Thin-layer Polyacrylamide Gel Research Articles

Transferrin (Tf) polymorphism: An analysis by isoelectric focusing

The polymorphism of the transferrin (Tf) system was studied in a total of 300 unrelated Japanese individuals from Miyagi prefecture, the northern part of Japan, using isoelectric focusing in thin-layer polyacrylamide gel. In our population samples three common phenotypes and nine variants were observed. The calculated allele frequencies were Tf C 1 = 0.773, Tf C 2 = 0.212, Tf D chi (Chinese) = 0.008, and the combined frequencies for the Tf B var (variants) = 0.007. Family data ( n = 44) were in accordance with an autosomal codominant fashion of inheritance. The use of isoelectric focusing procedure among Japanese will raise the probability of excluding a man falsely accused of paternity to 15.8% as compared with 1.5% when the conventional electrophoretic methods are used.

Purification and characterization of multiple forms of mutarotase from hog kidney cortex.

The enzyme mutarotase [aldose 1-epimerase, EC 5.1.3.3] from hog kidney cortex was separated into four fractions (designated types I, II, III, and IV in order of elution) by column chromatography on DEAE-cellulose. Two major forms, types I and II, were purified to homogeneity as judged by polyacrylamide gel electrophoresis and isoelectric focusing on thin layer polyacrylamide gel. Types I, II, III, and IV had isoelectric points of 5.78, 5.48, 5.23, and 5.10, respectively. The following physicochemical properties were common to all four types: molecular weight, 41,000; Km for alpha-D-glucose at pH 7.4 and 25 degrees C, 19 mM; optimum pH, 6.5-7.5; optimum temperature, 30-37 degrees C; heat stability, up to 50 degrees C. On double immunodiffusion, the four types of mutarotase gave single precipitin lines, which fused completely with each other, against the antibody to purified type II enzyme. Types I and II had an identical amino-terminal residue, arginine, and an identical carboxyl-terminal sequence, -(Phe-Phe-Ser-Val)-Val-Ala. The amino acid composition of type I was almost identical with that of type II. Very similar tryptic peptide maps were obtained from types I and II, with only a few points of variance. These results suggest that the four types of hog kidney mutarotase are quite similar but not identical.

Separation of intermediates in the refolding of reduced erabutoxin b by analytical isoelectric focusing in layers of polyacrylamide gel.

Isoelectric focusing in a thin layer of polyacrylamide gel is shown to be a suitable method for the resolution of intermediates trapped during the refolding process of reduced cystine-containing proteins. The method has been applied to the well-characterized snake neurotoxin erabutoxin b. An explanation is offered for the relatively low rate of refolding of this polypeptide.

Antibodies against viruses and structural brain components in oligoclonal IgG obtained from multiple sclerosis brain.

Immunoglobulin G (IgG) was isolated from three multiple sclerosis (MS) and two control brains by Protein A Sepharose affinity chromatography and was characterized by thin-layer polyacrylamide gel isoelectric focusing (PAG IEF) and antiserum immunofixation. The three MS brains contained oligoclonal IgG. Immunofixation with measles, herpes simplex, varicella, rubella, mumps and cytomegalovirus as antigens and autoradiography revealed that some of the oligoclonal IgG bands separated by PAG IEF contained antibodies against herpes simplex in one, measles in two and varicella virus in all three MS brains. No antibodies were detected with this technique against structural human (crude saline, lipid-proteolipid, ganglioside, and myelin basic protein extracts of MS and normal human brain) and bovine (purified myelin myelin basic protein and oligodendrocytes of bovine brain) brain components. The finding of viral antibodies and the absence of antibodies against structural brain proteins in oligoclonal MS brain IgG is similar to that previously recorded in MS cerebrospinal fluid (CSF).

Isoelectric focusing studies on the PGM1 subtypes in the northern Japanese population.

The distribution of the human red cell phosphoglucomutase (PGM1) subtypes in samples from Japanese population (n = 277) living in the Miyagi Prefecture, the northern part of Japan, was investigated by applying the thinlayer polyacrylamide gel isoelectric focusing. In our population sample all the ten common phenotypes were demonstrated, and the estimated allele frequencies for the genes PGM1+1, PGM1-1, PGM2+1, and PGM2-1 were 0.671, 0.107, 0.161, and 0.061, respectively. Family studies (n = 40) indicated an autosomal codominant inheritance and confirmed the four alleles. The new system will increase the probability of exclusion in paternity cases among Japanese to 29.4% compared with 14.3% if the two allele system is used.

