Polyacryamide Gel Electrophoresis Research Articles

The predominant circular form of avocado sunblotch viroid accumulates in planta as a free RNA adopting a rod-shaped secondary structure unprotected by tightly bound host proteins.

Avocado sunblotch viroid (ASBVd), the type member of the family Avsunviroidae, replicates and accumulates in chloroplasts. Whether this minimal non-protein-coding circular RNA of 246-250 nt exists in vivo as a free nucleic acid or closely associated with host proteins remains unknown. To tackle this issue, the secondary structures of the monomeric circular (mc) (+) and (-) strands of ASBVd have been examined in silico by searching those of minimal free energy, and in vitro at single-nucleotide resolution by selective 2'-hydroxyl acylation analysed by primer extension (SHAPE). Both approaches resulted in predominant rod-like secondary structures without tertiary interactions, with the mc (+) RNA being more compact than its (-) counterpart as revealed by non-denaturing polyacryamide gel electrophoresis. Moreover, in vivo SHAPE showed that the mc ASBVd (+) form accumulates in avocado leaves as a free RNA adopting a similar rod-shaped conformation unprotected by tightly bound host proteins. Hence, the mc ASBVd (+) RNA behaves in planta like the previously studied mc (+) RNA of potato spindle tuber viroid, the type member of nuclear viroids (family Pospiviroidae), indicating that two different viroids replicating and accumulating in distinct subcellular compartments, have converged into a common structural solution. Circularity and compact secondary structures confer to these RNAs, and probably to all viroids, the intrinsic stability needed to survive in their natural habitats. However, in vivo SHAPE has not revealed the (possibly transient or loose) interactions of the mc ASBVd (+) RNA with two host proteins observed previously by UV irradiation of infected avocado leaves.

Characteristics of Phenotype and Isozyme Variation in Salvia miltiorrhiza after Spaceflight

To investigate the mechanism of spaceflight induced mutations, the dried seeds of Salvia miltiorrhiza, carried by the Shenzhou-7 spaceship of China in 2008 for 68h, were studied. After spaceflight, a mutant of salvia (SP) with large leaves and dwarf stem, whose phenotype was different from the ground control (CK), was obtained from the seedlings of the recovered spaceship-flown germinated seeds. Major characteristics of the SP phenotype included the large leaves with dark green color, deeply folded leaf surface, dwarf stems and less stem section space. Polyacryamide gel electrophoresis (PAGE) was used for analyzing the soluble protein, CAT, POD and SOD isozyme of SP1 and CK. The results revealed that space flight led to changes in the plant's soluble protein, CAT, POD and SOD isozymes in terms of bands, numbers and vigor of SP. The content of soluble protein in SP was higher than that of CK and the activities of isozyme increased, which indicated that seeds of S. miltiorrhiza appeared to be significantly affected after being exposed to space conditions. These experimental results demonstrated that space flight has the influence to the phenotype and physiological characteristics of S. miltiorrhiza. The space condition could bring inheritable mutagenic effects on S. miltiorrhiza seeds and may be used as a tool for accelerating the progress in S. miltiorrhiza breeding. Acknowledgements: This work is supported by the Science and Technology Committee of Hunan Province numbers 2010sk3013 and State Administration of Traditional Chinese Medicine of Hunan province numbers 201105.

