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At least 19 records
All sourcesNASA NTRSDOE OSTI
NASA NTRS1986

An explanation for the plateau phenomenon in isoelectric focusing

Isoelectric focusing of mixtures of simple ampholytes occurs in two phases, an initial rapid separation phase and a second relatively slow stabilizing phase. Transient and steady-state computer simulation data are shown to predict the development of pH plateaus around neutrality during the stabilizing phase of the focusing of such mixtures. This occurs because a nonzero electrophoretic flux is present in a pure zone of focused ampholyte, which is a function of both its isolectric point (pI) and its Delta-pK value. For an ampholyte with a pI greater than 9 or less than 5, this flux causes the development of a significant concentration gradient within its focused zone which is accompanied by a contraction of this zone along the focusing axis. Acidic and basic ampholytes are thereby displayed toward the anode and cathode respectively, creating a pH plateau in the neutral region. Thus, there will be regions of the focusing column, closer to the electrodes and containing more acidic or basic pH values, within which the resolution of samples will reach a maximum and then decrease.

Mosher, Richard A.↗
NASA NTRS1982

Hormone purification by isoelectric focusing in space

The performance of a ground-prototype of an apparatus for recycling isoelectric focusing was evaluated in an effort to provide technology for large scale purification of peptide hormones, proteins, and other biologicals. Special emphasis was given to the effects of gravity on the function of the apparatus and to the determination of potential advantages deriveable from its use in a microgravity environment. A theoretical model of isoelectric focusing sing chemically defined buffer systems for the establishment of the pH gradients was developed. The model was transformed to a form suitable for computer simulations and was used extensively for the design of experimental buffers.

Bier, M.↗
NASA NTRS1979

Recycling isoelectric focusing with computer controlled data acquisition system

Isoelectric focusing is a high-resolution technique for separating and purifying large peptides, proteins, and other biomolecules. The apparatus described in the present paper constitutes a new approach to fluid stabilization and increased throughput. Stabilization is achieved by flowing the process fluid uniformly through an array of closely spaced filter elements oriented parallel both to the electrodes and the direction of the flow. This seems to overcome the major difficulties of parabolic flow and electroosmosis at the walls, while limiting the convection to chamber compartments defined by adjacent spacers. Increased throughput is achieved by recirculating the process fluid through external heat exchange reservoirs, where the Joule heat is dissipated.

Egen, N. B.↗
NASA NTRS1986

Rotating Apparatus for Isoelectric Focusing

Remixing of separated fractions prevented. Improved isoelectric focusing apparatus helps to prevent electro-osmosis and convection, both of which cause remixing of separated fractions. Fractionating column segmented and rotated about horizontal axis: Only combined effects of both features fully effective in making good separations. Improved apparatus slowly rotated continuously or rocked (at rotational amplitude of at least 180 degrees) about its horizontal axis so average gravitational vector experienced by fluid is zero and convection is therefore suppressed. Electro-osmosis suppressed and convection further suppressed by separating column into disklike compartments along its length with filters. Experiments have shown dimensions of apparatus not critical. Typical compartment and column volumes are 2 and 40 ml, respectively. Rotation speeds lie between 3 and 30 rpm.

Bier, M.↗
NASA NTRS1986

Rotating apparatus for isoelectric focusing

This disclosure is directed to an isoelectric focusing apparatus, wherein stabilization of the fluid containing the isolated proteins is achieved by carrying out the separation in a rotating cylinder with the separation cavity of the cylinder being segmented by means of filter elements. The filter elements are constituted of a material offering some degree of resistance to fluid convection, but allowing relatively free and unhindered passage of current and transport of proteins. The combined effect of segmentation and rotation has been found to be superior to either segmentation or rotation alone in maintaining the stability of the migrated fractions.