Isoelectric focusing of multiple forms of DNase in thin layers of polyacrylamide gel and detection of enzymatic activity with a zymogram method following separation

Multiple forms of bovine pancreatic DNase (DNases A, B, C, and D) are separated by isoelectric focusing in thin layers of polyacrylamide gel with a carrier ampholyte in the pH range 4–6. The isoelectric points of DNases A, B, C, and D are 5.22, 4.96, 5.06, and 4.78, respectively. A zymogram method for detecting DNase activity as bands in the gel following isoelectric focusing is described. The method detects microgram amounts of DNase and has only one step. It can be used with the parified cazyme as well as with crude extracts of tissues containing DNase. By this method, two major components of DNase in ovine pancreas and at least three in malted barley as well as two previously unideatified forms of DNase in bovine pancreas with isoelectric points of 5.12 and 5.48 (DNases E and F) are observed.

Application of capillary thin layer isoelectric focusing in polyacrylamide gel to the study of alkaline proteinases in stored-product insects

1. 1. Isoelectric focusing in horizontal thin layers of polyacrylamide gel in combination with selected synthetic substrates and inhibitors was used to analyze the alkaline midgut proteinases from larvae of 5 species of stored-product insects. 2. 2. Partially purified proteinases from the black carpet beetle, Attagenus megatoma, had isoelectric points ranging from 3.9 to 9.0. About 15 proteinases were detected when focused gels were incubated on a casein-agar layer. 3. 3. A complex of 17 anionic and cationic proteinases was found in the hide beetle, Dermestes maculatus. Six trypsin-like and two chymotrypsin-like enzymes, one aminopeptidase, and one carboxypeptidase were demonstrated with artificial substrates and inhibitors. 4. 4. Two distinct, strongly anionic proteinases (pI 2.8 and 2.9) were found in Tribolium castaneum, while a single cationic proteinase was demonstrated in the furniture carpet beetle, Anthrenus flavipes. Compared with the other species, there were no strong proteinases in the midguts of larvae of the Indian meal moth, Plodia interpunctella. 5. 5. The use of horizontal isoelectric focusing in polyacrylamide gel for the analysis of proteinases in insects is discussed.

Novel and sensitive activity stains on polyacrylamide gel of serine and threonine dehydratase and ornithine aminotransferase.

A sensitive activity stain for serine and threonine dehydratase is described. After electrophoresis on thin-layer polyacrylamide gel, a reaction mixture containing L-serine or L-threonine as a substrate, NADH, lactate dehydrogenase and agar was solidified on the gel. The oxidation of NADH was recorded by contact printing on photographic paper with UV light. A novel activity stain for ornithine aminotransferase in the gel is also described. A reaction mixture containing substrates, o-aminobenzaldehyde and agar was solidified on the gel. A yellow color developed in a reaction between pyrroline-5-carboxylate and o-aminobenzaldehyde. The photograph was taken by contact printing. Both of the methods are highly sensitive and can detect less than 0.01 unit (mumol product/min at 37 degrees C) of each enzyme.

Hemoglobin A1C by isoelectric focusing.

We separated the hemoglobins in an unwashed erythrocyte hemolysate according to their isoelectric points on a thin-layer horizontal polyacrylamide gel containing ampholyte (40 g/L) over a pH gradient of 6-8. We scanned the fixed, unstained gels by microdensitometry and calculated the percentage of hemoglobin A1C. The overall CV (between-run imprecision) for a normal hemolysate (mean, 5.2% of total hemoglobin) stored at -70 degrees C was 12.6%. An above-normal pooled specimen (mean 8.5%) showed an overall CV of 8.4%. We confirmed that hemoglobins S, C, and F do not interfere; acetylated F co-migrates with A1C. The mean A1C percentage in non-diabetic adults was 4.9%, the reference interval 3.9-6.4%. The mean values for diabetics in various degrees of control were 7.6% in the group rated "good," 9.9% in the "fair" group, and 12.2% in the "poor" group. Results for patients' samples (y) were compared with results by cation-exchange chromatography (x). The slope was 1.0 and the intercept was 2.2%. The percentage of hemoglobin A1C in erythrocytes remains constant for seven days in samples stored at 30 degrees C.

