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PubMed1990

Affinity electrophoresis in agarose gels. Theory and some applications.

Affinity electrophoresis is the electrophoresis of components which interact during the electrophoresis procedure. Among the numerous modifications of the basic principle, affinity electrophoresis in agarose gels combined with subsequent immunochemical detection (crossed affinity immunoelectrophoresis) has emerged as a useful means of characterizing biospecific macromolecular interactions. Demonstration of ligand-binding proteins, enumeration of binding sites, evaluation of microheterogeneous forms, and estimation of binding constants can be achieved with this procedure even when analyzing small amounts of unpurified material. With an emphasis on interactions between lectins and glycoproteins as model systems, the principles and applicability of crossed affinity immunoelectrophoresis are exemplified. This includes examples of modifications developed for screening for lectins in plant extracts, for estimation of the binding capacity of affinity matrices, and for the calculation of dissociation constants.

Antibodies↗
PubMedDate not supplied

[The role of pulsed gel electrophoresis in the study of biological macromolecules].

We present modern conceptions concerning movement of biopolymer molecules in a gel under the action of static and pulsed electric fields, and we basically analyse some mostly used techniques of pulsed electrophoresis and the results yielded by using them. Pulsed procedures are shown to essentially widen the possibilities of analytical electrophoresis and electrophoretic transblotting are elaborated. Cameras and buffer systems used are the same as in classical methods involving the constant electric field. Promising results were collected while using sine-mode voltage in the constant and pulsed variants of electrophoresis. It is stated that the exceptionally wide application of pulsed methods in laboratory practice requires development of adequate theoretical conceptions concerning the movement of linear and globular polymers in gel under alternating field. In this connection the investigation of potentials of pulsed electrophoresis with inversions of field direction as the most simple and universal process of DNA division in a wide range of molecular masses and the use in electrophoretic techniques of sine-mode voltage obtained directly from the industrial circuit are most significant.

Biopolymers↗
PubMed1990

HydroLink gel electrophoresis: rapid electroblotting of dsDNA.

Blotting and probing of DNA, RNA and proteins after electrophoresis is a powerful technique for the study of the structure and function of biomolecules. Key factors in successful blotting experiments are efficiency of transfer, maintenance of the resolution obtained during gel electrophoresis, accuracy of the probes used and sensitivity of the detection method. We have recently developed a system for the high performance resolution of DNA with 10-fold greater capacity for sample loads than agarose or polyacrylamide. In the present study, we describe conditions for the rapid (less than one hour) and quantitative electrotransfer of DNA in the 100-23,000-base pair range, with subsequent conditions for probing of transfer membranes using radioactive or biotinylated probes. Our results suggest complete maintenance of the high-resolution characteristic of HydroLink gel electrophoresis and potentially increased sensitivity due to the high loading capacity in HydroLink gel electrophoresis.

Autoradiography↗
PubMed1979

Quantitative determination of high-density lipoprotein cholesterol by agarose gel electrophoresis.

We have developed a procedure for the determination of high-density lipoprotein cholesterol by agarose gel electrophoresis. Only 2 micro L of sample was applied to the gel. After electrophoresis at 90 V for 35 min, an enzymatic cholesterol reagent was applied. After a 30-min incubation, the high-density lipoprotein cholesterol was quantified by densitometry. Precision for this measurment approaches that reported for the heparin-manganese/Abell-Kendall method (Clin. Chem, 25: 596--609, 1979). We evaluated accuracy by comparing high-density lipoprotein cholesterol concentration measured by electrophoresis to that determined in the Framingham Heart Study procedure (J. Biol. Chem. 195: 357, 1952). The resulting correlation was excellent. By the paired Student's t-test, there was no significant difference between the two methods. The proposed method gives a linear standard curve when the concentration of total cholesterol is between 1.0 and 3.5 g/L. By accurate quantitation of high-density lipoprotein cholesterol, agarose gel electrophoresis can aid in assessment of coronary heart disease risk for a large segment of the population.

Cholesterol↗
PubMed1990

Measurement of radiation-induced DNA double-strand breaks by pulsed-field gel electrophoresis.

