Isoelectric focusing of proteins in rewetted ultrathin polyacrylamide gel layers

Ultrathin layers of polyacrylamide gel bound to glass can be washed, air-dried, and stored for at least 1 year before rewetting in ampholyte solutions for isoelectric focusing. Short-term drying affects neither fluorescent banding of the ampholytes (not evident in conventional gels) nor resolution of complex protein mixtures while prolonged storage seems to have no deleterious effects. Layers are fully functional after soaking for 10 min in solutions that may contain 8 M urea and 10% sorbitol. Rewetting allows the rapid survey of different ampholytes, gradient stabilizers, separator compounds, or protein reagents and is adaptable to concentration modification of the pH gradient (alone or with a gel overlay), to focusing in a transverse urea gradient, and to electrophoresis across a preformed pH gradient. The procedure avoids protein modification by residual polymerizing reagents while adding to the convenience and economy of using ultrathin layers in relatively small formats.     

Isoelectric focusing in polyacrylamide gel using buffer electrode solutions.

Isoelectric focusing in ampholytes of pH 6–8 range has been carried out in polyacrylamide gels using ammonia buffer at pH 10.0 and acetate buffer at pH 4.0 for the cathode and anode solutions, respectively. This system requires low voltages but compares well with isoelectric focusing using strong acid and strong base electrode solutions. The advantages of this method are the less extreme pH's in the electrode solutions and lower resistance in the neutral region of the pH gradients.     

Isoelectric focusing of fibrinogens on polyacrylamide gels

The thiol-oxidizing agent, diamide, has been used to convert glutathione to glutathione disulfide within the cells of a stringent strain of Escherichia coli (CP 78), leading to a cessation of 14C-leucine incorporation (protein synthesis) and 3H-uracil incorporation (RNA synthesis). Parallel experiments with an isogenic relaxed strain (CP 79) gave similar results, providing evidence that glutathione is closely linked to RNA synthesis indepently of the link previously shown to protein synthesis.     

A new device of preparative polyacrylamide gel electrophoresis and its application to analysis of cellular RNA

The apparatus for preparative polyacrylamide gel electrophoresis has been devised, by which eluted materials can be sampled continuously and quantitatively in a drop-scale.     

Isoelectric focusing of alkaline phosphatases in agarose gel

An isoelectric focusing technique in agarose gel is presented which is suitable for alkaline phosphatases from both serum and tissue sources. An anomaly in the literature about isoelectric focusing of serum alkaline phosphatase from liver origin is discussed and a possible explanation is proposed. The presented technique is used to demonstrate some differences in behaviour of serum liver and bone isoenzymes towards neuraminidase treatment.     

Isoelectric focusing of horse acidic prealbumins on thin-layer polyacrylamide gels

The paper describes a technique of thin-layer polyacrylamide gel isolectric focusing of horse serum, within a pH range of 4.0--6.0, which permits the improved resolution of the acidic prealbumin protein bands. The increased heterogeneity of the Pr prealbumin antiprotease allele products apparent using this technique is described and discussed in detail, and the potential use of the technique in routine Pr phenotyping is considered.     

Isoelectric focusing of human granulocyte lysosomal elastase in thin-layer polyacrylamide gels

A technique is described which allows isoelectric focusing of human granulocyte elastase in thin-layer polyacrylamide gels, with retention of between 70 and 90% of the elastolytic activity. Digestion of elastin was observed to occur between pH 8.13 and 8.3 as well as between pH 8.77 and 9.15. Detection of elastolytic activity and visualization of protein bands following staining of the gels, has been successfully attempted with as little as 20 μg of lysosomal extract (about 2 to 3 μg of elastase).     

Purification of tetanus toxin and its peptide components by preparative polyacrylamide gel electrophoresis

A discontinuous preparative gel electrophoresis system has been devised and used successfully to separate the different tetanus toxin forms and fragments into highly purified preparations. A major feature of the system is the interaction of toxin, a suitable reducing agent and a critical concentration of denaturant during electrophoresis. With this procedure, filtrate (nicked) toxin has been separated into two distinct, but closely related molecular species. They appear to be nicked close to but on either side of an interchain disulfide bond, yielding heavy and light chains. The heavy- and light-chain components of each form of nicked toxin have been prepared and characterized. The system was also used to prepare extract (unnicked) toxin to a degree of purity not previously achieved in this laboratory. Nicked and unnicked toxin as well as the two forms of both heavy and light chain can consistently be prepared in sufficient purity and quantity to allow extensive biological, chemical, and physical characterization of each.     

Isoelectric focusing of horse acidic prealbumins on thin-layer polyacrylamide gels

The paper describes a technique of thin-layer polyacrylamide gel isolectric focusing of horse serum, within a pH range of 4.0 - 6.0, which permits the improved resolution of the acidic prealbumin protein bands. The increased heterogeneity of the Pr prealbumin antiprotease allele products apparent using this technique is described and discussed in detail, and the potential use of the technique in routine Pr phenotyping is considered.