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PubMed1987

Comparison of latex and polyacrylamide beads for detecting sperm antibodies.

The immunobead test is excellent for the routine screening of semen for sperm antibodies but the polyacrylamide beads currently used are of variable diameter (3-10 micron) which tends to obscure details of regional binding. Thus the use of smaller latex beads of more uniform diameter has been investigated. Latex beads were coated with IgG by exposure to anti D serum for a mixed antiglobulin reaction (MAR). Rabbit antihuman IgG and IgA antibodies were covalently bound to carboxylated latex microspheres (CLM) by the carbodiimide reaction. Polyacrylamide immunobeads (IBT) with antihuman IgG or IgA antibodies covalently attached were obtained commercially. The IgG IBT and MAR were compared in 55 samples. The MAR was positive in 15 of the 31 IBT positive samples but the proportion of sperm with attached beads was lower. CLM and IBT were compared in 106 samples. Both tests were positive in 34 samples with virtually identical proportions of motile sperm with beads attached and the patterns of attachment were similar. These results show that CLM microspheres give equivalent results to IBT and that as they are smaller and have a more uniform size they provide advantage in ease of detection of positive reactions and in determination of the regional blinding pattern.

Acrylic Resins↗
PubMed1986

Electroblotting onto activated glass. High efficiency preparation of proteins from analytical sodium dodecyl sulfate-polyacrylamide gels for direct sequence analysis.

We have developed a new method for the isolation of proteins for microsequencing. It consists of electrophoretic transfer (electroblotting) of proteins or their cleavage fragments onto activated glass filter paper sheets immediately after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins are immobilized on the glass fiber sheets by ionic interactions or by covalent attachment. A wide range of proteins can be prepared in this fashion with no apparent restriction due to solubility, size, charge, or other intrinsic properties of the proteins. As little as 50 ng of the transferred proteins can be detected using Coomassie Blue or fluorescent dye staining procedures and even smaller amounts of radiolabeled proteins by autoradiography. After detection, the protein-containing bands or spots are cut out and inserted directly into a gas-phase sequenator. The piece of glass fiber sheet acts as a support for the protein during the sequencing. Amounts of protein in the 5- to 150-pmol range can be sequenced, and extended runs can be obtained from the blotted samples because of improved stepwise yields and lower backgrounds. The method has been successfully applied to the sequencing of a variety of proteins and peptides isolated from one-dimensional and two-dimensional polyacrylamide gels.

Amino Acid Sequence↗
PubMed1985

Discontinuous polyacrylamide gel electrophoresis for typing haptoglobin in bloodstains.

A routine method is described for obtaining reproducible haptoglobin patterns from bloodstains by discontinuous polyacrylamide gel electrophoresis. By employing a stacking gel, proteins from bloodstain extracts are concentrated into narrow zones, before entering the resolving gel. This effect yields highly resolved haptoglobin patterns. Therefore, laboratories without the specialized equipment and expertise for polyacrylamide gradient gel preparation can still obtain highly resolved haptoglobin patterns from bloodstains.

Blood Grouping and Crossmatching↗
PubMed1979

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of bovine serum albumin oligomers produced by lipid peroxidation.

The oligomers of bovine serum albumin were produced by controlled reaction with peroxidizing linoleic acid to examine their possible utility as calibration proteins insodium dodecyl sulfate-polyacrylamide gel electrophoresis. The polymerization was effected in reaction mixtures containing linoleic acid undergoing peroxidation in the presence of ascorbic acid, and conditions that yield soluble oligomers with a wide molecular weight distribution were established. The interaction of these soluble oligomers with sodium dodecyl sulfate exhibited a binding isotherm indistinguishable from that obtained with bovine serum albumin. Furthermore, sodium dodecy sulfate-polyacrylamide gel electrophoresis of the albumin oligomers conformed to the empirical relation of molecular weight to mobility that pertains to the use of these oligomers as standard molecular weight markers.

