions, and separated cell populations are needed for research.

Bier, M.↗

Differential electrophoretic separation of cells and its effect on cell viability

An electrophoretic separation method was applied to the separation of cells. To determine the efficiency of the separation, it was necessary to apply existing methodology and develop new methods to assess the characteristics and functions of the separated subpopulations. Through appropriate application of the widely used isoelectric focusing procedure, a reproducible separation method was developed. Cells accumulated at defined pH and 70-80% remained viable. The cells were suitable for further biologic, biochemical and immunologic studies.

Leise, E. M.↗

Electrophoresis of biological materials

The selection of biological products was studied for electrophoresis in space. Free flow electrophoresis, isoelectric focusing, and isotachophoresis are described. The candidates discussed include: immunoglobulins and gamma globulins; isolated islet of langerhans from pancreas; bone marrow; tumor cells; kidney cells, cryoprecipitate; and column separated cultures.

Source record↗

Electrophoretic separation of proteins in space

Commercially available and synthetic wide range and short range ampholytes used in the isoelectric focusing of proteins was analyzed by ion exchange chromatography. A pH gradient over the pH range 3.8 to 11.0 was used to elute the ampholytes from a column of a sulfonated polystyrene resin. The wide range ampholytes were resolved into some 60 to 70 ninhydrin positive components. The recovery obtained with the method was quantitative. Acid short range ampholytes have approximately 35 components which elute readily from the ion exchange resin. Basic short range ampholytes gave about 50 components, most of which eluted at alkaline pH.

Brown, R. K.↗