One-dimensional Slab Gel Research Articles

Automated lanes detection and comparison of bacterial electrophoretic protein fingerprints using fast Fourier transformation

A method of computer-automated analysis of bacterial fingerprints produced by electrophoresis of proteins in a one-dimensional slab gel system is described. Proteins were visualized by silver staining, Western blotting, or autoradiography. Gels were recorded with a CCD camera, and after initial manual removal of the unwanted image margins, track margins were identified and extracted and a normalized trace was produced automatically using Fourier routines to smooth plots required for this process. Normalized traces were then compared by Fourier correlation after application of a high-pass step filter.

Nematode chromosomal proteins—IV. The nonhistones of Caenorhabditis elegans

1. 1. A method has been developed which yields reasonably clean nuclei from the free-living nematode Caenorhabditis elegans. Nonhistones were prepared from these nuclei by gentle extraction with 2M NaCl and analyzed by one- and two-dimensional electrophoresis using a nonequilibrium pH gradient electrophoresis in the first dimension after removal of nucleic acids by phenol extraction. 2. 2. The extraction procedure yielded nonhistone chromatin proteins and proteins associated with nuclear RNA and a background of polypeptides derived from the nuclear matrix and pore-lamina complex. 3. 3. Over 40 polypeptides were distinguished on silver stained one-dimensional slab gels. 4. 4. The presence of actin, tropomyosin and tubulin was demonstrated by immunoprobing after transfer of the polypeptides to nitrocellulose. 5. 5. Bands co-migrating with myosin and α-actinin gave no specific when probed with antibodies raised against the respective proteins from vertebrate tissues. 6. 6. Over 200 spots were visible on two-dimensional gels stained with silver nitrate.

Changes of sertoli cell glycoproteins induced by removal of the associated germ cells

Sertoli cell glycoproteins were studied in culture where these cells were in contact with germ cells (Sertoli cell enriched cultures, SCEC) and in pure Sertoli cell cultures. Sertoli cell only cultures (SCOC) were prepared by a short treatment of SCEC with hypotonic solution or by culturing seminiferous epithelium fragments from prenatally irradiated rats. After metabolic labeling with [3H]fucose. [14C]N-acetylglucosamine or [3H]leucine, SCEC and SCOC particulate fractions (105 000 g pellet) were analyzed by one-dimensional slab gel electrophoresis and fluorography. The comparison of the electrophoretic patterns obtained, demonstrated that a glycoprotein of MW 48 000, undetectable in SCEC, was present in SCOC after labelling with both sugar precursors. The MW 48 000 glycoprotein was also present in the electrophoretic profile of particulate fraction from [3H]fucos-labelled Sertoli cell cultures from prenatally irradiated rat. Such difference was not observed after labelling with [3H]leucine; in this experimental condition a MW 48 000 band was present in the electrophoretic profile of polypeptides from SCEC as well from SCOC. The synthesis of this glycoprotein represented a specific and stable cell response, since it occurred only a few hours after germ cell removal, and it was still detectable 3 days later. FSH stimulation did not influence the synthesis of the MW 48 000 glycoprotein, whereas it increased the synthesis of high MW glycoproteins. The hypothesis is discussed that the appearance of a new glycoprotein when Sertoli cells have lost their contact with germ cells could represent a product of glycosylation of preexisting molecules and their possible location in the Sertoli cell membrane. The results presented here provide additional evidence that Sertoli cell functions may be dependent on the association with the germ cell.

Demonstration of alpha 1-acid glycoprotein (orosomucoid) by double one-dimensional slab gel electrophoresis: evidence for intra- and interindividual variability of the microheterogeneity pattern in health and disease.

This double one-dimensional slab gel electrophoresis technique, in the sequence polyacrylamide gel electrophoresis followed by isoelectric focusing in polyacrylamide gels, permits the selective demonstration and comparison of the micro-heterogeneity pattern of alpha 1-acid glycoprotein (orosomucoid) in as many as 96 human plasma or serum samples on one gel. The electrophoretic analysis is performed on 8 microL of serum or plasma without prior purification. Several hundred samples can be analyzed by one investigator during a working day. Densitometric evaluation of the patterns revealed two new findings: 1) The microheterogeneity pattern can be described by a parameter that is independent of the concentration of orosomucoid in plasma: the center of density of the pattern, with the peak number as coordinate. 2) There is a considerable average shift of the center of density towards more basic components among the patterns obtained for samples from hospital patients. The results suggest that followup of the intra-individual variation of the orosomucoid pattern in health and disease might help in studying the still-uncertain function of this protein as well as the diseases affecting the pattern.