We have examined the use of pulsed-field gel electrophoresis (PFGE) to measure DNA double-strand breaks induced in CHO cells by ionizing radiation. The PFGE assay provides a simple method for the measurement of DNA double-strand breaks for doses as low as 3-4 Gy ionizing radiation, and appears applicable for the measurement of damage produced by any agent producing double-strand breaks. The conditions of transverse alternating field electrophoresis determined both the sensitivity of the assay and the ability to resolve DNA fragments with different sizes. For example, with 0.8% agarose and a 1-min pulse time at 250 V for 18 h of electrophoresis, 0.39% of the DNA per gray migrated into the gel, and only molecules less than 1500 kb could be resolved. With 0.56% agarose and a 60-min pulse time at 40 V for 6 days of electrophoresis, 0.55-0.90% of the DNA per gray migrated into the gel, and molecules between 1500 and 7000 kb could be resolved.

Animals↗
PubMedDate not supplied

Analysis of brain neuropeptides by capillary electrophoresis: determination of luteinizing hormone-releasing hormone from ovine hypothalamus.

The use of capillary electrophoresis to analyze in vivo release of luteinizing hormone-releasing hormone from the ewe median eminence and tissue content of luteinizing hormone-releasing hormone from discrete brain areas (within and immediately adjacent to the median eminence) is described. Results were compared with those obtained with a specific luteinizing hormone-releasing hormone radioimmunoassay. In vivo release samples were obtained by a push-pull cannula procedure. The exact position of the median eminence in each animal was determined from lateral roentgenographs after infusion of a radiopaque dye into the third ventricle. Each ewe median eminence was perfused with an artificial cerebrospinal fluid solution and sampled at 20-min intervals. Brain tissues for luteinizing hormone-releasing hormone content were obtained after each animal was killed. Samples were either analyzed by capillary electrophoresis and UV detection at 210 nm or assayed with a specific luteinizing hormone-releasing hormone radioimmunoassay. Tissue homogenate content and release samples had several peaks resolved by capillary electrophoresis and UV detection. Luteinizing hormone-releasing hormone was identified as one of these peaks. Luteinizing hormone-releasing hormone was also shown to be present in these samples when evaluated by radioimmunoassay. A good correlation between capillary electrophoresis and radioimmunoassay was observed for luteinizing hormone-releasing hormone content values obtained from discrete hypothalamic areas.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Preparative free flow electrophoresis for the isolation of two populations of proximal cells from the rabbit kidney.

High voltage free flow electrophoresis is a carrier-free method used for analytical and preparative cell separation, based on charge surface properties of cells. Two cell populations from the proximal tubule of the rabbit kidney were isolated by free flow electrophoresis from a suspension of pure proximal cells. This single-cell suspension was obtained through an original method by the combination of a Ca-binder action and gentle mechanical treatment associated with several shifting steps, on a pure suspension of isolated proximal tubules. Before the electrophoretic separation, the proximal cell origin was confirmed by enzymatic marker measurements, and the metabolic capacity was assessed by the cell respiratory activity. The isolated cells were very poor in distal tubule marker enzymes and were enriched in proximal tubule marker enzymes. Respiratory measurement showed a high cell metabolic capacity. After the electrophoretic separation, the origin of the cell populations was assessed by measuring specific marker enzymes. The cells in the slow-moving electrophoresis fractions had a high gamma-glutamyl transpeptidase activity and a low glucose-6-phosphatase activity. The fast moving cells showed a high glucose-6-phosphatase content and a poor gamma-glutamyl transpeptidase activity. Cells isolated by free flow electrophoresis were shown to possess long microvilli. This new methodology, allowed for the first time, the separation of a fast-moving cell population originating from the convoluted portion of the proximal tubule and a slow-moving cell population originating from the straight part of the proximal tubule of the rabbit kidney.

Animals↗
PubMed1989

The determination of high density lipoprotein cholesterol separated by electrophoresis and sodium phosphotungstate/Mg2+ precipitation: a physical evaluation.