Isolation and characterization of zinc-binding proteins of canine seminal plasma

Zinc-binding proteins from seminal plasma (ZnBPs) originate in the secretions of different accessory sex glands and are implicated in key events associated with sperm-egg fertilization processes. This study describes the isolation and characterization of the ZnBPs of canine seminal plasma. Ejaculates were collected from three crossbred dogs for a 2-week period. The ZnBPs as well as non zinc-binding proteins (nZnBPs) were isolated by zinc-dependent affinity chromatography. The isolated fractions were subjected to native gel electrophoresis (one-dimensional polyacryamide gel electrophoresis, PAGE) and sodium dodecyl sulphate polyacryamide gel electrophoresis (SDS-PAGE), using denaturing and reducing conditions. Zinc-elution profile using affinity chromatography displayed two protein fractions represented by the nZnBPs and ZnBPs, respectively. Using native gel electrophoresis, it was found that both the nZnBPs and ZnBPs occurred in their native state as aggregates, ranging from 140 to 669 kDa. The nZnBPs were disaggregated into 8 protein bands, with molecular weights ranging from 10.7 to 79.7 kDa, following SDS-PAGE analysis. By contrast, SDS-PAGE analysis of the ZnBPs revealed 13 protein bands, with molecular weights ranging from 11.6 to 152.3 kDa. Densitometric analysis showed that 46-48% of nZnBPs could be accounted by protein fractions with molecular weights of 10.7 and 14.2 kDa. Also, 2 protein fractions with molecular weights of 11.6 and 14.3 kDa, were predominant in ZnBPs, accounting for approximately 28-30% of the total proteins. These results demonstrate the zinc-binding capacity of proteins secreted by the canine prostate. The findings of this study indicate that ZnBPs of canine seminal plasma comprise several protein fractions, which might be implicated in the reproductive processes in the dog.

Seed storage protein polymorphism in ten elite rice (Oryza sativa L.) genotypes of Sindh

Seed protein profiling is the most promising tool in determining the molecular polymorphism and genetic homology. Seed storage proteins help in cultivar identification by avoiding the external environmental influences. Electrophoretically detectable proteins in rice grains possess the potential of characterizing the germplasms by their taxonomic and evolutionary aspects. This study was aimed at exploiting the genetic variations among ten elite rice genotypes of Sindh through electrophoretical separation of grain proteins by sodium dodecyl sulphate polyacryamide gel electrophoresis (SDSPAGE). In conclusion, the investigation revealed negligible polymorphism, with reference to the total seed protein profiles, among the rice genotypes of Sindh, Pakistan used in this study. Hence, it is highly important to include a significant number of rice genotypes to explore their existing genotypic diversity for future rice breeding programs. The SDS-PAGE in combination with 2-D electrophoresis is further suggested for documenting contrasting variations of isoforms of protein peptides. Key words : Seed storage proteins, polymorphism, Oryza sativa , Sindh, SDS-PAGE

Preparation of genome-wide DNA fragment libraries using bisulfite in polyacrylamide gel electrophoresis slices with formamide denaturation and quality control for massively parallel sequencing by oligonucleotide ligation and detection

Bisulfite sequencing is widely used for analysis of DNA methylation status (i.e., 5-methylcytosine [5mC] vs. cytosine [C]) in CpG-rich or other loci in genomic DNA (gDNA). Such methods typically involve reaction of gDNA with bisulfite followed by polymerase chain reaction (PCR) amplification of specific regions of interest that, overall, converts C→T (thymine) and 5mC→C and then capillary sequencing to measure C versus T composition at CpG sites. Massively parallel sequencing by oligonucleotide ligation and detection (SOLiD) has recently enabled relatively low-cost whole genome sequencing, and it would be highly desirable to apply such massively parallel sequencing to bisulfite-converted whole genomes to determine DNA methylation status of an entire genome, which has heretofore not been reported. As an initial step toward achieving this goal, we have extended our ongoing interest in improving bisulfite conversion sample preparation to include a human genome-wide fragment library for SOliD. The current article features novel use of formamide denaturant during bisulfite conversion of a suitably constructed library directly in a band slice from polyacryamide gel electrophoresis (PAGE). To validate this new protocol for 5mC-protected fragment library conversion, which we refer to as Bis-PAGE, capillary-based size analysis and Sanger sequencing were carried out for individual amplicons derived from single-molecule PCR (smPCR) of randomly selected library fragments. smPCR/Capillary Sanger sequencing of approximately 200 amplicons unambiguously demonstrated greater than 99% C→T conversion. All of these approximately 200 Sanger sequences were analyzed with a previously published web-accessible bioinformatics tool (methBLAST) for mapping to human chromosomes, the results of which indicated random distribution of analyzed fragments across all chromosomes. Although these particular Bis-PAGE conversion and quality control methods were exemplified in the context of a fragment library for SOLiD, the concepts can be generalized to include other genome-wide library constructions intended for DNA methylation analysis by alternative high-throughput or massively parallelized methods that are currently available.