Electrophoresis, Polyacrylamide Gel↗
PubMedDate not supplied

Polyacrylamide gel electrophoresis of the RNAs of new influenza virus strains: an epidemiological tool.

Recently, we have shown by separating the RNAs and proteins of different influenza A viruses on polyacrylamide gels, that influenza A viruses contain 8 genes corresponding to 8 virus specific proteins. By analyzing the RNA patterns and the protein patterns of different recombinant viruses we, subsequently, succeeded in establishing a complete genetic map for influenza A viruses. These techniques were extended to characterize the genome of new influenza virus isolates. Thus it was found that the human isolate A/Wi/263/76 and the two swine virus isolates, A/swine/Wi/49/76 and A/swine/Wi/50/76 possess identical RNA patterns, which differ from the RNA patterns of other recent swine virus isolates. The human (Wi/263) and the two animal isolates (Wi/49 and Wi/50) were obtained on the same farm suggesting that the animal virus was transmitted from swine to man. This proves that swine viruses may occasionally infect humans without causing a pandemic. A comparative analysis of the different RNA patterns of other swine influenza viruses isolated around the same time at different locations and of those of recent human isolates showed that our polyacrylamide gel technique is a valuable epidemiological tool to identify and and characterize different influenza virus isolates.

Animals↗
PubMed1977

Transformation in Naegleria gruberi: patterns of RNA synthesis examined by polyacrylamide gel electrophoresis.

Naegleria gruberi were grown on bacteria and methods were devised to free the cellular RNA from bacterial RNA contamination. Use of actinomycin D and cycloheximide showed that the transformation of Naegleria from amoeba to flagellate required RNA synthesis for 30 min and protein synthesis for 40 min after the initial stimulus of distilled water. Comparison of the patterns of RNA synthesized during transformation with those during growth indicated a considerable amount of new RNA produced during the phenotypic change. Most marked was the increase in RNA co-migrating on polyacrylamide gels with the small ribosomal sub-unit RNA, together with RNAs between the latter and transfer RNA. These results were compared with other published results using axenically-grown cells cells and sucrose density gradient centrifugation. Cells placed in 80 mM NaCl instead of distilled water fail to transform but the pattern of newly-synthesized RNAs was not significantly different from that seen in transforming cells. This suggested that high salt concentrations inhibit transformation by inhibiting synthesis and/or assembly of certain proteins rather than RNA synthesis. Eluted material from various regions of polyacrylamide gels containing RNA extracted from transforming cells was used in a cell-free system. Incorporation of 3H-glutamic acid but not 3H-tryptophan was stimulated by material extracted from the 18S regions of the gels.

Amoeba↗
PubMed1984

Polyacrylamide gel electrophoresis of the Kemerovo virus RNA.

Ribonuclease (RNase)-resistant RNA was isolated from partially purified Kemerovo virus by gel chromatography and or sucrose density gradient centrifugation. Double-stranded (ds) RNA only was found in the purified viral cores. The RNAs from both sources exhibited the same pattern of distribution in polyacrylamide gels. Ten dsRNA segments were identified. According to the results of coelectrophoresis of the Kemerovo virus and reovirus dsRNAs, the size of Kemerovo virus genome was estimated to be of about 11.7 X 10(6). The grouping of Kemerovo virus double-stranded segments according to their size in polyacrylamide gels corresponded to the 2:4:3:1 pattern.

Electrophoresis, Polyacrylamide Gel↗
PubMed1984

Phosphorylation-induced mobility shift in phospholamban in sodium dodecyl sulfate-polyacrylamide gels. Evidence for a protein structure consisting of multiple identical phosphorylatable subunits.