Epidemiologic studies show that the risk of clinically evident atherosclerosis correlates negatively with concentrations of high density lipoprotein (HDL) and the influence of different factors on variations in the concentration of HDL cholesterol. We measured HDL cholesterol by both the electrophoresis and the precipitation method, and then proposed reference intervals for HDL cholesterol, adjusted for age and sex. Our findings show that the HIDL cholesterol concentration was lowest in cord blood. The concentration increased between the ages of 1 to 15 years, but lowered between the ages of 16 to 20 years. After this age period our study shows that the HDL cholesterol levels began to rise again before leveling off between the ages of 21-30 years. There was a significant difference in concentrations of HDL cholesterol between males and females. Our findings also show that the precision of the precipitation method is better than that of the electrophoresis method. The C.V. of the within-run analysis was 4.8% vs. 10.7%, while the C.V. of the between-run analysis was 4.3% vs. 14.6%. However, a significant correlation (r = 0.77) was noted between the precipitation method and electrophoresis. The electrophoresis method can be recommended as an aid in lipoprotein phenotyping, so we suggest it as a routine clinical method for HDL cholesterol analysis.

Adolescent↗
PubMed1989

Capillary electrophoresis.

The combined techniques that comprise capillary electrophoresis are under rapid development for a wide range of molecules and sample types. The capillary format provides fast, highly efficient separations and makes automation possible. At present, systematic approaches to separation methodology are still in the development stages and better detection schemes need to be developed. There is no doubt that electrophoresis in a capillary tube will take its place as a substitute for HPLC in specific applications. This is especially apparent when analysis time, efficiency, or simplicity is of greatest concern. Capillary electrophoresis will prove extremely useful for separations of nucleotides and proteins for analytical purposes in biotechnology. Finally, the ability to use ultrasmall-volume samples makes capillary electrophoresis ideal for the development of separations-based sensors for the analysis of microenvironments.

Electrophoresis↗
PubMed1989

Use of high-resolution two-dimensional gel electrophoresis and affinity labeling to probe glucocorticoid receptor structure and function.

The possible role of posttranslational modification in glucocorticoid receptor regulation was investigated. Glucocorticoid receptor (GR), prepared from the human B-cell line IM-9 and affinity labeled with [3H]-dexamethasone 21-mesylate, was examined by a combination of one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high-resolution two-dimensional gel electrophoresis. Two-dimensional electrophoresis of immunopurified [3H]dexamethasone 21-mesylate-labeled GR revealed the presence of two isoelectric species (apparent pI approximately to 5.7, and 6.0-6.5). Both forms were present in preparations of unactivated receptor. After GR activation, the pI of neither isoform was altered, indicating that activation does not involve covalent charge modification of the steroid-binding protein. However, only the pI 6.0-6.5 isoform bound to DNA, suggesting that covalent charge modification of the GR can alter its ability to bind to DNA. Two-dimensional electrophoresis of tryptic and chymotryptic fragments showed that the charge heterogeneity responsible for the two GR isoforms is located in a Mr 26,500 tryptic fragment derived from the steroid-binding domain of the protein. In addition, analysis of [3H]dexamethasone 21-mesylate-labeled tryptic fragments suggests that the Mr 26,500 fragment corresponds to residues 499-743 of the human GR. These results demonstrate that posttranslational modification of the steroid-binding domain may regulate the ability of the protein to bind to DNA.

Affinity Labels↗
PubMed1988

The mechanism of DNA's fractionation during pulsed-field agarose gel electrophoresis: a hypothesis.

The resolution by length of linear double-stranded DNA longer than 10-20 kb is increased by periodically changing direction of the electrical field during agarose gel electrophoresis (pulsed field gel, or PFG, electrophoresis). Previously proposed mechanisms for this effect include viscoelastic relaxation of the DNA and DNA length-dependent path lengths through the gel. Several data are not explained by these mechanisms. To better explain the data, here is proposed: (a) the existence of flexible projections from the more rigid fibers that form the network of agarose gels; these projections slow DNA's motion by threading loops of DNA; (b) the bending of these projections in the direction of DNA's motion during electrophoresis; (c) hysteresis in the re-orientation of these projections when the direction of the electrical field is changed, and (d) increase in resistance to DNA's motion in a direction opposite to that of the projections' bending. By use of (a)-(d), the facts known about DNA fractionation during PFG electrophoresis are qualitatively explained.