Analysis of Membrane Protein Complexes by Blue Native PAGE

Blue native polyacryamide gel electrophoresis is a special case of native electrophoresis for high resolution separation of enzymatically active protein complexes from tissue homogenates and cell fractions. The method is powerful between 10 and 10,000 kDa. Also membrane protein complexes are separated well after solubilization of complexes with mild neutral detergents. The separation principle relies on binding of Coomassie blue G250 which provides negative charges to the surface of the protein. During migration to the anode, protein complexes are separated according to molecular mass and/or size and high resolution is obtained by the decreasing pore size of a polyacrylamide gradient gel. The principles of 2-dimensional blue native sodium dodecyl sulfate polyacrylamide gel electrophoresis are presented here together with a practical step-by-step guide to performing the method in the laboratory.

Purification and characterization of an exochitinase fromBacillus thuringiensissubsp.aizawaiand its action against phytopathogenic fungi

A chitinolytic enzyme from Bacillus thuringiensis subsp. aizawai has been purified and its molecular mass was estimated ca. 66 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was able to hydrolyze chitin to chitobiosides but not carboxymethylcellulose, cellulose, pullulan, and laminarin. Optimal pH and temperature were detected at 6 and 50 degrees C, respectively. Stability, in the absence of substrate, was observed at temperatures less than 60 degrees C and pH between 5 and 8. Enzyme activity was significantly inhibited by K+ and EDTA and completely inhibited by Hg2+. Purified chitinase showed lytic activity against cell walls from six phytopathogenic fungi and inhibited the mycelial growth of both Fusarium sp. and Sclerotium rolfsii. The biocontrol efficacy of the enzyme was tested in the protection of bean seeds infested with six phytopathogenic fungi.

IDENTIFICATION OF FOUR COMMON Culex (Culex) (DIPTERA: CULICIDAE) SPECIES FROM FLORIDA WITH ISOENZYME ANALYSIS

Females of four common Culex (Culex) species from Florida were analyzed for isoenzymes using polyacryamide gel electrophoresis. Ten enzymes that yielded 11 putative loci were studied. Most of the loci showed diagnostic characteristics in the four species, but four of the loci (glycerol-3-phosphate dehydrogenase [Gpd-2], hexokinase [Hk], isocitrate dehydrogenase [Idh-1], and malate dehydrogenase [Mdh]) could be used in sequence to identify the four Culex species. Culex salinarius and Cx. p. quinquefasciatus could be separated from Cx. restuans andCx. nigripalpus by Mdh locus. Culex salinarius could be distinguished from Cx. p. quinquefasciatus by Hk locus and Cx. nigripalpus could be distinguished from Cx. restuans, by Idh-1 and/or Gpd-2 loci. Randomly combined specimens of these four Culex species were identified accurately by using these enzyme loci.

Degradation and inactivation of photosystem I complexes during linear heating

The heat effect on photosytem I (PSI) was characterized by visible and fluorescence spectroscopy, oxygen electrode polarography, sodium dodecyl sulfate–polyacryamide gel electrophoresis (SDS–PAGE) and Fourier transform infrared (FTIR). During linear heating, the absorption maximum at 680 nm of PSI decreased and blue shifted, of which pigments and polypeptides in PSI complexes degraded at high temperature. The chlorophyll a (Chla) molecules with maximum absorption at 683 nm in the fourth derivative absorption spectra, which are mainly attributed to light-harvesting complex of PSI (LHCI), were more sensitive to high temperature. So it was suggested that Chla (absorbing at 683 nm) was destroyed firstly by elevated temperature. The characteristic peak at 728 nm and the ratio of F728–F720 to F680 in 77 K fluorescence spectra decreased apparently when temperature increased, showing that high temperature blocked energy transfer in LHCI and the energy transfer from LHCI 680 to LHCI 730 and PSI reaction center was inhibited. SDS–PAGE displayed that PsaA/B subunits and LHCI subunits were degraded and aggregated at different levels upon linear heat treatment. However, at high temperature especially at 90–100 °C, the PsaA/B subunits disappeared completely while LHCI subunits remained, indicating that the core complex subunits were more sensitive to heat treatment than LHCI subunits. The probable reason for the above results is that during linear heating the disconnection of the antenna from the reaction center occurred and then followed by the inhibition of the photochemical charge separation in reaction center. The oxygen uptake rate decreased with the increase of temperature, and it was almost completely lost at around 70 °C, demonstrating that the PSI complexes were destroyed seriously when the temperature reached 70 °C. FTIR analysis indicated that a major conformational transition of PSI complexes occurred above 60 °C, which was accompanied by unfolding of α-helical to the β-sheet structures.

