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At least 19 recordsLinked to original sources
PubMed1983

Quantitation of hemoglobin biosynthesis with agarose isoelectric focusing.

Isoelectric focusing in polyacrylamide gels has been widely used to separate fetal (HbF) and adult (HbA) hemoglobins, and the relative synthesis of HbA and HbF has been estimated by fluorography or autography of dried gels. In order to measure the absolute synthesis of hemoglobins, we developed a system that utilizes isoelectric focusing in agarose and quantitates the total radioactivity of separated hemoglobins. After isoelectric focusing, the protein bands are individually eluted from the agarose and the radioactivity measured by liquid scintillation counting. We used this technique to study the synthetic capabilities of erythroid precursors at sequential times in culture. As previously reported, the relative ratio of HbF decreased over time in culture. However, our results clearly revealed that the absolute synthesis of HbF did not decrease until there was a parallel decrease in hemoglobin A, and that changes in the relative ratio occur because of disproportionate increases in HbA. This methodology, allowing independent evaluation of the radioactivity in synthesized hemoglobin, enabled us to gain new insight into the biosynthetic capabilities of erythroid precursors in clonal cell culture.

Cells, Cultured↗
PubMed1975

Evidence for multiple forms and partial resolution of rabbit reticulocyte alpha- and beta-globin messenger RNA by gel isoelectric focusing.

Isoelectric focusing in polyacrylamide gels has been used to fractionate and characterize RNAs from rabbit reticulocytes with major emphasis on globin mRNA. Reticulocyte 18S and 28S RNAs banded essentially as single components, well separated from each other and from the multiple forms of tRNA. By contrast, mRNA was resolved into a number of major and minor components. These bands were shown to contain intact globin mRNA by translation in a messenger-dependent cell-free protein synthesizing system. One major band was enriched slightly in alpha-globin mRNA and a second major band was enriched considerably in beta-globin mRNA. Reticulocyte supernatant mRNA, containing predominantly alpha-globin messenger, demonstrated only one major component which banded at the same position as the alpha-enriched band from total mRNA. Little of this material behaved as beta mRNA either by its focusing profile or by its translation products. Globin mRNA fractions with high and low 3' poly(A) contents also demonstrated differences in focusing distribution profiles. Although the basis for separating RNA by this technique has not been established, our results suggest that isoelectric focusing may offer a new approach to fractionation and characterization of specific mRNA species.

Animals↗
PubMed1986

Transferrin variants in sheep: separation and characterization by polyacrylamide gel electrophoresis and isoelectric focusing.

Isoelectric focusing with carrier ampholytes in ultrathin polyacrylamide gels and polyacrylamide gel electrophoresis in a discontinuous buffer system were used for the separation of sheep transferrin variants. For identification of the different iron-binding sites of transferrin a stepwise urea gradient, different degrees of iron saturation and double one-dimensional electrophoresis were used. Isoelectric focusing results in an increased resolution of the Fe0-transferrin, Fe1-transferrin and Fe2-transferrin region. At the level of Fe0-transferrin and Fe1-transferrin the variants I, A, G, B, C, D, M, E, Q, P can be identified. The method is especially suitable for genetic studies. For screening purposes up to 108 samples can be separated within one run in an ultrathin gel.

Animals↗
PubMed1989

Differentiation of Sarcocystis species in European sheep by isoelectric focusing.

Isoelectric focusing was carried out on total proteins derived from cystozoites of Sarcocystis tenella, S. arieticanis and S. gigantea. The overall protein profile of each species was distinct and reproducible and enabled the unequivocal differentiation of the three Sarcocystis species. In total, approximately 60 bands could be counted for S. tenella and S. arieticanis and approximately 45 bands for S. gigantea cystozoites in the pH range of 3-9. Isoelectric points (pIs) were determined for dominant peaks of densitometric scans of gels in the pH range of 3-9 (cathodal and anodal sample application) and arbitrary identification numbers were assigned to 25 (5 and 20) peaks of S. tenella, 25 (8 and 17) peaks of S. arieticanis and 21 (7 and 14) peaks of S. gigantea; similar assignments were also used for 11 peaks of S. tenella, 18 peaks of S. arieticanis and 12 peaks of S. gigantea observed on densitometric scans of gels in the pH range of 4-6.5.

Animals↗
PubMed1972

Anomalous behavior of yeast isocitrate dehydrogenase during isoelectric focusing.

