Free-flow Apparatus Research Articles

The role of receptor internalization in CD95 signaling

Activation of the cell surface CD95 receptor triggers a cascade of signaling events, including assembly of the death-inducing signaling complex (DISC), that culminate in cellular apoptosis. In this study, we demonstrate a general requirement of receptor internalization for CD95 ligand-mediated DISC amplification, caspase activation and apoptosis in type I cells. Recruitment of DISC components to the activated receptor predominantly occurs after the receptor has moved into an endosomal compartment and blockade of CD95 internalization impairs DISC formation and apoptosis. In contrast, CD95 ligand stimulation of cells unable to internalize CD95 results in activation of proliferative Erk and NF-kappaB signaling pathways. Hence, the subcellular localization and internalization pathways of CD95 play important roles in controlling activation of distinct signaling cascades to determine divergent cellular fates.

Preparative isoelectric focusing of proteins using binary buffers in a vortex-stabilized, free-flow apparatus.

Recombinant proteins are often produced as isoforms with different kinds and amounts of post-translational modifications that alter their function. Isoelectric focusing in shallow pH gradients, less than 0.5 pH/cm, might be capable of fractionating these isoforms. The synthetic carrier ampholyte mixtures typically used to generate these pH gradients are expensive and may adversely interact with proteins. Using defined buffers instead of synthetic carrier ampholytes reduces these problems. We tested two defined buffer systems in a vortex-stabilized electrophoresis device to see if they could form shallow pH gradients useful for separating isoforms. These pH gradients were formed by pouring a two-component concentration gradient. The poured gradients were smooth, reproducible, and stable for at least 1.5 h at 5 kV. One poured gradient focused 20 mg of cytochrome c. A second poured gradient separated glucose oxidase from amyloglucosidase. The breadth of the amyloglucosidase band indicates that the shallow, poured pH gradients can only partially separate protein isoforms at 10 kV. Proteins with pI < 0.2 pH units apart will have overlapping bands in these shallow, poured pH gradients.

Electrophoretic mobilities of proteins and protein mixtures

Electrophoretic mobilities of bovine serum albumin (SA), haemoglobin (Hb) and ovalbumin (OA) have been determined by direct ultramicroscopic observation of the velocities of individual particles in a particle electrophoresis apparatus, and of SA and Hb in a free-flow apparatus of the type described independently by Hannig and by Strickler. Agreement between the results obtained by the two methods for SA and Hb over wide pH ranges covering both sides of the respective isoelectric points was surprisingly good. Runs were also conducted in the free-flow cell in which SA was transported in buffer containing Hb, and vice versa; these showed clear evidence of interactions between the motions of the two proteins, as occurs in the parallel case of multicomponent diffusion, but the results could not be quantified in terms of the Maxwell-Stefan theory. Finally, the single protein data were compared with predictions made on the basis of the Debye-Huckel-Henry theory, and showed reasonable agreement.

Peculiar features of application of pH gradients formed in borate buffer with a polyhydroxy compound for separation of proteins in a free-flow electrophoretic apparatus.

The use of pH gradients in borate buffer with a polyhydroxy compound for separation of proteins in a free-flow electrophoretic apparatus is outlined. The composition of the pH gradients, the free-flow apparatus design and its operation, and the main causes of failures in the protein separation are considered.

Protein separation in pH gradients using free‐flow electrophoretic apparatus II. The pH gradients formed by the concentration gradient of boric acid in solutions of borax and mannitol

AbstractProtein separation was investigated in the free‐flow apparatus using pH gradients generated by a concentration gradient of boric acid in a solution of borax. The linear pH gradient covering the range pH 8.2–9.2 was obtained in a solution of 2.5 mM borax due to the formation of the logarithmic concentration gradient of 0–45 mM boric acid. These pH gradients lacked a conductivity gradient in contrast to borate mannitol pH gradients, described in the joint report. By addition of mannitol into the gradient, at constant concentration, the pH was shifted toward more acidic values in proportion to the amount of mannitol added, without changing the range and shape of the pH gradient. According to this procedure of gradient formation the pH gradients can be obtained within the range from pH 3.5 to 9.2. The gradients were rapidly decaying under the experimental conditions. Thus, isoelectric focusing of proteins could not be achieved, and the separation resulted from electrophoresis in the pH gradient. The results of protein separation in the gradients practically did not depend on the point of injection of the samples into the pH gradient, at least in the cases when the proteins were injected near their isoelectric points.

Free-flow electromigration separations

A continuous free-flow apparatus for thin-layer zone electrophoresis and adaptations for separations by isotachophoresis and by isoelectric focusing are described. The isotachophoresis and the isoelectric fractionation of an amylase concentrate is shown as an example. The concentration profiles of the leading electrolyte, KCl, and of the terminator, Tris, under conditions of continuous isotachophoresis are demonstrated.