Zsuzsanna Buzás

Direct vertical electroelution of protein from a PhastSystem band for mass spectrometric identification at the level of a few picomoles.

An electroelution apparatus prototype of a new design was constructed. In that design, the electric field passes vertically through the protein band located on a horizontal (PhastSystem) minigel polymerized on a net of Gel‐Fix (Serva). A simple, home‐made apparatus allows for electroelution of protein bands at the level of a few picomoles and their identification, after concentration, by matrix‐assisted laser desorption/ionization‐time of flight mass spectrometry. The technique is applicable to one‐dimensional (1‐D) or two‐dimensional (2‐D) gels of any size, but has been exemplified only by application to 1‐D minigels to demonstrate the lower limits of protein load of the method. When in the course of further development of the prototype it will be combined with a modification to two dimensions of the electroelution mechanism under computer control of the high‐performance gel electrophoresis apparatus**** (formerly of LabIntelligence), the new design appears uniquely qualified for an automated spot elution from 2‐D gels under avoidance of gel sectioning.

Novel application of PhastSystem polyacrylamide gel electrophoresis using restriction fragment length polymorphism--internal transcribed spacer patterns of individuals for molecular identification of entomopathogenic nematodes.

A relatively rapid and economic way of identifying and assigning nematodes to taxons, which had already been determined either by comparative sequence analysis of nuclear rDNA internal transcribed spacer (ITS) region or by other methods of molecular or conventional taxonomy, is provided. Molecular identification of entomopathogenic nematodes (EPN) can be upgraded by basing it on PhastSystem polyacrylamide gel electrophoresis (PAGE) analysis of restriction fragment length polymorphism (RFLP) patterns of polymerase chain reaction (PCR)‐amplified DNA derived from single nematodes of Steinernema or Heterorhabditis spp. Although analysis from single worms has previously been made on agarose gel, the resolution on PhastSystem PAGE gel is much higher. The DNA sequences selected for analysis were those constituting the internal transcribed spacer region between the 18S and 26S rDNA genes within the rRNA operon. RFLP analysis was carried out by gel electrophoresis on the PhastSystem (Pharmacia) as detailed elsewhere (Triga et al., Electrophoresis 1999, 20, 1272—1277). The downscaling from conventional agarose to PhastSystem gels resulted in pattern of DNA fragments differing from those obtained with agarose gel electrophoresis under conventional conditions by increasing the number of detected fragments. The approach supported previous species identifications and was able to identify several unclassified isolates, such as those from Hungary and Ireland, and provides a method for identification of previously unclassified strains. We confirmed that Heterorhabditis “Irish Type”, represented by two strains of different geographical origin, comprise a species different from H. megidis. We also confirmed that strain IS5 belongs to the species H. indicus rather than to H. bacteriophora, as had been suggested previously.

Gel electrophoretic restriction fragment length polymorphism analysis of DNA derived from individual nematodes, using the PhastSystem

The DNA sequences constituting the internal transcribed spacer region, located between 18S and 26S rDNA genes within the rRNA operon, derived from single nematodes of two genera (Steinernema and Heterorhabditis) were amplified by polymerase chain reaction (PCR) and subjected to digestion by four restriction enzymes. The digests were analyzed by restriction fragment length polymorphism (RFLP) gel electrophoresis on the PhastSystem, using 7.5%T, 5%C(Bis) polyacrylamide. The downscaling from conventional agarose to PhastSystem gels permitted the analysis to be done on individual nematodes, rather than on mixed samples with average properties. The analysis time was reduced so as to allow for the electrophoretic separation on 200 samples/workday. The resulting patterns of DNA fragments differed from those obtained by agarose gel electrophoresis under conventional conditions by an increased number of detected fragments. The PhastSystem gel analysis provides the basis for taxonomical revisions (Pamjav et al., Electrophoresis 1999, 20, 1264—1271.)

PhastSystem electrophoresis in β-octylglucoside containing gels with immunodetection of a nondenatured vesicle-associated membrane protein

Recombinant vesicle‐associated membrane protein (rVAMP), implicated as a participant in membrane exocytosis and fusion (a “SNARE protein”), was subjected to gel electrophoresis in the miniaturized gels of the PhastSystem (Pharmacia) containing the nondenaturing, nonionic detergent β‐octylglucoside (OG), followed by immunodetection of the protein. Three major components of nondenatured rVAMP are detected by Western blotting both in 0.5% OG and in the absence of detergent. Their separation increases with increasing gel concentration above 7%T. Ferguson plot analysis indicates that the three species of VAMP are size isomers (i.e., they differ in size but share a common surface net charge density), the common point of intersection of the plots (μ‐point) being a measure of their common free mobility. By the criteria of size and free mobility (related to surface net charge), VAMP components I, II and III in 0.5% OG‐containing buffer are indistinguishable from components II, III and IV, respectively, observed in the absence of the detergent. The feasibility of immunodetection of nondenatured rVAMP on gels containing nondenaturing detergents opens up the possibility of gaining biochemical information regarding nondenatured SNARE protein complexes and SNARE proteins linked to membrane fragments.

Detection of DNA curvature by transverse pore gradient gel electrophoresis on PhastSystem gels.

It has been known since 1990 that DNA curvature can be recognized on transverse pore gradient gels by an intersection of “Ferguson curves” with those of DNA size standards. The miniaturized PhastSystem polyacrylamide gels allow one to detect DNA curvature effortlessly and fast and at great economy of sample relative to alternative methods of electrophoresis. Using the transverse gradient gel electrophoresis method, it was found that the 660 bp length subfragment of the matrix attachment region (MAR) sequence of the chicken lysosyme gene migrates as a fragment of 800—900 bp length. When subjected to digestion with the restriction enzyme HaeIII, the fragment gives rise to two species of 248 and 412 bp length, respectively. The Ferguson curves of both species intersect with those of DNA size standards, indicating that both exhibit curvature. Only the curvature of the 412 bp fragment conforms to prediction. Ethidium bromide abolishes the effect of curvature on the fragment, reducing its apparent size from 900 to 660, the value obtained by agarose gel electrophoresis.