Bier, Milan↗
NASA NTRS1987

Quality control of murine monoclonal antibodies using isoelectric focusing affinity immunoblot analysis

The quality control of murine hybridoma secretory products has been performed using two approaches for isoelectric focusing affinity immunoblot analysis: (1) a method in which antigen-coated nitrocellulose is placed on top of an acrylamide gel containing isoelectrically focused ascites to bind the antigen specific monoclonal antibody; and (2) a method in which focused ascite proteins were passively blotted onto nitrocellulose and specific monoclonal antibodies were detected with enzyme-conjugated antigen. Analysis by both methods of batches of ascites containing antihuman IgG antibodies that were produced by six hybridomas permitted effective monitoring of immunoreactive antibodies for pI microheterogeneity.

Hamilton, Robert G.↗
NASA NTRS1981

Mathematical modeling and computer simulation of isoelectric focusing with electrochemically defined ampholytes

A mathematical model of isoelectric focusing at the steady state has been developed for an M-component system of electrochemically defined ampholytes. The model is formulated from fundamental principles describing the components' chemical equilibria, mass transfer resulting from diffusion and electromigration, and electroneutrality. The model consists of ordinary differential equations coupled with a system of algebraic equations. The model is implemented on a digital computer using FORTRAN-based simulation software. Computer simulation data are presented for several two-component systems showing the effects of varying the isoelectric points and dissociation constants of the constituents.

Palusinski, O. A.↗
NASA NTRS1986

Experimental and theoretical dynamics of isoelectric focusing - Elucidation of a general separation mechanism

The transient states in isoelectric focusing were monitored using a potential gradient array detector. Electric field profiles are presented which show the formation of transient moving boundaries, as well as the approach of the steady-state distribution, during the focusing of two- and three-component systems. The results are completely consistent with corresponding computer-simulation data. The focusing process is comprised of two sequential phases, a relatively rapid separation phase and a much slower stabilizing phase. It was found that the second phase can last as much as 20 times longer than the separation of the constituents.

Thormann, W.↗
NASA NTRS1982

Theoretical studies in isoelectric focusing

A mathematical model has been developed which describes the steady state in an isoelectric focusing (IEF) system with ampholytes or monovalent buffers. The model is based on the fundamental equations describing the component dissociation equilibria, mass transport due to diffusion and electromigration, electroneutrality, and the conservation of charge. The validity and usefulness of the model has been confirmed by using it to formulate buffer systems in actual laboratory experiments. The model has been recently extended to include the evolution of transient states not only in IEF but also in other modes of electrophoresis.

Mosher, R. A.↗
NASA NTRS1986

Scale-up of isoelectric focusing

The paper describes some applications to large scale protein fractionation using a recycling isoelectric focusing apparatus. Separation is achieved in free solution without the use of supporting media. Various alternatives for the formation of the pH gradient are discussed and results of a computer simulation are presented.

Bier, Milan↗
NASA NTRS1981

Computer simulation and experimental validation of isoelectric focusing in ampholine-free systems

An abbreviated version of a mathematical model of the steady state in isoelectric focusing is presented. Details of the mathematical transformations leading to a model suitable for computer implementation and numerical solution are given in the Appendix. The model describes the structure of concentration, pH, conductivity and potential gradients arising from the focusing of electrochemically defined ampholytes. Some of the results of computer simulations of two and three component systems are of particular interest to the experimentalist and are presented together with experimental validation of this model.

Bier, M.↗
NASA NTRS1985

The formation of stable pH gradients with weak monovalent buffers for isoelectric focusing in free solution

Two methods which utilize simple buffers for the generation of stable pH gradients (useful for preparative isoelectric focusing) are compared and contrasted. The first employs preformed gradients comprised of two simple buffers in density-stabilized free solution. The second method utilizes neutral membranes to isolate electrolyte reservoirs of constant composition from the separation column. It is shown by computer simulation that steady-state gradients can be formed at any pH range with any number of components in such a system.