Phosphorylation of purified phospholamban isolated from canine cardiac sarcoplasmic reticulum vesicles decreased the electrophoretic mobility of the protein in sodium dodecyl sulfate (SDS)-polyacrylamide gels. Different mobility forms of phospholamban in SDS gels were visualized both by direct protein staining and by autoradiography. Unphosphorylated phospholamban migrated with an apparent Mr = 25,000 in SDS gels; maximal phosphorylation of phospholamban by cAMP- or Ca2+-calmodulin-dependent protein kinase increased the apparent Mr to 27,000. Partial phosphorylation of phospholamban by either protein kinase gave intermediate mobility forms of molecular weights between 25,000 and 27,000, suggesting that more than one phosphorylation site was present on the holoprotein for each activity. Boiling of phospholamban in SDS dissociated the holoprotein into an apparently homogeneous class of low molecular weight "monomers." Only two mobility forms of monomeric phospholamban were observed in SDS gels after phosphorylation by cAMP-dependent protein kinase, corresponding to 9-kDa dephospho- and 11-kDa phosphoproteins. All of the 9-kDa protein could be phosphorylated and converted into the 11-kDa mobility form, suggesting the presence of only one site of phosphorylation on a single type of monomer for cAMP-dependent protein kinase. Simultaneous phosphorylation of monomeric phospholamban by cAMP-dependent protein kinase and Ca2+-calmodulin-dependent protein kinase gave an additional mobility form of the protein, suggesting that different sites of phosphorylation were present for each activity on each monomer. Incomplete dissociation of the holoprotein by boiling it in a relatively low concentration of SDS facilitated the detection of five major mobility forms of the protein in SDS gels, and the mobilities of all of these forms were decreased by phosphorylation. We propose that the high molecular weight form of phospholamban is a multimer of electrophoretically indistinguishable monomers, each of which contains a different phosphorylation site for cAMP-dependent protein kinase activity and Ca2+-calmodulin-dependent protein kinase activity. Phosphorylation of phospholamban at multiple sites is responsible for the various mobility forms of the holoprotein detected in SDS-polyacrylamide gels.

Adenosine Triphosphatases↗
PubMedDate not supplied

[A device for protein preparative separation in polyacrylamide gel].

A device of simple design is suggested for proteins preparative separation in polyacrylamide gel. The design of the device include glass and plexiglas elements and ensures bilateral cooling of polyacrylamide gel and continuous elution of protein fractions. Separation of the bull brain neurospecific antigens S-100 and D was performed by the device.

Animals↗
PubMed1980

Phosphohydrolase activity of the isolated, brush-border membrane of Hymenolepis diminuta (Cestoda) following sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis.

Following electrophoresis of isolated, brush-border membranes of Hymenolepis diminuta on SDS-polyacrylamide gels, three distinct areas of alpha-naphthyl phosphate (NP) hydrolysis were detected; these corresponded to proteins with molecular weights of 106,800, 172,700, and greater than 340,000 Daltons. Hydrolysis of NP was inhibited by adenosine triphosphate, adenosine;5'-monophosphate, p-nitrophenyl-phosphate, glucose-1-phosphate, glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-diphosphate, molybdate, ethylenediaminetetraacetate (EDTA), and ethyleneglycol-bis-(beta-amino-ethyl)-N,N'-tetraacetate (EGTA), but not by fluoride. Inhibition of NP hydrolysis by EDTA was relieved in the presence of Mg++ or Ca++. Heating the isolated, brush-border membrane in the presence of SDS for 5 min at 95 C destroyed all enzymatic activity. These characteristics indicated that the enzyme(s) responsible for NP hydrolysis (following separation of membrane proteins by SDS-polyacrylamide gel electrophoresis) were the same enzymes responsible for the phosphohydrolase activity associated with intact and solubilized, brush-border membrane preparations of H. diminuta.

Animals↗
PubMed1983

Further characterization of filarial antigens by SDS polyacrylamide gel electrophoresis.

