Andreas Chrambach

A procedure for rapid and sensitive staining of protein fractionated by polyacrylamide gel electrophoresis

Abstract Protein zones may be stained by Coomassie blue without any destaining of the background and with a sensitivity at least as high as that of amido black. A vastly increased sensitivity may be achieved with Coomassie blue at the price of background destaining.

Estimation of molecular radius, free mobility, and valence using polyacrylamide gel electrophoresis☆

Relative mobility values for macromolecules in polyacrylamide gel electrophoresis at various gel concentrations are used to compute the retardation coefficient, KR, molecular radius, R, free mobility, M0, and valence, V. Automated data processing and formal statistical analysis are applied. Results obtained with 29 proteins in 9 electrophoretic systems illustrate the utility of this approach, and also provide estimates of radius and total length of the acrylamide polymer.

Polyacrylamide gel electrophoresis in sodium dodecyl sulfate-containing buffers using multiphasic buffer systems: properties of the stack, valid Rf- measurement, and optimized procedure.

SDS-proteins can be stacked in sharp starting zones in SDS-PAGE in multiphasic buffer systems, Stacking of SDS-proteins has been possible with a lower stacking limit of up to 0.300, above neutral pH in “nonrestrictive” gels. Under the same conditions, SDS is stacked. Stacking of SDS, derived from both the SDS content of the sample and the upper buffer, broadens the stack in proportion to SDS load and duration of electrophoresis. Such broadening produces an inordinately wide starting zone in the stacking gel, leading to impaired resolution, unless one limits the SDS load. In the resolving gel, this zone broadening makes the trailing edge of the stack, and the tracking dye migrating there [e.g., bromphenol blue at low gel concentration (%T)] unsuitable as a valid reference for Rf. However, the leading edge of the stack and the tracking dye migrating there (e.g., pyronin-Y-SDS at low %T) are a valid Rf reference. At high %T, both SDS and pyronin-Y-SDS unstack, whereas bromphenol blue now moves to the leading edge of the stack and becomes, for that %T range, a valid reference of Rf. An optimized system for SDS-PAGE is described which contains no SDS in the gel, 0.03% SDS in the upper buffer, 150 μg or less SDS in the sample, and a single set of gel buffer constituents in stacking and resolving gels. Valid Rf measurement is a requisite for linear Ferguson plots (log Rf vs %T). Since the slope of these plots, KR, is a measure of molecular weight the procedure of SDS-PAGE proposed in this study should provide improved reliability of melecular weight estimates.

Isoelectric focusing in polyacrylamide gel and its preparative application

Abstract A method for isoelectric focusing in polyacrylamide gels (IFPA) has been described. A 3.7% T polyacrylamide gel allowed for isoelectric focusing of hemoglobin and cyanogen bromide fragments of both collagen and hemoglobin in the absence of significant molecular sieving and provided satisfactory mechanical stability. Support of the gels was enhanced by polyacrylamide coating of tubes, by use of a membrane support, and by hydrostatic equilibration. Runs for 8 hr at 200 V/6 cm at 5°C provided optimal separation. Preparative IFPA was conducted in tubes of up to 18 mm diameter with loads of up to 2 mg per band. Protein was recovered quantitatively from the gel slices.

A highly crosslinked, transparent polyacrylamide gel with improved mechanical stability for use in isoelectric focusing and isotachophoresis

Polyacrylamide gels highly (10–50%) crosslinked with N,N′-methylenebisacrylamide (BIS) provide large pore sizes but are mechanically labile. By substituting N,N′-diallyltartardiamide (DATD) for BIS, mechanical stability and firm adherence to glass walls is conferred to these gels, with only a minor decrease in pore size compared to highly BIS-crosslinked gels. The highly DATD-crosslinked gels have the further advantage of being transparent. It appears that highly DATD-crosslinked gels combining large pore size with mechanical stability are superior to presently used gel types in isoelectric focusing on polyacrylamide gel and isotachophoresis on polyacrylamide gel.

Pore gradient electrophoresis.