Chemical Fractionation↗
PubMed1986

[Electrophoresis applied to the analysis and separation of cells].

The present paper describes the theory and practice of cell/particle electrophoresis: the migration of cells/particles free to move in suspension under the influence of an applied electrical field, without any undue damage of cell membranes. The technique is quite different from high voltage electrophoresis on gels and paper. The quantitative biophysical parameter, never likely to be zero, is the electrophoretic mobility, experimentally determined by the cell electrophoresis apparatus, and mathematically related to the electrokinetic or the zeta potential. A detailed description of the analytical and preparative electrophoresis is given. Data are presented on the electrophoretic properties of bone tissue cells and cells of the immune system.

Animals↗
PubMed1985

Incubation with neuraminidase and affinity electrophoresis with wheat-germ lectin compared for separating and quantifying alkaline phosphatase isoenzymes in plasma.

Of 98 patients' specimens examined for alkaline phosphatase (EC 3.1.3.1) isoenzymes by electrophoresis on cellulose acetate membrane after incubation with neuraminidase, 50 showed only a single liver or bone isoenzyme staining band; in 15 of these, the tissue origin of the fraction could not be accurately identified from its electrophoretic location. In the remaining 48 specimens, both liver and bone fractions were identifiable, but in only 25 of these was the electrophoretic resolution sufficient to yield separate peaks on densitometry. In contrast, both liver and bone alkaline phosphatase isoenzymes were identified in 95 of the 98 specimens by affinity electrophoresis involving wheat-germ lectin, the detection of both fractions being in agreement with the results of sequential heat inactivation. The tissue origin of the enzyme bands was readily ascertainable from their consistent electrophoretic location in this medium, and in 89 of the specimens the isoenzyme fractions could be resolved into separate peaks on densitometry. We conclude that resolution of liver and bone alkaline phosphatase by incubation with neuraminidase followed by cellulose acetate electrophoresis is greatly inferior to that obtained by wheat-germ lectin affinity electrophoresis.

Alkaline Phosphatase↗
PubMedDate not supplied

Antibiotic identification by high voltage electrophoresis bioautography.

The high voltage electrophoresis bioautography method is applicable to meat, milk, and animal feeds. Meat is freeze-dried, powdered, and extracted with acetonitrile-water (9 + 1), and the extract is concentrated by evaporation at room temperature. Milk is examined directly or following acetonitrile-water extraction. Feed is extracted with acetonitrile-water. Samples or extracts are applied to preliminary assay plates of antibiotic medium No. 1 at pH 6 and 8, seeded with Micrococcus luteus (ATCC 9341), M. luteus DHSR (ATCC 9341A), Bacillus cereus (ATCC 11778), or B. cereus K250 TR (NCIB 11183), and nutrient agar at pH 7 seeded with B. subtilis BGA. Inactivation of penicillinase indicates beta-lactam antibiotics. Addition of trimethoprim increases sensitivity to sulfonamides. After 18-24 h incubation at 30 degrees C, plates yielding clear inhibition zones guide selection of conditions for subsequent electrophoresis bioautography. Extracts are applied (5-100 microL) to 10 mm diameter wells on electrophoresis plates 60 cm long and 40 cm wide, with a gel depth of 1.6 mm. The support medium is 1% agar and 1% agarose in Tris/succinic acid buffers pH 6 and pH 8. A potential of 1500 V is applied for 1.5 h at 15 degrees C. Following electrophoresis, the migrated antibiotics are visualized by over-layering with antibiotic medium No. 1, pH 6 or 8, seeded with M. luteus or B. cereus spore suspension; plates are incubated for 18-24 h at 30 degrees C. Identification is based on results of preliminary screening together with electrophoretic migration distances and inhibition zone appearances compared with standards.

Animal Feed↗
PubMed1979

[Isoelectric focusing and agar electrophoresis of the cerebrospinal fluid in neurologic patients].