Isoelectric focusing of yeast isocitrate dehydrogenase apparently reveals a number of ;isoenzymes'. These have isoelectric points near pH5.5 in crude material, but during purification the mean isoelectric point progressively rises to pH7.0 and the band pattern changes. The shift in isoelectric point during purification is apparently genuine, since it is also manifested in the electrophoretic and chromatographic properties of the enzyme. The multiple forms, however, are an artifact, generated by exposure of the enzyme to Ampholine, since their activities vary with the protein/Ampholine ratio and they cannot be observed in any system from which Ampholine is excluded. There are no detectable isoenzymes of yeast isocitrate dehydrogenase.

Buffers↗
PubMed1975

Microheterogeneity of Immunoglobulin G from plasmocytomas. Identification of two types of IgG by isoelectric focusing.

Isoelectric focusing of serum immunoglobulin G (IgG) revealed that the microheterogeneity, which is expressed in the isoelectric points (IEP), partly is caused by differences in the content of N-acetyl-neuraminic acid (NANA), partly by other effects, probably including deamidation. Two different types of Plasmocytoma-IgG, which differ in IEF-pattern, i.e. population distribution, in sensitivity towards neuraminidase and in carbohydrate content, are described. The contents of carbohydrates in these 2 IgG-types are up to 30% greater and 50% less, respectively, than that of normal IgG. A precise correlation was found between the total content of NANA in moles per mole IgG-monomer and the shift in band-IEP on hydrolysis catalyzed by neuraminidase. This may be used for a rapid estimate of NANA in IgG. The results do not permit a discrimination between an anabolic and a catabolic origin of the heterogeneity in the carbohydrate moiety of IgG.

Antibodies, Heterophile↗
PubMed1981

Human thyroxine-binding globulin (TBG): heterogeneity within individuals and among individuals demonstrated by isoelectric focusing.

Isoelectric focusing of human plasma samples labeled in vitro with [125I]-thyroxine reveals considerable biochemical and genetic variation in thyroxine-binding globulin. (1) In all individuals tested, at least three primary isoelectric bands are seen in the pH range of 4.2 to 4.5, with additional bands at lower pH ranges. Similar patterns are produced by plasma from nonhuman primates. These band differences appear to be the result of differences in sialic acid content. TBG produces a single electrophoretic band on standard polyacrylamide gel electrophoresis. (2) Genetically determined, X-linked differences in electrophoretic mobility of TBG are observed in several human populations. Female homozygotes or male hemizygotes for the TBG slow variant (TBG-S) produce band patterns shifted by 0.5 pH unit cathodal to the common pattern (TBG-C). Female heterozygotes produce patterns with six-plus bands, representing the simple sum of the common and slow types. This difference is not the result of differences in sialic acid content. The gene frequency of this variant is 10% in American Blacks. (3) In pregnant women additional anodal bands are observed, giving the impression of a "shift," by integral steps, in the pattern relative to the nonpregnant type. This shift is abolished by mild neuraminidase treatment.

Animals↗
PubMed1991

Demonstration of murine pancreas elastase and its interstrain variation by isoelectric focusing.

Isoelectric focusing between pH 9 and 11 was used for separation of murine pancreas proteases. One of these proteases is characterized by its preference for N-acetyl-L-alanine-alpha-naphthylester as substrate and by its genetic linkage to bt, a coat color marker of chromosome 15. This protease was identified as elastase, and is probably elastase-1 (ELA-1). Because of the simple procedure and the excellent reproducibility of the focusing pattern, ELA-1 is recommended as a useful marker for mouse chromosome 15.

Animals↗
PubMed1983

Characterization of normal and abnormal variants of galactose-1-phosphate uridylyltransferase (EC 2.7.7.12) by isoelectric focusing.

Isoelectric focusing (IEF) in polyacrylamide gels has been used to study the isozymes of human galactose-1-phosphate uridylyltransferase (GALT) in erythrocytes and fibroblasts. In addition to the usefulness of IEF in differentiating normal, Duarte variant, and galactosemic homozygotes and heterozygotes, the ability of IEF to distinguish the residual GALT activity in two different galactosemic fibroblast lines and in revertants from them is demonstrated.

Cells, Cultured↗
PubMed1984

A new alpha 2HS-glycoprotein typing by isoelectric focusing.