Abstract Gel gradient electrophoresis has been proposed as a method for improving resolution and for measuring molecular size. Margolis and Slater have suggested that each macro-ion will reach a “pore limit” or “dead stop” and that the protein pattern would remain essentially stable thereafter. However, no quantitative treatment of the migration of charged macromolecules in a gel gradient has been reported to date. The present report provides an analysis of the behavior of macro-ions in both linear and nonlinear gel gradients. This is made possible by combining Fergusons relationship for electrophoretic mobility versus gel concentration with the equation describing the gel gradient. Solution of these two equations yields the instantaneous velocity and position for each band as a function of time. This analysis provides insight into situations in which pore gradients may improve resolution and conditions for optimal resolution of multicomponent systems (macromolecular mapping). Position and instantaneous velocity of migration for any macromolecule on gel concentration (“pore”) gradient electrophoresis may be calculated for a linear gradient: χ= log e (ba 2 u o t + exp (bT o )) − bT o ba 2 (iva) ν= u o (ba 2 u o t + exp (bT o )) (va) This makes it possible to calibrate or standardize pore gradient electrophoresis. This analysis indicates that the proteins do not come to a “pore limit” or “dead stop.” Pore gradient electrophoresis is not indicated for analysis or separation of one, two, or three components, nor for charge fractionation. It is useful for simultaneous analysis of components of multicomponent mixtures (macromolecular mapping); and a “transverse” gradient presents a promising approach to obtaining Ferguson plots.

Separation and characterization of sub‐μm‐ and μm‐sized particles by capillary zone electrophoresis

The analytical separation and characterization of particles in the size range of sub‐νm and νm diameters by capillary zone electrophoresis (CZE) has been reviewed. The theoretical basis, on which the mobility can be interpreted to provide information regarding characteristics of particle surface, has shortly been presented. Particular emphasis was put on the model dependence of that interpretation and the need in most applications to forego the classical idealized model of spherical particles with “smooth” surfaces and to apply more realistic models, which take the “hairy” surface of real particles into account. Some highlights of the literature on the CZE of polystyrene latex microspheres, organic and inorganic colloids, lipoprotein particles, viruses, liposomes, biological membrane vesicles, and biological cells have been discussed. Also summarized are the reports on the particle size dependence of mobility and peak broadening in CZE and on electrophoretic behavior of rodlike particles and particle aggregates. Finally, the effects of neutral polymers in the background electrolyte on particle mobility and peak width are reviewed.

Anti-estradiol antibodies: Isoelectric focusing in polyacrylamide gel

Abstract Conditions for isoelectric focusing in polyacrylamide gel of IgG globulins have been refined. Using undenatured antibodies against estradiol, preliminary information about structural and functional properties of antibodies was obtained.

Analytical and Preparative Polyacrylamide Gel Electrophoresis

Biochemistry may be viewed as the attempt to fragment living matter to its component molecular species, and to elucidate the chemical reactions of these species first in two-component systems, then in ever more complex systems and finally to reconstitute life as the sum of its component chemical reactions. The first task, i.e., the isolation of nature’s building-block molecules, remains a formidable task. This chapter is concerned with the instrumentation and procedures available for isolation of any given charged species according to a generally applicable fractionation strategy.

Nonisoelectric focusing in buffers.

Abstract Stable pH gradients were formed and focusing of proteins was carried out in polyacrylamide gels containing mixtures of simple, amphoteric buffers, replacing the Ampholine hitherto used in isoelectric focusing (IF). Stable pH gradients can also be formed between acid anolyte and basic catholyte if Ampholine is replaced by nonamphoteric buffers. The fact that focusing can be carried out with nonampholytes shows that focusing in this case is, and in all other cases may be, nonisoelectric. It is postulated that the pH gradient in IF forms by steady-state stacking (isotachophoresis) and forms within the stack. In distinction to ordinary steady-state stacking, however, the stack remains confined within the gel (or density gradient) since the strong acid and base in the electrolyte reservoirs bar by deprotonation or electrostatic repulsion migration into the electrode chambers.