Cloning and mapping of variety-specific rice genomic DNA sequences: amplified fragment length polymorphisms (AFLP) from silver-stained polyacrylamide gels.

An efficient technique for cloning DNA from silver-stained denaturing polyacrylamide gels was developed to allow the isolation of specific bands obtained from selective restriction fragment amplification (SRFA). This method proved as reliable as cloning radioactively labelled SRFA bands from the same gels. Rice DNA was used as a template, both with and without [32P]dCTP, using the same PCR profiles. Amplified products were separated using denaturing polyacryamide gel electrophoresis and visualized either by silver staining of gels or by autoradiography of 32P-labelled products. We cloned specific polymorphic SRFA bands directly from the denaturing polyacrylamide gels with one round of PCR amplification and confirmed that the sequences of the bands from silver-stained gels were identical to the corresponding 32P-labelled bands. The bands that were chosen represented amplified fragment length polymorphisms (AFLPs) between japonica and indica rice varieties. We studied the ability of two cloned AFLP bands to serve as heritable genetic markers by mapping them as RFLPs in an interspecific rice population and found that they represented single-copy DNA at unique loci in the rice genome.

CNBr-cleavage of proteins on blots and subsequent separation of the fragments by polyacryamide gel electrophoresis (PAGE) directly from those blots

CNBr-cleavage of proteins on blots and subsequent separation of the fragments by polyacryamide gel electrophoresis (PAGE) directly from those blots

Purification and immunological properties of vanadate sensitive Mg: ATPase from plasma membrane of maize roots

The vanadate-sensitive ATPase from maize ( Zea mays L.) root plasma membrane was partially purified from KI-washed microsomes by discontinuous sucrose gradient centrifugation followed by deoxycholate (DOC) treatment, l-l-lysophosphatidyl-choline (lyso-PC) solubilization and glycerol gradient centrifugation. The plasma membrane preparation had a relatively high specific activity of ATPase activity (182 μmol P i/h (mg proteins). During lyso-PC solubilization, the presence of Mg:ATP and vanadate substantially improved the recovery of active ATPase. The 100-kDa polypeptide of the plasma membrane ATPase was isolated by sodium dodecyl sulfate polyacryamide gel electrophoresis (SDS-PAGE) and used to raise a specific polyclonal antiservm. The addition of antiserum to the lyso-PC solubilized plasma membrane resulted in a loss of 90% of the total ATPase activity from the supernatant fraction after centrifugation. The antiserum also inhibited the proton transport activity of reconstituted plasma membrane vesicles. Western blot experiments showed that a single band of 100 kDa reacted with the anti-ATPase antibodies. Enzyme-linked immunosorbant assay (ELISA) analysis indicated that the antiserum exhibited specificity to plasma membrane as compared to other subcellular membranes. Thus, the antiserum obtained in this study is specific for the 100-kDa polypeptide of the plasma membrane ATPase.

Genetic variation in parthenogeneticArtemia from the Shandong Peninsula, P.R.C.

