Peptide analysis by isoelectric focusing in polyacrylamide gels

We have examined the use of isoelectric focusing in polyacrylamide gels for the analysis of heterogeneous mixtures of cyanogen bromide peptides. High resolution, sensitivity, and reproducibility are obtainable under conditions which are described. Peptides having molecular weights above 1000 or 2000 can be visualized by fixation and staining. The presence of urea in the gels is important to the procedure; formation of carbamylated derivatives from this cause occurs at most in trace amounts in unfavorable cases. No artifactual heterogenelty from any other cause was apparent.     

Heterogeneity and specificity of human anti-insulin antibodies determined by isoelectric focusing

Anti-insulin antibodies are present in the majority of insulin treated diabetics, and in some cases these antibodies have been found to be highly specific for limited epitopes on the molecule. To determine how the human response differs from that seen in inbred animals, we have examined the heterogeneity and specificity of human anti-insulin antibodies by isoelectric focusing (IEF). In addition, we have used human insulin to examine the extent of autoreactivity in the serum of subjects treated with animal insulins. The majority of diabetic sera exhibited complex IEF spectra that were composed of discrete bands and unresolved smears. Autoradiography using 125I-beef, pork, and human insulin revealed some affinity differences; however, the predominant antibodies were capable of binding all insulins, including human. These specificity studies were extended by comparing competitive inhibition with excess cold insulins, and sera with highly specific binding of the A chain loop of beef insulin were identified. The spectra by IEF of these highly specific sera were found to be variable. Our results indicate that the majority of insulin-treated diabetics develop a heterogeneous antibody response that is more complex than the response of inbred animals and includes reactivity with autologous insulin. Although infrequent, individuals having antibodies directed at limited regions of the molecule can be identified and will provide valuable tools for dissecting this complex response.     

Ultrathin-layer isoelectric focusing in polyacrylamide gels on cellophane.

A method of ultrathin-layer isoelectric focusing in 0.12-, 0.24-, or 0.36-mm polyacrylamide gel layers polymerized on a sheet of cellophane as support is deseribed. The gel adheres firmly to the cellophane during all operation steps, is protected from fracture, and can be handied very conveniently. Resolution is markedly improved in ultrathin gels in comparison with the conventional 1- to 2-mm-thick gels. Staining and destaining are completed in a substantially shorter time than so far achieved. The ultrathin gels can be easily dried on the cellophane, a perfectly transparent record being obtained for future reference and for densitometric evaluation. Results are presented for a number of commercial proteins and legume seed proteins. The advantages of ultrathin-layer isoelectric focusing are discussed.     

Phophoglucomutase first locus polymorphism as revealed by isoelectric focusing in Southern Africa

Eleven Southern African populations (representing European, Asian and Negroid populations) have been typed for the first locus phosphoglucomutase (PGM1) using isoelectric focusing (pH range 5.0-8.0) in acrylamide gels. The gene frequencies of the four common alleles at this locus in these populations were compared to those found previously in European and Negroid populations. Marked differences in gene frequencies were observed: Negroes have a lower PGM1(2-) compared with Caucasoids due to a lower PGM1(2-) frequency, Indians a relatively high PGM1(2) due to a higher frequency of the PGM1(2+) allele. The Afrikaans and Ashkenazim do not differ appreciably from their European counterparts. The appearances of the rarer PGM1(6) and PGM1(7) alleles on isoelectric focusing are described and some kinetic properties examined. The PGM2(2-1), or 'Atkinson' phenotype, can also be detected with this technique.     

Isoelectric focusing in layers of granulated gels. II. Preparative isoelectric focusing.

A method for preparative isoelectric focusing of 0.1-10 g amounts of proteins is described. For anticonvective stabilization of the pH gradient, layers of granulated gels (E.G. Sephadex or Bio-Gel) of variable length, width and thickness were used either on glass plates or in troughs. Load capacity, defined as the amount of protein per ml gel suspension, was determined to be 5-10 mg per ml for total protein, irrespective of the pH range of the carrier ampholytes. For single proteins load capacities of 0.25-1 mg per ml were found for pH 3-10 carrier ampholytes, and 2-4 mg per ml for narrow pH range ampholytes. Experiments on a quartz plate followed by densitometric evaluation in situ at 280 nm have demonstrated that it is possible to proceed from analytical thin-layer isoelectric focusing to preparative separations without loss of resolution, just by changing the dimension of the gel layer and increasing the protein load. Improved resolution which facilitates isolation of isoelectrically homegenious components could be achieved on a 40 cm long separation distance. The geometry of a layer is favourable to heat dissipation and this permits the use of high voltage gradients. Recovery of the focused proteins is high an elution simple. The efficiency of the method is illustrated by examples showing separations of single proteins and protein mixtures.     