SDS (sodium dodecyl sulfate)-polyacrylamide gel electrophoresis of an antigen isolated from sera of Wuchereria bancrofti-infected patients and Setaria digitata antigen SD2-4 is reported. Both antigens showed carbohydrate (glycoprotein) staining. The W. bancrofti antigen had an apparent relative molecular mass of 35 000 while the S. digitata antigen SD2-4 migrated at the marker dye position on SDS-polyacrylamide gel electrophoresis. SDS treatment of these antigens did not abolish the precipitation reaction with antibody. In the case of W. bancrofti antigen, SDS treatment probably exposed hitherto hidden antigen epitopes.

Animals↗
PubMed1984

Immunochemical characterization of Mycobacterium leprae antigens by the SDS-polyacrylamide gel electrophoresis immunoperoxidase technique (SGIP) using patients' sera.

In this study the SDS-polyacrylamide gel electrophoresis immunoperoxidase (SGIP) assay was used for characterizing the antigenic components of Mycobacterium leprae using patients' sera. This technique involved the separation of mycobacterial sonicates on SDS-polyacrylamide gels, longitudinal sectioning of the gels, incubation with patients' sera and visualization of the antigen-antibody complexes by the indirect immunoperoxidase technique. A number of antigens present in M. leprae sonicates were recognized by leprosy patients' sera, some of which were seen in other mycobacteria as well. Antibody binding to a 33 kD antigen, present in both M. leprae and BCG sonicates, was reduced only in the latter after 6 months of multiple drug treatment of one patient. It is suggested that this is a common mycobacterial antigen with one or more M. leprae specific determinants. Several antigens were identified only in M. leprae sonicates, only by leprosy patients: a 12, 22, 28, 36, 41 and 86 kD component. These antigens lost their antigenicity after trypsin treatment, but were heat stable. Such M. leprae specific antigens may be useful for immunodiagnosis.

Antigens, Bacterial↗
PubMed1984

Localization of a light-chain binding site on smooth muscle myosin revealed by light-chain overlay of sodium dodecyl sulfate-polyacrylamide electrophoretic gels.

The interactions of smooth muscle myosin and its light chains have been examined by incubating sodium dodecyl sulfate-polyacrylamide gels of myosin with radioactively labeled regulatory or essential light chains. The technique involves sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fixation with methanol and acetic acid followed by an extensive series of washes. The gel is incubated overnight with labeled light chains in the presence of bovine serum albumin and then washed extensively to remove unbound protein. Following staining and destaining, the gel is autoradiographed to reveal which protein bands have bound light chain. The myosin heavy chain was able to rebind labeled regulatory or essential light chains despite the harsh procedure described above. By fragmenting the myosin heavy chain proteolytically, we were able to determine the binding site for both types of light chains to be within the 26,000-Da COOH-terminal segment of smooth muscle subfragment 1 (S-1) or the 20,000-Da COOH-terminal segment of skeletal muscle S-1. The extent of binding was 0.1-0.4 mol of light chain/mol of S-1 heavy chain. No binding was observed to portions of the myosin molecule which do not contain this segment such as myosin rod, light meromyosin, S-2, or the NH2-terminal 75,000-Da segment of S-1.

Animals↗
PubMed1984

Biochemical and polyacrylamide gel electrophoretic analyses of vaginosis-associated anaerobic curved rods.

Morphologic, biochemical, and sodium-dodecyl sulfate polyacrylamide gel electrophoresis characteristics of twenty-six anaerobic curved rods isolated from vaginal discharge or transcervical uterine cultures were compared with each other and with those of five known control strains of anaerobic bacilli. The curved rods could be divided into two distinct groups, based on shared morphological characteristics and similar patterns of major cell protein bands. The protein band pattern exhibited by Group I organisms, which were short, Gram-variable, comma-shaped bacilli, did not resemble the protein band pattern of Group II organisms, which were seen as long, thin, Gram-negative curved rods. Polyacrylamide gel electrophoretic analysis of protein band patterns of these curved rods failed to show any strong similarities to the patterns of the five known bacterial strains that were tested. Although the role of these organisms in the etiology of bacterial vaginosis is not known at present, characterization studies such as this should facilitate the further recognition and identification of these bacilli as a prerequisite to determining their significance in human infection.