Fish Species Identification by Thin Layer Agarose Isoelectric Focusing and Densitometric Scanning

Examination of sarcoplasmic proteins of fish by thin layer polyacrylamide gel isoelectric focusing as a means of fish species identification is a powerful and reliable technique, but it displays a number of disadvantages. These problems include the care required when handling acrylamide monomer (a neurotoxin), the mechanical skill needed in molding the gel, the difficulties in ensuring correct gel polymerization, and the extensive destaining periods. Specially treated agarose has been used to obtain protein patterns for an interval of pH 5-8. The patterns so produced were scanned by a densitometer in the visible range after completing staining and destaining. Most of the problems associated with polyacrylamide gels have been overcome by using agarose as a support medium.

Alpha-1-antitrypsin (PiM) subtypes in Japanese

Using isoelectric focusing (IEF) in thin-layer polyacrylamide gel, the polymorphism of the serum alpha-1-antitrypsin (Pi system) was investigated in 335 healthy unrelated Japanese individuals living in Miyagi prefecture, the northern part of Japan. Six common and five rare variant phenotypes were identified in our population samples, and the estimated allele frequencies for the genes Pi M1, Pi M2 and Pi M3 were 0.718, 0.238 and 0.044, respectively. Family studies ( n = 46) showed an autosomal codominant inheritance, and no exclusion was found in 23 mother-child pairs.

Viral antibodies in oligoclonal and polyclonal IgG synthesized within the central nervous system over the course of mumps meningitis

The occurrence of viral antibodies in relation to IgG separated by thin-layer polyacrylamide gel isoelectric focusing was studied in CSF and serum from 24 patients with mumps meningitis by immunofixation with viral antigens and autoradiography. Eleven of the patients displayed on the autoradiograms evidence of locally in the central nervous system synthesized mumps virus antibodies which were related to oligoclonal IgG bands in all 5 patients who displayed this CSF abnormality, otherwise to polyclonal IgG bands. Local synthesis of mumps virus antibodies was detectable in 43% of specimens obtained 1–13 days after onset, and in 75% obtained 27–47 days after onset. Only one patient displayed local synthesis of antibodies to other viruses (measles and herpes simplex) which could then be traced to polyclonal IgG bands.

Isoelectric focusing of hemoglobins on thin-layer agarose.

Isoelectric focusing on thin layers of agarose was used to separate several human hemoglobin variants in narrow pH range (pH 6-9). Problems with gel flooding and distortions due to electroendosmotic flow were solved by altering the casting and processing of the gel, by modifying the focusing apparatus, and by utilizing commercial agarose that had been chemically modified to reduce electroendomosis. Hemoglobins C, O-ARAB, A2, and E were distinguished from one another, as were hemoglobins S, D-LOS ANGELES, G-PHILADELPHIA, F. A, I, and J. The technic is rapid, simple, and relatively inexpensive. The agarose is nontoxic, has excellent gelling properties, and possesses large pores, yet gives resolution equivalent or superior to that obtained on thin layers of polyacrylamide gel, making it preferable to polyacrylamide for thin-layer isoelectric focusing.

Two new methods for pH gradient engineering in isoelectric focusing illustrated by increased resolution between hemoglobin A I and A Ic

It was shown that pH gradients in thin-layer polyacrylamide gel isoelectric focusing can be extended by decreasing the gel thickness in the region of interest, thickness modified pH gradients, or by using a relatively lower concentration of carrier ampholytes in the same region, concentration modified pH gradients. The two new methods for gradient expansion were compared with gradient expansion by the use of a so-called “chemical spacer,” β-alanine. All three methods were found to give pH gradient flattening around pH 7 and increased resolution of hemoglobins A I and A Ic. The effect of the three methods on field strength distribution was also compared. As expected the concentration modified gradient and the thickness modified gradient methods were found to give an increased field strength in the region of interest. An additional advantage with the two new methods is their general applicability to any desired pH region.

Viral antibody activity of oligoclonal and polyclonal immunoglobulins synthesized within the central nervous system in multiple sclerosis.

