Two-Dimensional Electrophoresis and Western-Blotting Analyses with anti Ara h 3 Basic Subunit IgG Evidence the Cross-Reacting Polypeptides ofArachis hypogaea,Glycine max,andLupinus albusSeed Proteomes

The aim of this study was to evaluate the effect of heat stress in in vitro conditions on the induction of heat-shock protein (Hsp)70 by Escherichia coli cells, and to determine the localization of Hsps in cell fractions. The material consisted of wild strains of E. coli isolated from the digestive tract of calves, suspended in an exponential-phase culture and subjected to 41.5 °C for 2 h. Individual fractions were analysed by SDS-PAGE and two-dimensional electrophoresis. Western blotting with mouse anti-Hsp70 and anti-Hsp60 mAbs was used to identify the proteins. Electrophoretic analysis of the heat-treated cells detected Hsp70 in all three fractions, cytoplasmic, periplasmic and membrane, which was confirmed by Western blotting. The proteins obtained had diverse localizations in the pH gradient in two-dimensional electrophoresis, which may indicate changes in their conformation and physical properties leading to stabilization and protection of intracellular structures in stress conditions. The presence of these Hsps in different cell fractions indicates a very strong protective adaptation in the bacteria in unfavourable conditions, which is critical for the organism infected by them.

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Direct end labelling of telomeres

Genome ◽

10.1139/g93-031 ◽

1993 ◽

Vol 36 (2) ◽

pp. 224-229 ◽

Cited By ~ 5

Author(s):

Alexander Kolchinsky ◽

Peter M. Gresshoff

Keyword(s):

Glycine Max ◽

Gel Electrophoresis ◽

Pulsed Field Gel Electrophoresis ◽

Telomeric Sequence ◽

Two Dimensional ◽

Pulsed Field ◽

Two Dimensional Electrophoresis ◽

Restriction Nuclease ◽

Novel Approach ◽

Dimensional Electrophoresis

A novel approach of direct end labelling of telomeres is presented. Chromosome-sized, agarose-embedded DNA was treated with T4 DNA polymerase to remove protruding 3′ end of telomeres and to generate single-stranded 5′ ends. The DNA was then labelled by the same enzyme in the presence of [α-32P|dGTP and cold dATP and dTTP. Labelled yeast chromosomes separated by pulsed field gel electrophoresis maintained their integrity. Digestion of yeast chromosomes separated in pulsed field gels with a restriction nuclease (HinfI), followed by conventional electrophoresis in the second dimension, resulted in a fingerprint-like pattern of labelled telomeres. This was very similar to the hybridization pattern of a similar two-dimensional gel probed with cloned yeast telomeric sequence. The same approach enabled us to label telomeres in soybean, determine their size, and to reveal polymorphisms in the length of telomeres between the closely related subspecies Glycine max (soybean) and Glycine soja.Key words: telomeres, Saccharomyces cerevisiae, Glycine max, two-dimensional electrophoresis, DNA polymorphism.

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Sialic Acid Dependent Polypeptide Chain Heterogeneity of Human Fibrinogen Demonstrated by Two-Dimensional Electrophoresis

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657116 ◽

1982 ◽

Vol 47 (01) ◽

pp. 019-021 ◽

Cited By ~ 13

Author(s):

Cemal Kuyas ◽

André Haeberli ◽

P Werner Straub

Keyword(s):

Sialic Acid ◽

Polypeptide Chain ◽

Two Dimensional ◽

Charge Heterogeneity ◽

Human Fibrinogen ◽

Two Dimensional Electrophoresis ◽

Isoelectric Points ◽

Polypeptide Chains ◽

Dimensional Electrophoresis ◽

Chain Type

SummaryHuman fibrinogen was compared with asialofibrinogen by two-dimensional electrophoresis to evaluate the contribution of sialic acid to the heterogeneity of the γ- and Bβ-polypeptide chains.Reduced fibrinogen showed three major variants for both the γ- and Bβ-chains. In addition two minor γ-bands with a more acidic isoelectric point than the normal γ-chains were observed. Electrophoresis in the second dimension (SDS) suggests that these most acidic bands are γ-chain-variants with a higher molecular weight. In asialofibrinogen only two predominant variants with more alkaline isoelectric points were present in each chain type.It is concluded that enzymatic removal of sialic acid partially reduces the heterogeneity of the γ- and Bβ-polypeptide chains of human fibrinogen, but additional sources producing charge heterogeneity must be sought.

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