Biomedical subjects

K Ek

Publications and source records attributed to K Ek.

5 recordsLinked to original sources

PubMed1983

Preparative isoelectric focusing in immobilized pH gradients. I. General principles and methodology.

A new method for preparative protein purification is described, based on the use of Immobiline matrices. After electrofocusing, the protein zone of interest is recovered by electrophoretic transfer to a hydroxyapatite gel, from which it is eluted with 0.2 M phosphate buffer, pH 6.8, with yields for the proteins studied in the range 76-98%. For six different proteins, the focusing step gives a common upper limit of approximately 45 mg protein/ml gel as mean concentration in a focused protein zone. It is demonstrated that in practical preparative work, components with a pI difference of 0.007 pH units can be completely resolved, and that on a 5-mm-thick gel of dimensions 240 X 110 mm, samples containing as much as 400 mg of the major protein component can be applied. Focusing of large amounts of a salt-containing sample is demonstrated with the aid of human serum. A theoretical expression is given relating the concentration distribution and maximum protein concentration within a focused zone to the applied voltage, the pH slope used and the zone width. Based on this expression and the finding of an upper concentration limit for a protein we shown how to optimize the parameters in preparative work with immobilized pH gradients in relation to the separation power needed. Finally, it is shown that, in comparison with conventional preparative electrofocusing in polyacrylamide gels, immobilized pH gradients allow a ten-fold increase in load, whilst still giving a resolution comparable to that of analytical isoelectric focusing.

Animals↗

PubMed1982

Isoelectric focusing in immobilized pH gradients: principle, methodology and some applications.

A new technique for generating pH gradients in isoelectric focusing is described, based on the principle that the buffering groups are covalently linked to the matrix used as anticonvective medium. For the generation of this type of pH gradient in polyacrylamide gels, a set of buffering monomers, called Immobiline (in analogy with Ampholine), is used. The pH gradient gels are cast in the same way as pore gradient gels, but instead of varying the acrylamide content, the light and heavy solutions are adjusted to different pH values with the aid of the Immobiline buffers. Available Immobiline species make it possible to generate any narrow linear pH gradient between pH 3 and 10. The behaviour of these types of gradients in isoelectric focusing is described. Immobilized pH gradients show a number of advantages compared with carrier ampholyte generated pH gradients. The most important are: (1) the cathodic drift is completely abolished; (2) they give higher resolution and higher loading capacity; (3) they have uniform conductivity and buffering capacity; (4) they represent a milieu of known and controlled ionic strength.

Acrylic Resins↗

PubMed1980

Affinity titration curves: determination of dissociation constants of lectin-sugar complexes and of their pH-dependence by isoelectric focusing electrophoresis.

By performing electrophoresis perpendicular to a stationary pH gradient (pH-mobility curves) in polyacrylamide gels containing a specific ligand either covalently fixed or entrapped in the gel matrix, it is possible to measure dissociation constants (Kd) and their pH-dependence in the pH range 3.5-10. The present technique, called 'affinity titration curves', is an extension of 'affinity electrophoresis'. This system has been applied to the study of the interaction between lectins and sugars: lectin from Ricinus communis seeds and alpha-D-galactose, and lectin from Lens culinaris seeds and alpha-D-mannose. The pH-dependence of Kd values indicated a more rapid decrement of affinity of both lectins for their ligands at acidic pH as compared to alkaline pH. For both lectins, maximum affinity was found in the pH range 7-8. Since the ionic strength of focused carrier ampholytes is 100-200-times lower than in conventional electrophoresis, the Kd values found by the present method are generally lower than the same values obtained by affinity electrophoresis.

Carbohydrates↗

PubMed1979

Use-effectiveness and client satisfaction in six centers teaching the Billings Ovulation Method.

Use-effectiveness of the Billings Ovulation Method (OM) is reported for 1139 clients from six centers who were followed for at least 24 months. Method failure rates were 1% when calculated by either Pearl formula or life table analysis. Combined method and user failure rates were 20% when calculated with the Pearl formula, and 16% at 12 months and 23% at 24 months by life table analysis. Pregnancy rates were higher for couples using OM alone, than for couples using OM in combination with additional fertility awareness methods or together with barrier methods. The difference was significant when estimated by the Pearl formula but not when analyzed by the life table. The emotional implications underlying the apparent contradiction between a desire to avoid pregnancy and the deliberate coital use of fertile days are reflected in the differences between method and user failure rates, the high client satisfaction levels reported with "user failure," and 56% continuation rate at 24 months. The will require in-depth exploration.

Abortion, Legal↗

PubMed1979

Agarose isoelectric focusing of native human immunoglobulin M and alpha 2-macroglobulin.

The resolution of native 19S IgM and alpha 2-macroglobulin by agarose isoelectric focusing is described. The agarose used was practically charge free, thus avoiding disadvantages of electroendoosmosis, and gives a very large network gel, with minimal molecular sieving effects. The resolving power is comparable to that obtained in thin-layer isoelectric focusing in polyacrylamide gel. Clones of human IgM showed a microclonal heterogeneity, similar to IgG antibody heterogeneity, alpha 2-macroglobulin gave a pattern of seven bands in the pI range of 4.1 to 4.9. The flexibility of the agarose isoelectric focusing (IEF) system with regard to immunodetection techniques is illustrated by the use of immunofixation and two-dimensional crossed immunoelectrofocusing. The agarose IEF method has several advantages, viz., non-toxicity, simple handling, uniform and rapid gel formation, and considerably shortened fixing and staining times. The value of the new method is discussed, in particular its usefulness in detecting and isolating IgM antibodies of known specificity produced by cells in culture.

Humans↗