Isoelectric focusing (pH 4.0-5.0) of serum alpha 2HS-glycoprotein on polyacrylamide gels has been found to be a useful tool in population genetics and forensic science. Using this method, we isolated three common types, alpha 2HS 1-1, alpha 2HS 2-1 and alpha 2HS 2-2, and showed that alpha 2HS types are determined by two autosomal codominant alleles, alpha 2HS1 and alpha 2HS2. The method is simple, fast and easy to perform. Results of typing for the two alleles, alpha 2HS1 and alpha 2HS2, are described for a Japanese population sample (n = 1003).

Alleles↗
PubMed1981

Transferrin (Tf) subtyping on agarose: a new technique for isoelectric focusing.

Isoelectric focusing (IEF) of the serum protein transferrin (Tf) on polyacrylamide gels has been found to be a useful tool in population genetics and forensic medicine. Three autosomal codominant alleles can be subtyped from the TfC allele, TfC1, TfC2, and TfC3. In this report the authors describe the use of agarose as an alternative technique for Tf subtyping. The method is faster, easier to perform and eliminates the use of toxic chemicals used with polyacrylamide gels. Results of subtyping for the three alleles Tfc1, Tfc2, and Tfc3 are described for a population of U.S. whites (n = 392) and U.S. blacks (n = 194).

Acrylic Resins↗
PubMed1992

Distribution of cerebrospinal fluid oligoclonal IgM bands in neurological diseases: a comparison between agarose electrophoresis and isoelectric focusing.

Isoelectric focusing (IEF) and agarose gel electrophoresis were used to detect oligoclonal IgM bands in cerebrospinal fluid and serum samples from 850 patients with diverse neurological diseases. Oligoclonal IgM bands in cerebrospinal fluid were mainly detected in patients with infectious and inflammatory disorders of the nervous system. Both IEF and agarose electrophoresis revealed similar frequencies of oligoclonal IgM bands. Bands detected by IEF were mainly seen in the anodal range. Despite higher resolving capacity, IEF was less specific than agarose gel electrophoresis. It is concluded that oligoclonal IgM bands have important diagnostic significance and that agarose gel electrophoresis is more suitable for their detection in routine clinical work and use in differential diagnosis.

Blood Protein Electrophoresis↗
PubMedDate not supplied

Analysis of human alcohol- and aldehyde-metabolizing isozymes by electrophoresis and isoelectric focusing.

Isoelectric focusing and electrophoresis were used to identify the various isozymes of alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH), aldehyde oxidase (AOX), and xanthine oxidase (XOX). ADH types I, II, and III were located primarily in the cytosol fraction of liver, but some activity was found also in the small granule fraction. The ALDH-I and -IV isozymes were found in the large granule fraction, while ALDH-II and -III were present in the cytosol and ALDH-V in the small granule fraction. AOX and XOX each appeared as a single cytosolic form with some small granule activity. The tissue distribution of these isozymes is presented and the physiological role of each enzyme is discussed.

Adult↗
PubMed1981

Demonstration of oligoclonal immunoglobulin G in Guillain-Barré syndrome and lymphocytic meningoradiculitis by isoelectric focusing.

Isoelectric focusing (IEF) of serum and CSF revealed oligoclonal IgG in 13 out of 16 patients with the Guillain-Barré syndrome (GBS), in 10 of them in serum only and in 3 in serum and CSF. Seventeen out of 19 patients with lymphocytic meningoradiculitis (LMR) showed oligoclonal IgG, 12 of them in CSF only. These findings, together with additional results, mean that in GBS oligoclonal IgG is synthesized mainly outside the CNS and in LMR within the CNS. Follow-up studies revealed changes in the oligoclonal IgG during the course of GBS and LMR. After treatment by plasma exchange the disappearance of oligoclonal IgG bands was followed by an improvement of GBS symptoms. The oligoclonal IgG bands returned in correlation with worsening of the disease. We were not able to elucidate the antibody character of oligoclonal IgG in GBS or LMR. No antibodies against the myelin basic protein (fragment 89-169) were detectable in the sera and CSF in any of the patients tested.

Adolescent↗
PubMed1983

Identification of Pacific rockfish (Sebastes) species by isoelectric focusing.