Parvalbumins for calibrating acidic isoelectric focusing gels

Partially purified fish and amphibian parvalbumins are compared to several proteins commonly used in commercial standard mixtures for calibrating isoelectric focusing gels. Parvalbumins are proffered as useful standards for acidic ranges on the basis of conformity to a set of five criteria.     

Analysis of colloidal gold probes by isoelectric focusing in agarose gels

Colloidal gold particles of different size (3-20 nm in diameter) were prepared by tannic acid-citrate and citrate reduction methods. From these colloids, different probes were prepared using sheep anti-rabbit antiserum, sheep anti-rabbit IgG, bovine serum albumin, polyethylene glycol, and protein A as the primary stabilizers and polyethylene glycol and/or bovine serum albumin as secondary and tertiary stabilizers, in different combinations. The probes were analyzed by isoelectric focusing in agarose gels, which allow the migration of particles in the size range 3-20 nm. (P. Sewer and S. J. Hayes, 1986, Anal. Biochem. 158, 72-78). Isoelectric focusing revealed that the surface charge of colloidal gold probes is dependent upon the size of the gold particle, the reduction method used, the primary ligand, and the pH at which this is adsorbed, as well as upon the secondary and tertiary stabilizers used. It is proposed that such differences in surface charge may underlie the different results which may sometimes be observed in colloidal gold labeling, especially when novel ligands are used.     

Staining protein in isoelectric focusing gels with fast green

A rapid, simple technique for staining proteins in isoelectric focusing polyacrylamide gels was demonstrated using fast green in 10% acetic acid. Fast green has the distinct advantage of not binding to ampholytes, thus staining only protein. Maximum staining was achieved within 5 min, and bands were visible after 3 to 6 h of destaining. Background stain removal, however, was not complete until 72 h after placing gels in a diffusion destainer. Gel quantitation was demonstrated with actin using fast green and Coomassie brilliant blue R-250. A standard curve prepared with fast green was linear from 0.5 to 8 μg of actin in contrast to Coomassie brilliant blue R-250 which provided linearity from 0.1 to 2.5 μg actin. Application of fast green staining to quantitation of α-actin from cultured muscle satellite cells has been demonstrated.     

Segregation of membrane components using isoelectric focusing in polyacrylamide gels

A method for efficiently fractionating human erythrocyte membranes into their individual components is presented. This procedure employs isoelectric focusing in polyacrylamide gels after solubilization of the membranes in 8M urea, 0.02M EDTA, 0.2% 2-Me. Electrofocusing of membranes affords higher resolution than previous membrane separation procedures. Also, this method is rapid, requiring 72 hours at most to separate, stain and destain the membrane components in the polyacrylamide gels.     

An environmentally determined polymorphism in malate dehydrogenase isozymes ofMyrmica rubra as revealed by isoelectric focusing

Summary A polymorphism in the enzyme malate dehydrogenase in Dorset populations ofMyrmica rubra was detected using isoelectric focusing (IEF). The polymorphism was not detected on native polyacrylamide gels. Two forms, with pI values of 4.9 and 5.7, were resolved.Several lines of evidence show that the polymorphism has an environmental rather than a genetic basis. The cause of the change from one phenotype to the other may be related to a seasonally varying factor. The results indicate that whilst IEF has great potential for revealing isozyme polymorphisms in ants, care should be taken in interpreting results.     

Selective elution of zones from preparative isoelectric focusing gels by ampholytes or buffers.

