Two-dimensional electrophoresis of small-molecular-weight proteins

We have developed a procedure for two-dimensional separation of small-molecular-weight (9000–30,000), acidic (pI 4–6) proteins that allows the use of strips cut from horizontal isoelectric-focusing slab gels for the first dimension, and discontinuous gels containing sodium dodecyl sulfate and high concentrations of urea in the second dimension. This technique facilitates the screening of large numbers of samples and the evaluation of electrofocusing artifacts. We emphasize measures to prevent major problems encountered in the use of this technique, particularly those caused by diffusion and aggregation. We also describe an extension of the method which allows the two-dimensional comparison of many samples in a selected narrow pH zone of interest.     

Surface inactivation of bacterial viruses and of proteins

1. The seven bacterial viruses of the T group active against E. coli, are rapidly inactivated at gas-liquid interfaces. 2. The kinetics of this inactivation whether brought about by shaking or by bubbling with nitrogen are those of a first order reaction. 3. This inactivation may be prevented by the addition of enough protein to maintain the gas-liquid interface in a saturated condition. 4. The analogy between this phenomenon and the surface denaturation of proteins is pointed out and discussed.     

Genome-linked proteins in DNA and RNA viruses

General principles of the organization of viral nucleic acid--protein covalent complexes are formulated. Participation of the genome-linked proteins in the initiation of viral nucleic acid replication is discussed.     

Agarose-acrylamide gradient gel electrophoresis of proteins

A new agarose-acrylamide gradient slab gel electrophoresis system is described. The preparation of this new gel has been facilitated by the use of agarose with a relatively low gelation temperature. Fractionation of marker proteins and crosslinked proteins from a subcellular cytoskeletal preparation on agarose-acrylamide gradient gels is compared to that found using other acrylamide gel electrophoresis systems.     

Semipreparative electrophoresis with prestained proteins.

A simple method for prestaining proteins with Coomassie blue G prior to acrylamidegel electrophoresis is described. Many, although not all, undenatured proteins can be stained. This prestaining technique can be conveniently coupled with a second electrophoresis step, using Sephadex as a supporting medium, to yield isolated protein in about 4 h.     

Assembly of flexuous plant viruses and their proteins.

Recent experiments on the disassembly and assembly of some flexuous plant viruses and their proteins are described. The properties of reconstituted potato virus X and those of assembled potato virus Y protein are considered as well as the suitability of other flexuous viruses for reconstitution studies.     

Fractionation of liver proteins by preparative electrophoresis

Summary. Proteomics offers unique possibilities to investigate changes in the levels and modifications of proteins involved in the pathomechanisms of diseases and toxic events. However, search for potential drug targets and disease or toxicity markers is limited by the fact that mainly the high-abundance, hydrophilic proteins are visualized in two-dimensional gels. Here we studied the enrichment of rat liver cytosolic proteins by preparative electrophoresis. Preparative electrophoresis was performed with the PrepCell apparatus in the presence of 0.1% lithium dodecyl sulfate. Lithium dodecyl sulfate was exchanged against agents compatible with isoelectric focusing prior to the two-dimensional gel electrophoresis. Proteins were identified from two-dimensional gels by matrix-assisted laser desorption ionization time-of-flight mass specrometry. Low- and middle-size proteins and low-abundance proteins, which had not been found before, were enriched by preparative electrophoresis. The present study represents a contribution of proteomics in the quantification of differences in the levels of low-abundance liver proteins in toxicity studies.