Characterization and Comparison of Mycobacterial Antigens by Two-Dimensional Polyacrylamide Gel Electrophoresis

Abstract

Cell extracts and culture filtrates were prepared from several mycobacterial species and subjected to two-dimensional acrylamide gel electrophoresis. The cell preparations were first electrophoresed in a 7% homogeneous gel followed by a second electrophoresis, at right angles to the first separation, in a 2 to 30% linear gel gradient slab. Because the stained slab resembled a "fingerprint" or "peptide map," this procedure has been called "electroprotein mapping." More than 200 proteinstaining spots were detected in several of the cell extracts, each map being characteristic of the species examined. The single most characteristic portion of each map was the region between the 16 and 30%;c gel concentrations. This region consisted of several small-molecular-weight components (many of peptide proportions) which together appeared as the most anodic band in the 7%" gel column (first separation). The number and location of these components in the linear gel slab (second separation) were specific for each species tested. By electrophoresis of molecular-weight markers (i.e., albumin, myoglobin, cytochrome c) with and across (transverse electrophoresis) the gel gradient, the relative molecular weights of the unknown cell extract components could be estimated. These results suggest that two-dimensional acrylamide gel electrophoresis is a useful tool for the characterization of mycobacterial cell constituents. 7.5 cm). Electrophoresis was carried out at room temperature at 5 ma, 40 v/column, until the bromophenol tracking dye front reached 1 cm from the end of the gel column (approximately 60 min). Electrophoresis was terminated, and the gel column was removed from the glass cylinder and laid on the upper edge of a 2 to 30%x, linear acrylamide gel gradient slab. A second electrophoresis was then carried out at right angles to the first separation for 18 hr at 20 ma, 180 v, and 10 C. In some experiments, two 482

Comparison of antigens in sonic and pressure cell extracts of Mycobacterium tuberculosis

“…(3), who showed that the IEP patterns of bacillary extracts were constant for a strain of tubercle bacilli regardless of the age of the culture. Similarly, the results of previous studies from one of our laboratories have indicated that sonic extracts prepared from different strains of a single mycobacterial species had essentially the same GAGE and 2-D IEP patterns; in contrast, the patterns of their culture filtrates varied markedly in most cases from strain to strain (1,22,(26)(27)(28). Also, as shown in the present study, the antigenic composition did not differ significantly when the disruption method was varied.…”

“…However, because they are autolytic products and subject to continuous enzymatic alteration and degradation during incubation, the antigenic composition of culture filtrate preparations can vary markedly, not only between strains of tubercle bacilli but also between identical cultures of the same strain (3, 21, 26). Preparations of mycobacterial cytoplasmic extracts, in contrast, have been reported to be uniform in composition (3,6,21,22,(26)(27)(28). In other studies, bacillary extracts have been demonstrated to be suitable materials for immunological study.…”

Mycobacterial antigens: a review of their isolation, chemistry, and immunological properties.

“…Provided that the physical conditions of acrylamide gel concentration, pH and buffer, and current flow are standardized, it provides a technique of powerful resolution that is readily adaptable to the identification and nomenclature of individual antigens. Moreover, the resolving power of gel electrophoretic techniques has been greatly increased by the two-dimensional procedures introduced by, among others, Augier and Augier-Gibory (9) and Wright and co-workers (202,203). As illustrated in Fig.…”

Serological studies of actionomyces israelii by crossed immunoelectrophoresis: standard antigen-antibody system for A. israelii

“…The chemical extraction methods, employed for the preparation of whole cell-associated antigens, were used because similar methods, had been previously applied (6,9,11,(22)(23)(24)(25). Cytoplasmic antigens, obtained by preparative ultracentifugation of water extracts of cell lysates were examined because of their successful usage as antigens with mycobacteria (42,43) and Candida albicans (1, 2) for CIE studies. Reported advantages of plasma antigens of Actinomycetales for serological taxonomic studies of these bacteria (24) also contributed to the choice of the cytoplasmic fraction of cell lysates.…”