Thin-layer polyacrylamide gel isoelectric focusing (PAG IEF), a very high capacity method for separating immunoglobulins (Ig), was performed on cerebrospinal fluid (CSF) and serum. It was followed by antigen immunofixation with measles, mumps, herpes simplex (HSV), and rubella virus antigens and anti-human Ig autoradiography in order to demonstrate viral antibodies in separated Ig zones. Two of 11 control patients and 21 of 25 patients with multiple sclerosis (MS) displayed one or more zones of viral antibodies in the CSF without any counterpart, or with distinctly fainter zones, in the serum. Such reaction patterns were taken to indicate the possibility of intrathecal antibody synthesis. Antibody synthesis to measles was found in one to five zones in 76% of the patients with MS; antibody zones were found to HSV in 36% of the patients, to mumps in 12%, and to rubella in 12%. In 36% of the patients, two or three different antibody specificities (of which one was always measles) were found simultaneously in individual autoradiogram zones. For all viral antibodies detected in the CSF autoradiograms, their counterparts in oligoclonal or polyclonal IgG zones (or both) were demonstrable by PAG IEF of the corresponding CSF. The majority of patients with MS also had one or more oligoclonal CSF IgG zones without known antibody specificity. Antigen immunofixation and autoradiography are mainly qualitative. It is not known whether the viral antibodies present in oligoclonal or polyclonal IgG zones in MS CSF reflect a polyclonal B cell activation, a disease-specific immune reaction, or both.

Improved method for ribonuclease zymogram

An improved method for ribonuclease zymogram in agarose overlays is described which enables significantly enhanced sensitivity and the avoidance of false-positive activity. Two critical aspects of the zymogram method—the fixation/staining technique and the RNA substrate concentration—were evaluated for the RNase activity in RNase A and in human serum and urine. The zymogram method is sensitive to at least 10 pg of RNase A and does not show a false-positive reaction from 1500 μg of non-RNase protein following electrophoresis in thin-layer polyacrylamide gel.

Generic Identification of Cooked and Frozen Crabmeat by Thin Layer Polyacrylamide Gel Isoelectric Focusing: Collaborative Study

Abstract Ten collaborators were required to focus the ureaextracted proteins from 14 crabmeat samples. Six of the samples were knowns and were labeled as to their species. Eight samples were unidentified as to species and were labeled with letters. The 8 unknowns were identified by comparing the focused patterns with the patterns of the 6 species-labeled crabmeat samples. Seventy-nine samples were identified with 97.5% accuracy. This method has been adopted official first action.

Oligoclonal immunoglobulins in cerebrospinal fluid in acute cerebrovascular disease.

Oligoclonal immunoglobulin (Ig) bands were found in cerebrospinal fluid (CSF) by agarose gel electrophoresis and thin-layer polyacrylamide gel isoelectric focusing in 10 patients with cerebral infarction and 2 patients with transient ischemic attacks. Immunofixation revealed that the oligoclonal Ig was of the G class. One patient also had a band of free lambda light chains. The appearance of oligoclonal IgG during the course disease was observed in one patient, and the disappearance in six patients. Only three patients had elevated CSF IgG levels or abnormal synthesis rate of IgG in the nervous system. The oligoclonal reaction observed in acute cerebrovascular disease may reflect a polyclonal B-cell activation within the central nervous system after brain tissue damage.

Detection of cystic fibrosis homozygotes and heterozygotes by serum isoelectrofocusing.

It is suggested that the majority of individuals with the cystic fibrosis (CF) gene have a unique serum protein (CFP) which can be demonstrated by means of isoelectrofocusing (IEF) in thin layer polyacrylamide gels. We have found the CFP in 90% of patients with cystic fibrosis, in approximately 80% of individuals heterozygous for the CF gene, and in 8% of normal control individuals. We conclude that CFP is a useful marker for the CF gene.

An improved procedure for isoelectric focusing of human salivary proteins

Isoelectric focusing in thin layers of polyacrylamide gel is a powerful technique for the separation of human salivary proteins. The method described involves prior de-salting and concentration, followed by isoelectric focusing, in a pH gradient of 4–8, of sample volumes up to 0.1 ml. With whole saliva, 30–40 protein bands were clearly evident on staining with Coomassie Brilliant Blue; and α-amylase zymogram procedure revealed 9 or 10 isozymes. Within 8 individuals, a number of reproducible protein variants were apparent.