Separator Isoelectric Focusing Research Articles

Orosomucoid (ORM) Typing by Isoelectric Focusing: An Analysis of ORM Haplotypes

A new separator isoelectric focusing method for typing of orosomucoid (ORM) was developed. This method provided a superior resolution of ORM patterns: two close bands of ORM1*5.2 products were clearly separated. A total of 364 subjects from Okinawa (Japan) were classified into 21 ORM phenotypes determined by 6 ORM1 and 7 ORM2 alleles including a polymorphic silent allele, ORM2*QO, and 2 new rare variants, ORM2*18 and ORM2*19. These phenotypes were also explained by 12 ORM haplotypes, half of which were polymorphic.

Group Specific Component Subtyping in Bloodstains by Separator Isoelectric Focusing in Micro-Ultrathin Polyacrylamide Gels Followed by Immunoblotting

The identification of group specific component (Gc) subtypes derived from blood-stains by separator isoelectric focusing in micro-ultrathin polyacrylamide gels (interelectrode distance: 50 mm) containing 4.5 to 5.4 pharmalytes is described. The separation achieved between Gc 1F and Gc 1S bands is compared favorably with that obtained using separator isoelectric focusing in conventional polyacrylamide gels dimensions (interelectrode distance: 110 to 120 mm). The technique is rapid and economical, and the immunoblotting method described is more sensitive than immunofixation followed by silver staining.

PI Mtoyoura: a new PI M subtype found by separator and hybrid isoelectric focusing.

In the course of a population study of alpha 1-antitrypsin polymorphism by separator isoelectric focusing, a variant phenotype having a somewhat narrower spacing than PI M1M3 was observed in a Japanese blood donor. Family studies by hybrid isoelectric focusing in a carrier ampholyte-supplemented immobilized pH gradient from 4.35-4.65 revealed that the products of the responsible gene, PI*Mtoyoura, were extremely close but slightly cathodal to those of PI*M1. The difference in isoelectric point between them corresponded to the resolving limit of isoelectric focusing. For this reason, although the propositus' father was deduced to have the genotype PI*M1/PI*Mtoyoura, the products of these two genes failed to form a double band pattern. Thus, the gene frequency for PI*Mtoyoura was unknown. These findings, however, indicate that a further microheterogeneity in the PI M subtype exists at least in the Japanese.

The Use of Separator Isoelectric Focusing in Micro-Ultrathin Polyacrylamide Gels in the Characterization of Some Polymorphic Proteins of Forensic Science Significance

The identification of phenotypes of erythrocyte acid phosphatase (EAP), esterase D (EsD), group specific component (Gc), and alpha-1-antitrypsin (PI) by separator isoelectric focusing in micro-ultrathin polyacrylamide gels (interelectrode distance: 45 mm) is described. The protein patterns obtained are compared favorably with the patterns seen by isoelectric focusing in conventional polyacrylamide gel dimensions (interelectrode distance: 110 to 120 mm). The technique described allows greater stability of pH gradients and is a fast and economic method.

Distribution of alpha-1-antitrypsin (Pi) phenotypes in Denmark determined by separator isoelectric focusing in agarose gel.

The distribution of phenotypes of alpha 1-antitrypsin (Pi) in 909 unrelated Danes was determined by the use of separator isoelectric focusing in agarose gel. The frequencies calculated were: PiM1 = 0.728, PiM2 = 0.136, PiM3 = 0.082, PiZ = 0.023, PiS = 0.022, PiF = 0.006, Pivar = 0.003. The segregation of phenotypes in 39 families with 94 children is presented. The advantages and disadvantages of the method are discussed.

Placental alkaline phosphatase types and subtypes determined by agarose gel electrophoresis and separator isoelectric focusing.

Two techniques for phenotyping the human placental alkaline phosphatase system were developed: high-voltage agarose-gel electrophoresis and thin-layer separator isoelectric focusing on agarose. These methods enabled a more rapid and sensitive phenotyping of all common phenotypes than the traditionally employed starch-gel electrophoresis. An extended polymorphism of placental alkaline phosphatase was revealed by isoelectric focusing. The existence of two suballeles of Pl1 allele and two suballeles of Pl2 allele was postulated.

A new alpha 1-antitrypsin allele PI Poki: isoelectric focusing with immobilized pH gradients as a tool for identification for PI variants.

A new rare genetic variant PI Poki in the protease inhibitor system found in two Japanese subjects, was compared with some PI P variants using several electrophoresis systems. Although this variant was slightly anodal to P by separator isoelectric focusing, it could be readily distinguished by isoelectric focusing with immobilized pH gradients: the mobility of Poki became cathodal to P. This method proved to be a powerful tool for identification of PI variants.

Application of separator isoelectric focusing within the pH range 4‐6

AbstractSeparator isoelectric focusing is a simple technique for creating narrow pH gradients (less than 1 pH unit wide). It has proved useful elsewhere for the enhanced separation of phosphoglucomutase, esterase D, group specific component (Gc) haemoglobin, transferrin and for distinguishing between seminal and vaginal acid phosphatases. The properties of 10 different zwitterionic buffers have been studied in order to determine pH gradient profiles, their stability, experimental reproducibility and the effect of temperature. The effect of adding non‐ionic constituents (glycerol or sucrose) to the gel has also been investigated. The buffers examined produced narrow pH gradients within the pH 4–6 range. It was shown that specific pH gradients could be produced by mixing different buffers and carrier ampholytes together.