Mosher, Richard A.↗
NASA NTRS1982

Isoelectric focusing in space

The potential of space electrophoresis is conditioned by the fact that all electrophoretic techniques require the suppression of gravity-caused convection. Isoelectric focusing (IEF) is a powerful variant of electrophoresis, in which amphoteric substances are separated in a pH gradient according to their isoelectric points. A new apparatus for large scale IEF, utilizing a recycling principle, has been developed. In the ground-based prototype, laminar flow is provided by a series of parallel filter elements. The operation of the apparatus is monitored by an automated array of pH and ultraviolet absorption sensors under control of a desk-top computer. The apparatus has proven to be useful for the purification of a variety of enzymes, snake venom proteins, peptide hormones, and other biologicals, including interferon produced by genetic engineering techniques. In planning for a possible space apparatus, a crucial question regarding electroosmosis needs to be addressed To solve this problem, simple focusing test modules are planned for inclusion in an early Shuttle flight.

Bier, M.↗
NASA NTRS1985

Hormone Purification by Isoelectric Focusing

Various ground-based research approaches are being applied to a more definitive evaluation of the natures and degrees of electroosmosis effects on the separation capabilities of the Isoelectric Focusing (IEF) process. A primary instrumental system for this work involves rotationally stabilized, horizontal electrophoretic columns specially adapted for the IEF process. Representative adaptations include segmentation, baffles/screens, and surface coatings. Comparative performance and development testing are pursued against the type of column or cell established as an engineering model. Previously developed computer simulation capabilities are used to predict low-gravity behavior patterns and performance for IEF apparatus geometries of direct project interest. Three existing mathematical models plus potential new routines for particular aspects of simulating instrument fluid patterns with varied wall electroosmosis influences are being exercised.

Bier, M.↗
NASA NTRS1989

Electrophoresis and isoelectric focusing of whole cell and membrane proteins from the extremely halophilic archaebacteria

The subunits from two purified halobacterial membrane enzymes (ATPase and nitrate reductase) behaved differently with respect to isoelectric focusing, silver staining and interaction with ampholytes. Differential behavior was also observed in whole cell proteins from Halobacterium saccharovorum regarding resolution in two-dimensional gels and silver staining. It is proposed that these differences reflect the existence of two classes of halobacterial proteins.

Stan-Lotter, Helga↗
NASA NTRS1986

Affinity immunoblotting - High resolution isoelectric focusing analysis of antibody clonotype distribution

A sensitive and specific method is proposed for the analysis of specific antibody clonotype changes occurring during an immune response and for comparing multiple sera for antibody clonotype similarities. Polyclonal serum antibodies separated by isoelectric focusing (IEF) were analyzed by an affinity immunoblotting method using antigen-coated nitrocellulose membranes. Antibodies present on the surface of the acrylamide gels following IEF bind the antigen on the nitrocellulose when the coated nitrocellulose is laid over the gels. The technique has been used to analyze Ig clonotypes specific for five protein antigens and two carbohydrate antigens. Optimal antigen concentrations for coating the nitrocellulose membranes were found to range from 10-100 microgram/ml.

Knisley, Keith A.↗
NASA NTRS1988

Isoelectric focusing analysis of antibody clonotype changes occurring during immune responses using immobilized pH gradients

Serum was collected from rabbits at 2-day intervals following a single injection with tetanus toxoid or at weekly intervals following multiple injections with Micrococcus lysodeikticus cell walls. These sera were analyzed for the presence of individual clonotypes of specific antitetanus or antimicrococcal antibodies by isoelectric focusing in immobilized pH gradients with added carrier ampholytes followed by affinity immunoblotting. The affinity immunoblots obtained clearly defined both the rapid disappearance and late appearance of distinct subsets of antibody clonotypes during the response. These data demonstrate the application of affinity immunoblotting combined with immobilized pH gradients for detecting the subtle changes in specific antibody clonotype patterns which occur during an immune response.

Knisley, Keith A.↗
NASA NTRS1979

Mathematical model for steady state, simple ampholyte isoelectric focusing: Development, computer simulation and implementation

The elimination of Ampholine from the system by establishing the pH gradient with simple ampholytes is proposed. A mathematical model was exercised at the level of the two-component system by using values for mobilities, diffusion coefficients, and dissociation constants representative of glutamic acid and histidine. The constants assumed in the calculations are reported. The predictions of the model and computer simulation of isoelectric focusing experiments are in direct importance to obtain Ampholine-free, stable pH gradients.

Palusinski, O. A.↗