Steven G. Shafer

Control of electroosmosis in coated quartz capillaries

Abstract Analytical particle microelectrophoresis was used to evaluate the effectiveness of various coatings for control of the electroosmotic fluid flow that hinders electrophoretic processes. Mobilities of 2-μm-diameter glass and polystyrene latex spheres, exhibiting both negative and zero effective surface charge, were measured in 2-mm-diameter quartz capillaries filled with NaCl solutions within the pH range of 3.5 to 7.8. Covalent coating of capillary inner surfaces with poly(ethylene glycol) caused a reduction in the degree of electroosmotic flow which was related to the poly(ethylene glycol) molecular weight. Poly(ethylene glycol) coatings of 5000 molecular weight, or higher, greatly reduced electroosmosis within the selected pH range, were stable for long periods of time, and appeared to be more effective than dextran, methylcellulose, or silane coatings. Because of these characteristics, poly(ethylene glycol) coatings should be of considerable use for improving various electrophoretic processes as well as in the production of standard particles exhibiting controlled electrophoretic mobilities.

Immuno-affinity partition of cells in aqueous polymer two-phase systems

Poly(ethylene glycol) (PEG) was covalently coupled to IgG antibody preparations directed against human red blood cells. This modification reduces the tendency of the antibody to agglutinate cells and increases its affinity for the upper phase in dextran-PEG aqueous two-phase systems. These effects are related to the molecular weight of the PEG used for modification and to the number of PEG molecules attached to the antibody. Exposure of human red blood cells to PEG-modified antibody causes a substantial and specific increase in cell partition into the PEG-rich phase in a number of PEG-dextran aqueous two-phase systems. Pertinent phase-system parameters were examined. Following a single incubation with PEG-derivatized antibody, a mixture of sheep and human red blood cells was completely separated in 100 min by a 30-transfer countercurrent extraction using a two phase system which normally offers little resolution.

Cell separation by immunoaffinity partitioning with polyethylene glycol-modified protein a in aqueous polymer two-phase systems

Previous work has shown that polyethylene glycol (PEG)-bound antibodies can be used as affinity ligands in PEG-dextran two-phase systems to provide selective partitioning of cells to the PEG-rich phase. In the present work we show that immunoaffinity partitioning can be simplified by use of PEG-modified Protein A which complexes with unmodified antibody and cells and shifts their partitioning into the PEG-rich phase, thus eliminating the need to prepare a PEG-modified antibody for each cell type. In addition, we provide a more rigorous test of the original technique with PEG-bound antibodies by showing that it is effective at shifting the partitioning of either cell type of a mixture of two cell populations.

Effects of Polyethylene Glycol Substitution on Enzyme Activity

The effects of polyethylene-glycol (PEG) substitution on protein activity, us ing alkaline phosphatase as a model, have been studied. Such variables as PEG molecular weight, degree of substitution, and PEG mono- and di-functionality have been examined. Modification with the monomethyl ether of PEG 1900 (M- PEG-1900) does not alter enzyme activity until greater than 40% of the protein lysine groups are substituted, at which point increasing the degree of modifica tion gives increasing deactivation. Substitution with M-PEG-5000 gives more deactivation than does substitution with M-PEG-1900. Interestingly, modifica tion with PEG itself gives active protein conjugates in which there is little de pendence on molecular weight or degree of substitution.

Cell Separation by Immunoaffinity Partition in Aqueous Polymer Two-Phase Systems

Two methods for immunoaffinity partitioning are described. In the first, poly(ethylene glycol) (PEG) was covalently coupled to immunoglobulin G antibody preparations directed toward surface antigens unique to either sheep or human erythrocytes. The modified antibody preparations exhibited a reduction in ability to agglutinate cells and increased affinity for the PEG-rich upper phase in Dextran-PEG aqueous two-phase systems. When either preparation was incubated with a mixture of target and control erythrocytes, the partition of the erythrocyte population bearing the complimentary antigen was selectively increased into the upper phase while the control cells remained unaffected. Effective separation of cell populations was obtained in 2 hours using 30-transfer countercurrent distribution.

Conformational analysis of tertiary cycloalkyl (C6,C7,C8) carbocations using molecular mechanics. Comparison with stable ion experimental data

Abstract Conformational calculations of the 1-methylcyclohexyl, 1-methylcycloheptyl, and 1-methylcyclooctyl cations agree with reported experimental observations.