Both isoelectric focusing and routine agar gel electrophoresis have been carried out on the CSF, and in some cases also on the corresponding serum, from 452 control subjects and patients with neurological diseases among whom cases with multiple sclerosis (MS) predominated. Comparison of the results confirms the superiority of electrofocusing over gel electrophoresis for the evaluation of oligoclonal immune reactions of the CNS. Over 90% of the MS patients showed discontinuous subfractionation of the gamma-globulins, mostly confined to the CSF, whereas with agar gel electrophoresis an identical effect could be seen only in about 60% of the same patients. Of special interest is the fact that by isoelectric focusing an additional 20% of MS patients whose gamma-globulin content, cell count and plasma cell findings in the CSF are within normal limits show oligoclonal CSF immune reactions. Thus, after appropriate screening by routine electrophoresis to reduce the number of analyses, the relatively laborious method of isoelectric focusing is an effective addition to the usual CSF investigations.

Cerebrospinal Fluid↗
PubMed1982

Separation of allelic variants by two-dimensional electrophoresis.

The resolving power of two-dimensional (2-D) gel electrophoresis has been tested using 17 allele products at five loci. Standard O'Farrell gels could separate 13 of these isozymes. The addition of a second pH gradient made it possible to separate all but one of the variant proteins. These results indicate that 2-D gel electrophoresis can resolve more than 90% of variants originally detected by one-dimensional (1-D) electrophoresis. The implications of these results for the low estimates of average heterozygosity obtained in surveys using 2-D gel electrophoresis are discussed.

Alleles↗
PubMed1982

Cell surface alterations of avian sarcoma virus B77- and 3,4-benzo(a)pyrene-transformed rat fibroblasts. I. Cell surface proteins characterized by two-dimensional electrophoresis.

Plasma membrane proteins of avian sarcoma virus B77-, 3,4-benzo(a)pyrene- and spontaneously transformed rat fibroblasts were metabolically- and cell surface radiolabeled and analyzed by electrophoresis under denaturing conditions (SDS-PAGE) or by two-dimensional electrophoresis. Most extensive series of protein alterations on transformed cells was detected by two-dimensional electrophoresis of cell surface proteins radiolabeled by lactoperoxidase catalyzed radioiodination: a fibronectin-like 220k protein with two isoelectric forms (pI 5.9 and 6.2-6.6), a 180k protein of pI 6.2-6.6 and two proteins of 48k and 50k with pI approximately 5.4-all decreased on examined transformed cells. In addition to these proteins, a series of proteins more markedly detectable on transformed cells was found: a 110-120k (pI 5.6) protein found particularly on 3,4-benzo(a)pyrene- and spontaneously transformed cells, a series of proteins in the region of molecular weights 46-54k with pI 5.5-6.0, two proteins of 35k (pI 6.2 and 6.4) and two further, basic proteins of 38k (pI approximately 7.2 and 7.8). Some of these changes were visualized also by further techniques for radiolabeling of proteins, i.e. reductive methylation of cell surface proteins, metaboling radiolabeling by 3H-leucine followed by plasma membrane isolation, as well as by protein staining after two-dimensional electrophoresis of isolated plasma membrane proteins. A 70k and a 120k radioiodinated proteins were immunoprecipitated from all transformed cells and, to a markedly lesser extent from untransformed fibroblasts by the immune serum against rat type-C endogenous virus. A similar 70k protein was immunoprecipitated from avian sarcoma virus B77-transformed cells also by the antiserum against Friend murine leukemia virus envelope glycoprotein gp70.

Animals↗
PubMed1981

The 16-hour-standing test and lipoprotein electrophoresis compared for detection of chylomicrons in plasma.

We compared the 16-hour-standing plasma test to lipoprotein electrophoresis on agarose gel for detection of chylomicrons in 129 patients' samples with triglyceride values greater than or equal to 4.00 g/L. Chylomicrons were observed in 12 samples (9.3%) by use of the standing-plasma test and in 58 samples (45.0%) by use of agarose-gel electrophoresis. Thus the standing-plasma test did not detect chylomicrons in 46 samples where they were observed by electrophoresis, or 79.3% of all cases where chylomicrons were present. Chylomicronemia was missed in the presence of lower triglyceride concentrations as well as at very high ones. We recommend that lipoprotein electrophoresis be routinely performed on plasma of patients with triglycerides concentrations greater than 4.00 g/L to distinguish between types IV and V hyperlipoproteinemia, as well as to detect failure of a patient to fast before sample collection.

Chylomicrons↗