Professor Volodymyr Oleksiiovych Shlyakhovenko (on the 80th birth anniversary).

In September 2018, Professor Volodymyr Oleksiiovych Shlyakhovenko, well-known Ukrainian scientist in the field of cancer biochemistry, celebrated his 80th anniversary.

Simian virus 40 and protein transfer.

Protein transfer to solid supports after polyacrylamide gel electrophoresis, and subsequent probing with specific antibodies, is one of the most important tools in modern molecular and cellular biology. Since its development in 1979, the improvement of the technique has been impressive, from new apparatus to streamline the electrophoresis step to different modalities of the transfer step or solid supports for the transfer. Perhaps most impressive has been the explosion of the production and availability of antibodies. In this chapter, I describe the environment and conditions that led to the development of this technique in George Stark's laboratory.

Electrophoresis of DNA in agarose gels, polyacrylamide gels and in free solution.

This review describes the electrophoresis of curved and normal DNA molecules in agarose gels, polyacrylamide gels and in free solution. These studies were undertaken to clarify why curved DNA molecules migrate anomalously slowly in polyacrylamide gels but not in agarose gels. Two milestone papers are cited, in which Ferguson plots were used to estimate the effective pore size of agarose and polyacrylamide gels. Subsequent studies on the effect of the electric field on agarose and polyacrylamide gel matrices, DNA interactions with the two gel matrices, and the effect of curvature on the free solution mobility of DNA are also described. The combined results suggest that the anomalously slow mobilities observed for curved DNA molecules in polyacrylamide gels are primarily due to preferential interactions of curved DNAs with the polyacrylamide gel matrix; the restrictive pore size of the matrix is of lesser importance. In free solution, DNA mobilities increase with increasing molecular mass until leveling off at a plateau value of (3.17 +/- 0.01) x 10(-4) cm2/V s in 40 mM Tris-acetate-EDTA buffer at 20 degrees C. Curved DNA molecules migrate anomalously slowly in free solution as well as in polyacrylamide gels, explaining why the Ferguson plots of curved and normal DNAs containing the same number of base pairs extrapolate to different mobilities at zero gel concentration.

Turning a PAGE: the overnight sensation of SDS-polyacrylamide gel electrophoresis.

The zonal separation of proteins on the basis of net charge was initially conducted on paper, then in columns of sucrose and later in gels of starch and polyacrylamide, with appropriate electric fields. Then, in 1964, a graduate student at MIT discovered the power of sodium dodecyl sulfate (SDS) to dissociate the envelope proteins of Escherichia coli and to dramatically enhance their electrophoretic resolution when the detergent was included in the gel. While this Ph.D. thesis work continued, a group at the Albert Einstein College of Medicine published in 1965 the use of SDS to disrupt poliovirus particles and to resolve the proteins in gels containing SDS. This group soon followed with a publication (1966) on the application of this new method to the study of immunoglobulin heavy and light chain synthesis. Because of concurrent advances in gel filtration and other methods of protein separation, SDS gel electrophoresis had its greatest impact not in biochemistry but in cell biology and virology. Ingenious devices were soon introduced that facilitated the application of this method to radioactive protein mixtures, followed by the introduction of slab gels for the simultaneous resolution of multiple samples in parallel lanes in a single run. As we today routinely perform "SDS PAGE" (as the method become known, to the great irritation of journal copyeditors and nomenclature committees at the time), it is fitting to pause--four decades later, and remember the pioneers who made SDS gel electrophoresis a reality, a true milestone that caught on almost overnight.

DNA sequencing: bench to bedside and beyond.

Fifteen years elapsed between the discovery of the double helix (1953) and the first DNA sequencing (1968). Modern DNA sequencing began in 1977, with development of the chemical method of Maxam and Gilbert and the dideoxy method of Sanger, Nicklen and Coulson, and with the first complete DNA sequence (phage X174), which demonstrated that sequence could give profound insights into genetic organization. Incremental improvements allowed sequencing of molecules >200 kb (human cytomegalovirus) leading to an avalanche of data that demanded computational analysis and spawned the field of bioinformatics. The US Human Genome Project spurred sequencing activity. By 1992 the first 'sequencing factory' was established, and others soon followed. The first complete cellular genome sequences, from bacteria, appeared in 1995 and other eubacterial, archaebacterial and eukaryotic genomes were soon sequenced. Competition between the public Human Genome Project and Celera Genomics produced working drafts of the human genome sequence, published in 2001, but refinement and analysis of the human genome sequence will continue for the foreseeable future. New 'massively parallel' sequencing methods are greatly increasing sequencing capacity, but further innovations are needed to achieve the 'thousand dollar genome' that many feel is prerequisite to personalized genomic medicine. These advances will also allow new approaches to a variety of problems in biology, evolution and the environment.

Discovering viroids--a personal perspective.

During 1970 and 1971, I discovered that a devastating disease of potato plants is not caused by a virus, as had been assumed, but by a new type of subviral pathogen, the viroid. Viroids are so small--one fiftieth of the size of the smallest viruses--that many scientists initially doubted their existence. We now know that viroids cause many damaging diseases of crop plants. Fortunately, new methods that are based on the unique properties of viroids now promise effective control.

Glycosylation analysis of gel-separated proteins.

Beyond the identification of proteins involved in a particular physiological situation, many aspects of proteomics require more detailed characterization of the proteins involved. Post-translational modifications (PTMs) of proteins are a common means to target proteins, regulate their activities and to mediate communication between proteins and cells. Owing to the much higher analytical complexity of glycan analysis compared to e.g. protein identification, PTM analysis in general and glycosylation analysis in particular is largely neglected in proteomics. In this review, the current technological status of global and site-specific glycosylation analysis of gel-separated proteins is described and the way in which the available technology can be employed in proteomics is critically discussed.