Protein zones formed by isoelectric focusing on polyacrylamide gels (IFPA) can be eluted without mechanical disruption of the gels. Specific elution is achieved by replacement of the original anolyte, a strong acid, with an ampholyte of a pI higher than that of the protein which is to be eluted. Alternatively, the anolyte may be a buffer of a pH higher than the pI of the focused protein zone. A rudimentary apparatus and procedures for the application of this method of zone elution are described but are not as yet sufficiently developed to provide a ready-to-use preparative IFPA procedure.     

Direct spectrophotometric analysis of hemoglobin in isoelectric focusing tube gels

Methods are described for the direct spectrophotometric analysis of human oxyhemoglobin, deoxyhemoglobin, and methemoglobin focused in polyacrylamide tube gels. Visible absorption spectra (350-650 nm) of electrofocused hemoglobin bands were recorded using a diode array rapid-scan spectrophotometer. Direct optical sampling of gels allowed the identification of focused hemoglobin valency hybrids which contain two oxidized monomers per tetramer.     

Two new Bf S subtypes revealed by isoelectric focusing and immunofixation

Summary The genetic types of the properdin factor B were analyzed by isoelectric focusing on polyacrylamide gels and subsequent immunofixation. Sera from 516 unrelated, healthy individuals from Southern Germany were examined. Two new subtypes of the Bf*S allele were observed. They were provisionally named Bf*Sb1 and Bf*Sb2 (b = basic), since the position of their bands is located slightly towards the cathode. Whereas Bf Sb2 has, thus far, been found only in a single individual, Bf Sb1 was found in five unrelated persons and in a mother and her child indicating a simple codominant mode of inheritance. The combined frequency of the Bf*Sb alleles was calculated to be 0.0067.     

Heterogeneity of staphylococcal enterotoxin A on isoelectric focusing and disc electrophoresis.

Heterogeneity of purified staphylococcal enterotoxin A, obtained from a culture supernatant of Staphylococcus aureus, strain 13N-2909, was demonstrated by isoelectric focusing. The toxin was composed of three immunologically identical fractions with isoelectric points of 6.5, 7.0 and approximately 8.0. Heterogeneity of the toxin was also shown by disc electrophoresis. At pH 8.0 and 9.4 two major bands and a faint minor band were observed, while at pH 4.3 only one band was observed. The faster-moving band for the anode in disc electrophoresis at pH 9.4 was found to correspond with the pI 6.5 component from isoelectric focusing, while the slower-moving band corresponded with the pI 7.0 component. From the results of the electrophoretic migration tests of the toxin, the components corresponding to the two major bands found in disc electrophoresis at pH 9.4 were considered to be charge isomers.     

Mapping of zein polypeptides after isoelectric focusing on agarose gels

Isoelectric focusing of zein in agarose gels gives sharp separations of at least 25 bands noted among 25 corn-belt inbreds. Six inbreds provided standard bands which were used to construct a pattern map. A method is provided for comparing bands, identified by distance from the cathode, which differ only slightly in position. The 25 inbreds were separated into five groups on the basis of pattern similarity. Some groups contained inbreds derived from widely different sources. Zein isoelectric focusing in agarose should be useful for genotype identification and for determination of varietal purity.     

Microheterogeneity of human kininogen by isoelectric focusing and crossed immunoelectrophoresis.

LMW kininogen was isolated from whole human plasma by gel filtration on Sephadex G-200 (Kav 0.34) followed by DEAE-chromatography according to earlier established methods. Further purification was performed with specific Sepharose-antibody columns to remove protein contaminants, avoiding procedures which may denature kininogen. The microheterogeneity was investigated by isoelectric focusing in column in the pH-gradients 3.5-10, 4-6 and 3.5-5. Kininogen components were determined by single radial immunodiffusion against monospecific anti-human kininogen serum, in comparison with focusing of whole plasma. 40% of isolated as well as whole plasma kininogen focused at pI 4.5; the respective focusing ranges were pI 4.4-4.7 (60--80%) and pI 4.3-4.6 (92%). The results were verified by crossed immunoelectrophoresis. The pI 4.5 component is apparently the main native form of human kininogen as shown by focusing of whole human blood bank plasma. Earlier described difficulty of separating kininogen and alpha2HS-glycoprotein was verified by crossed immunoelectrophoresis which showed approximately seven kininogen components after focusing in polyacrylamide gel electrophoresis at pI 4.5-5.0 and four alpha 2HS components at pI 4.2-4.6.