PI(alpha 1-antitrypsin) polymorphism in the Japanese: confirmation of PI*M4 and description of new PI variants.

PI phenotyping by separator isoelectric focusing (SIEF) was performed on a total of 1000 unrelated Japanese individuals from two different areas of Western Japan. The PI M1M4 subtype was observed together with the six common PI M subtypes. PI*M4 was confirmed to be present but rare in the Japanese. Several new PI variants were identified by comparison runs of each variant with previously reported genetic variants. The significance of treatment of serum with dithiothreitol (DTT) followed by iodacetic acid (IAC) in determination of PI variants is also described.

An improved method for phenotyping of alpha 1-antitrypsin variants using separator isoelectric focusing on thin-layer agarose gel

The previously described method for phenotyping of alpha 1-antitrypsin (alpha 1-protease inhibitor, Pi) that utilizes separator isoelectric focusing on thin-layer agarose gel (A. R. Qureshi and H. H. Punnett, in Electrophoresis '81, 3rd International Conference on Electrophoresis, pp. 83–87 (1981)) has been improved to give a better resolution of Pi pattern. A shallow pH gradient in the region of the isoelectric point of Pi pattern was obtained by the use of N-(2-acetamido)-2 aminoethanesulfonic acid (1%) and serine (0.8%). The present technique can resolve the Pi alleles. The patterns of Pi phenotypes were found to be similar to those observed on acrylamide gels. The method is fast, reliable, and reproducible.

The Gm--Pi linkage heterogeneity in view of Pi M subtypes.

In this study linkage between the loci for Gm (gamma-type heavy-chain immunoglobulin markers) and Pi (alpha 1-antitrypsin/alpha 1-protease inhibitor) has been shown in families segregating for the Pi M subtypes (M1, M2, M3 and Msal) as identified by separator isoelectric focusing . The estimate for the Gm--Pi (M type) recombination is 0.29 (95% limits 0.24--0.37) at a peak lod score of 4.31 and with no sex difference. This value is not significantly different from updated recombination frequency estimates for Gm--Pi in Pi MS (0.26) and Pi MZ, SZ and FZ families (0.21). The overall Gm--Pi recombination fraction estimate of 0.26 (95% limits 0.23--0.30) at a peak lod score of 20.75 must now be considered as solid. There is a significant heterogeneity within the male Pi MZ families in that the new Finnish families show a higher recombination between Gm and Pi. There is also a possible segregation distortion (Z:M = 23:8). The heterogeneity is discussed in terms of haplotypes, the behaviour of which could be determined by linked genes or chromosomal rearrangements. The possibility that the alpha 1-antitrypsin level influences recombination frequency has not been ruled out, but cannot explain the heterogeneity within Pi MZ families.

PH Gradient flattening in isoelectric focusing in long polyacrylamide gels

AbstractIsoelectric focusing in 60 cm long gels improves resolution in multiple‐component systems by providing very shallow, linear pH gradients. Broad range ampholytes available commercially or synthesized in our laboratory develop pH gradients of 0.1 pH/cm at voltages of 1500 to 4500 V, with running times of 25–30 h. Shallower gradients result with narrow range ampholytes or separator isoelectric focusing. Hemoglobin, human serum albumin, and an α‐N‐acetylgalactosaminidase have been used for defining this system.

Reliable Classification of Six Pi M Subtypes by Separator Isoelectric Focusing

For the first time, segregation of three common PiM alleles in family material is verified by application of separator isoelectric focusing. A new nomenclature system for the Pi M subtypes is used, whereby the common subtypes are designated according to their physicochemical properties; the most anodal type is called Pi M1, the intermediary one Pi M2, and the most cathodal variant Pi M3 (previously called Pi M2). Pi gene frequencies from Finnish, Dutch and Black populations are presented. The PiM2 allele was rather high in Finns (0.12) but low (0.04) in the West African Bozo. The PiM3 was found with a frequency of 0.13 in Dutch, 0.08 in Finns and 0.02 in Bozo. A previous Finnish sample was retested with the new subtyping method and the six-subtype distribution was found to be in good Hardy-Weinberg equilibrium. The validity of the Pi polymorphism for population genetics, linkage analysis and parentage testing is discussed.

Separator isoelectric focusing: An improved method of protein analysis and purification

Addition of small amphoteric substances or separators to the carrier ampholytes used in isoelectric focusing improves the separation of protein components. The technique has been applied to partially oxidized human hemoglobin, goat anti-bovine pancreatic oxidized ribonuclease, human γ-globulin, and barley β-amylase. Each of these proteins is a complex mixture of species which can be resolved selectively and reproducibly by this method. Superior separation is provided by this technique when compared to focusing in commercially available narrow-range ampholytes. Addition of separators usually results in the flattening of the pH profile of the gel. Inspection of the protein patterns indicates that no new protein bands arise, although different patterns of existing components are obtained by choice and concentration of separator as well as by concentration of carrier ampholytes. This technique, thus, offers the advantage of making conventional isoelectric focusing more versatile.