Bacteria, Anaerobic↗
PubMed1983

Polyacrylamide gradient gel electrophoretic studies of residual catalase in acatalasemia.

Erythrocyte catalse in a Japanese-type acatalasemia and a normal control subject was separated by chromatofocusing with or without prior partial purification with DEAE-cellulose. Fractions were analyzed by polyacrylamide gradient gel electrophoresis for catalse activity and protein stain. Chromatofocusing revealed no marked difference in pI values between normal and acatalasemic catalases with or without partial purification. In the gel electrophoresis, molecular weights were also similar; two bands of catalase activity with molecular weights of 290,000 and 350,000 for the acatalasemia and of 280,000 and 360,000 for the normal control were found in the partially purified preparations. The molecular weight of normal catalase in untreated hemolysate was 250,000. Normal catalse was identified as protein bands on polyacrylamide gradient gel after fractionation of hemolysate by chromatofocusing. A more sensitive method for protein stain is still required for demonstration of residual catalse protein on the gel.

Acatalasia↗
PubMed1983

An analysis of contractile proteins in developing chick heart by SDS polyacrylamide gel electrophoresis and electron microscopy.

Chick heart development was studied using transmission electron microscopy and SDS-polyacrylamide gel electrophoresis in combination with densitometry. Myosin heavy chain, alpha-actinin, actin and tropomyosin accumulations were analysed in developing hearts from preheartbeat stage 9 (Hamburger-Hamilton staging series) through 2 days after hatching. At the preheartbeat stage, electron microscopy revealed a significant number of thin filaments scattered throughout the cytoplasm of the myoblasts; however, very few thick filaments were seen. There was no obvious association between the two filament types. SDS-polyacrylamide tube gels of heart muscle homogenates demonstrated the presence of all five proteins in hearts at the preheartbeat stage. Further analyses of the proteins by gel densitometry indicated that both actin and myosin accumulated rapidly during heart development while alpha-actinin and tropomyosin levels remained relatively static. Our results show that detectable quantities of myosin heavy chain, alpha-actinin, actin and tropomyosin accumulate in myocardial cells prior to the appearance of myofibrils and initiation of the contractile function.

Actinin↗
PubMed1983

Mammalian cell propagation on derivatized polyacrylamide microcarriers.

A working system for studying the effects of factors involved in the chemical nature of microcarrier on cell attachment, spreading and growth, was established. The system is based on polyacrylamide beads, prepared by the emulsion polymerization technique. Sieved beads of desirable mean diameter were derivatized to generate controlled amounts of primary and tertiary amino groups. These microcarriers were used for propagation of vive different cell strains: BHK, MDCK, CEF, MRC-5 and FS. It was found that BHK cells attach and spread significantly faster on primary amino derivatized beads than those with tertiary amino groups, and at a lower degree of charging. As a result of introducing hydrophobicity to the side chain carrying the primary amino group, higher kinetic rates of BHK cell attachment were obtained. Optimal cell growth of all the tested cells was recorded for the butyl and the hexyl side chains. On the other hand the introduction of hydrophobicity into the polymeric backbone of the microcarrier led to lower cell yields. Primary amino derivatized MCs with an optimal degree of hydrophobicity city exhibit a higher cell yield of MDCK cells (with pronounced epithelial morphology), as compared to the tertiary amino derivatized MCs. CEF and FS cells (with pronounced fibroblast morphology) attained cell yields comparable to those obtained with the tertiary amino derivatized MCs. The diaminohexane derivatized polyacrylamide microcarriers seem to be a potential alternative for the commonly used tertiary amino derivatized micro-carriers.

Acrylic Resins↗