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JPC - Journal of Planar Chromatography - Modern TLC
Authors: Mitja Križman, Jernej Jakše, Mirko Prošek, Dea Baričevič, and Branka Javornik

Agarose gel electrophoresis is a basic separation tool used in molecular biology, mostly for qualitative DNA analysis. There are constraints limiting its use in quantitative analysis, namely low repeatability and a narrow linear range. However, by using an internal standard or internal normalization, repeatability and linear range could be significantly improved. In the work discussed in this paper it was shown that an approximately fivefold improvement in repeatability and an over threefold wider linear range could be achieved by applying internal normalization. Using the proposed approach, genetic markers, for example RAPD and PCR-RFLP, or even microsatellite markers, could be conveniently quantitatively assessed using agarose gel electrophoresis.

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Binding of fluorescein isothiocyanate-labeled concanavalin A to a series of molecular species of lipopolysaccharide (LPS), purified from pathogenic bacteria, was studied via agarose gel precipitation experiments and the results were compared with available structural data.The LPS species could be divided into ConA-reactive and non-reactive ones. Reactivity resided in the O-specific chain of LPS, and binding to the lipid A or core moieties of LPS could not be demonstrated by the present methods. The α-D-glucose or α-D-mannose residues of the repeating O-specific oligosaccharide units appeared to be recognized by ConA, except when blocked by steric hindrance. Specificity of the reaction was verified by inhibition with 2% D-glucose. Binding by bacterium-specific sugar-residues could not be demonstrated.For precipitation to occur, polyvalency was required both for LPS and ConA, and the resulting precipitation appeared to be promoted by hydrophobic interactions between the lipid A moieties of LPS molecules. The LPS species were differently retained by the agarose gel, which can be explained by differences in their micellar structure in aqueous solution. E. coli O83 LPS did not readily diffused in 1% agarose gel, but its precipitation with ConA could be demonstrated either at elevated temperature or mixing it previously with molten agarose (Mancini’s arrangement).

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. 11 31 915 Flores, N., Vale, F., Bolivar, F., Merino, E. (1992) Recovery of DNA from agarose gels stained with methylene blue. Biotechniques 13

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-plasma functionalized polyethylene and glass surfaces . J. Biomater. Sci. Polym. E. , 12 , 1027 – 1049 . Guisán , J.M. 1988 : Aldehyde-agarose

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Schaffer, H. E. and Sederoff, R. R. (1981): Improved estimation of DNA fragment length from agarose gels. Analyt. Biochem. 115, 113122. Improved estimation of DNA fragment length from agarose gels

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Shillito, R. D., Paszkowski, J., Potrykus, I. (1983) Agarose plating and a bead type culture technique enable and stimulate development of protoplast derived colonies in a number of plant species. Plant Cell Rep. 2 , 244

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Adhesion to target cells is an essential step in the pathogenesis of many protozoal infections. Some protozoa have been reported to have a lectin activity involved in their attachment to the cell surface. The ligand-receptor interaction involved in Theileria annulata infection is unclear at present, in spite of the fact that some aspects of the process have been investigated. To this end, T. annulata piroplasms have been screened for lectin activity. Blood taken from infected cattle was first depleted of leukocytes and then subjected to ammonium chloride lysis in order to isolate the piroplasms. The piroplasms were homogenised and a crude membrane extract was prepared by centrifugation. To investigate lectin activity in piroplasm proteins, a simple screening procedure was employed for analysing piroplasm proteins binding to various lectin ligands. Numerous immobilised lectin ligands (L-fucose-sepharose, N-acetyl-neuraminic acid-sepharose, N-acetyl-D-galactosamine-agarose, N-acetyl-D-glucosamine-agarose, D-mannose-agarose, β-D-glucose-agarose, α-methyl-D-mannoside-agarose) were incubated with T. annulata piroplasm crude membrane extract. The ligand-bound proteins were eluted and separated by a brief centrifugation and determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The present study suggests that a 32 kDa protein of piroplasm binds to D-galactosyl residues of the agarose matrix and that the binding is inhibited by galactose and not by the other monosaccharides tested.

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Species-specific PCR assay was used for the identification of Hungarian Fusarium graminearum isolates in pure mycelial culture. The Fg16F/Fg16R primer pair of the three known species-specific primers appeared to be the most appropriate one to identify F. graminearum .Two methods were used for comparative determination of the amplicon size of F. graminearum strains: traditional agarose gel electrophoresis, and chip electrophoresis. Our results have shown that the chip electrophoresis is an easy-to-use, time-efficient substitute for conventional agarose gel electrophoresis; moreover it provides a more precise size determination of amplicons. Amplicon size ranging from 415 bp to 421 bp in tested isolates may be associated with genetic diversity in the Hungarian population of F. graminearum .The PCR assay described in this study can be used for the routine detection and identification of F. graminearum without isolation and morphological investigation of this fungus.

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SmpB, a small tmRNA binding protein, is essential for trans-translation. 6His and FLAG tagged SmpB was cloned from Mycobacterium tuberculosis H37Rv. It was expressed in Escherichia coli using the T7 promoter-polymerase system. Anti-FLAG M2 agarose was used for its purification. Mycobacterial SmpB copurifies with other proteins. We identified elongation factor EF-Tu in the purified SmpB preparations.

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The purpose of this work was to study whether the bovine leukocyte adhesion deficiency (BLAD) allele is present in native cattle breeds and the Holstein breed in Turkey. Blood samples were obtained from 120 Holstein, 20 Brown Swiss, 20 Anatolian Black, 20 Turkish Grey, 20 South Anatolian Red and 20 East Anatolian Red cattle. The isolated DNA materials were multiplied in PCR using the primer developed by Kriegesmann et al. (1997). In order to determine the area of mutation in PCR products, the PCR products were digested with TaqI endonuclease enzyme. The resulting fragments were analysed on 2% agarose gel for the absence of a TaqI restriction site. It was found that two of the Holstein cattle (a bull and a cow) were heterozygote BLAD carriers. There was no homozygote BLAD animal. The BLAD allele was not found in the other breeds used in the study. The mutant BLAD allele frequency in the 120 Holstein cattle calculations was 0.0084.

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