Isoelectric focusing (IEF) is currently the most reliable method available for the identification of fish species. The high resolution of this method usually allows discrimination between even closely related species. One genus, the Sebastes, does present a problem however. Using both low and high resolution, IEF is unable to differentiate several species. Disc electrophoresis, used in an AOAC official final action method, does not differentiate the rockfish reliably. Using IEF, identical protein patterns were obtained for Pacific Ocean perch (Sebastes alutus), Bocaccio rockfish (S. paucispinis), and yelloweye rockfish (S. ruberrimus). A second group, comprised of silvergray rockfish (S. brevispinis), yellowtail rockfish (S. flavidus), black rockfish (S. melanops), and canary rockfish (S. pinniger), also has identical protein patterns. Widow rockfish (S. entomelas) and chilipepper rockfish (S. goodei) each had a unique pattern, different from the above 2 groups and from each other. The actual taxonomic relationships of these rockfish species are not clear and further work with IEF may help in this regard. Users of IEF and disc electrophoresis for identification purposes should be aware of this problem when working with the Sebastes.

Animals↗
PubMed1980

Determination of glycosylated hemoglobins in neonatal blood by isoelectric focusing.

Isoelectric focusing (IEF) with suitable "spacers" can be used to distinguish glycosylated hemoglobins from HbA and HbF in adults and infants, respectively. Using this technique we have shown that there is a good correlation between glycosylated hemoglobins measured by IEF and column chromatography (r = 0.778, P less than 0.001). There was no significant difference in the rate of glycosylation between adult and cord blood hemolysates following incubation with glucose in vitro: In both instances there was an increase from the normal levels of about 5% to 26% through 31% after 18 hours of incubation. The levels of glycosylated hemoglobins in infants of varying gestation, neonates, and normal adults was remarkably constant, varying from 4.7 +/- 1.1% to 6.1 +/- 1.6%. These results show that IEF can be useful in the quantitation of glycosylated hemoglobins, particularly in the presence of hemoglobins (eg, HbF) where standard chromatographic techniques cannot be used. IEF has the added advantage of using small quantities of blood, and allows a number of samples to be tested at the same time, thus facilitating the identification of abnormal hemoglobins.

Adult↗
PubMed1983

Isoelectric focusing and isoelectric points of aminoacyl-tRNA synthetases.

Isoelectric points and isoelectric focusing behaviour of 10 highly purified eukaryotic aminoacyl-tRNA synthetases from 3 sources, Saccharomyces cerevisiae, Euglena gracilis and Phaseolus vulgaris were examined. The pI-values measured on polyacrylamide gels under native conditions are situated between pH 5.0-7.5. A microheterogeneity was observed for 9 enzymes appearing otherwise homogeneous on gel electrophoresis. A compilation of the isoelectric points of aminoacyl-tRNA synthetases is given and literature data are compared with our experimental results.

Amino Acyl-tRNA Synthetases↗
PubMedDate not supplied

Capillary isoelectric focusing and isoelectric buffers: an evolving scenario.

The present review offers a new look at capillary isoelectric focusing (cIEF) by centering on the most troublesome aspects of the technique, namely: 1) how to modulate the slope of the pH gradient, for increasing resolution (equivalent to pH gradient engineering, as easily available in immobilized pH gradients); and 2) how to keep proteins in solution at (and in the proximity of) the pl value. A simple solution is offered in the first case: addition, to the standard 2-pH-units interval, of separators or spacers, i.e., of amphoteric molecules (either single or in combination) able to locally flatten the pH and increment resolution. Examples of the separation of fetal and glycated hemoglobins are provided. In the second case, a unique solubilization power (while maintaining full protein integrity and enzyme activity) is obtained if class I solubilizers are used. They consist of mixtures of sugars (e.g., sucrose and sorbitol) at ca. 1 M concentration, with zwitterions (up to 1 M) such as the class of nondetergent sulfobetaines, but also taurine and some of the Good's buffers (e.g., CAPS). In these solvents, the protein exists in a state of superhydration and its solubility is greatly augmented. The review ends with an excursus on the use of isoelectric buffers in zone electrophoretic separations. Such isoelectric buffers offer unique advantages: They permit very-high-voltage gradients (up to 1000 V/cm) and thus minimize analysis times (down to a few min in 30-35 cm long capillaries). This results in a marked increase in resolution, due to minimal diffusion-driven peak spreading. Such buffers are finding unique applications for generating peptide maps of tryptic digests of proteins and also in the analysis of oligonucleotides.

Animals↗