Results are presented of a survey of isozyme frequencies in parthenogeneticArtimia from six salterns in the Shandong Peninsula, P.R.C. Three of the eleven enzymes we scored using polyacryamide gel electrophoresis proved to be useful for analysis of electromorph frequency variation. These enzymes are tetrazolium oxidase (TO), lactate dehydrogenase (LDH), and malate dehydrogenase (MDH). Each of these systems showed extensive isozyme variation both within and between salterns. In addition, we have examined the possibility that electromorph frequencies may vary as the result of natural selection for adaptation to specific salinity, or salinity-associated environmental conditions. An indication of clinal variation was found in two series of ponds differing in salinity, however more extensive data are needed before it is possible to conclude that these patterns are the result of natural selection. Finally, the use of isozyme analyses such as ours, for unraveling taxonomic problems inArtemia is discussed.

Characterization of lotus tetragonolobus fucolectin Components for differences in hemagglutinating and macrophage activating activities

The fucose binding proteins (FBP) extracted from Lotus tetragonolobus seeds were isolatedby affinity chromatography and compared with affinity purified commercial preparations for physical, antigenic, and biological properties. All preparations contained three protein components as determined by polyacryamide gel electrophoresis, each with a subunit molecular weight of approximately 27 Kd. FBP preparations were also found to be antigenically identical by immunodiffusion analysis and possessed similar biological activities for hemagglutination of group 0 erythrocytes and macrophage activation in the migration inhibition assay. A reversible temperature dependent hemagglutination characteristic was found; FBP agglutinated erythrocytes at 4° and 22°C but not at 37°C, which was reversed by decreasing the incubation temperature from 37°C to 22°C. Temperature dependent binding of FBP for macrophages was also demonstrated. Adsorption of crude FBP by group 0 erythrocytes preferentially removed hemagglutinin without loss of macrophage activating properties. Similarly adsorption of FBP with macrophages preferentially removed macrophage activating component. Separation of the lectin components by DEAE cellulose chromatography yielded two major fractions: a potent hemagglutinin with weak macrophage activating properties and a potent macrophage activator with weak hemagglutinating activity. Separation of the crude lectin by ultrafiltration indicated that the macrophage activating component exists in a highly aggregated form which may determine its macrophage activating properties. Our results indicate that L. tetragonolobus consists of two distinct classes of components which correspond to tetrameric glycoproteins of 118–120 Kd with potent temperature dependent hemagglutinating activity and a highly aggregated dimeric component of 58 Kd with macrophage activating properties.

小児じん疾患におけるじん組織フィブリンとＦＤＰフラグメントについて

In order to elucidate the relation between intraglomerular fibrin deposition and types of serum and urinary fibrin/fibrinogen degradation products (FDP) in children with various renal diseases, the extent and distribution of fibrinogen related antigen (FRA) deposition in the kidney tissue which was perfesed with monochloacetic acid (MCA) solution was examind by immunofluorescent microscopy in comparison with deposition of factor XIII subunit-A (XIII-a). In addition to the immunofluorescent studies, the typing of FDP was made by SDS polyacryamide gel electrophoresis and crossed immunoelectrophoresis (SDS-PAGE-CIE).Intrarenal deposition of FRA after MCA treatment was observed mainly within the glomerular capillary walls, mesangium or vessels, and its extent and distribution showed a good agreement with that of XIII-a. Patterns of SDS-PAGE-CIE of MCA insoluble FRA products by plasmin were similar to those seen in cross-linked fibrin. These findings suggest that MCA insoluble FRA or XIII-a deposition reflects the extent and distribution of cross-linked fibrin.The presence of D-dimer in the urinary FDP was well seen in the children who had intrarenal fibrin deposition.

Purification of soluble glutaminase from pig brain

A relatively simple method for the purification of the soluble form of glutaminase from pig brain is described. Because the soluble and membrane-bound forms of the enzyme differ in their sensitivity to activation by phosphate ions and by preincubation in buffers containing phosphate and borate, the apparent yield and recovery of the purified enzyme varied with the assay and preincubation conditions. The recovery of the soluble enzyme was about 25 per cent and the purification was more than 49,000 fold, but the apparent yield and purification were about one order of magnitude less than this when glutaminase activity was assayed in the total absence of phosphate, because the activity of the soluble enzyme constitutes only some 10 per cent of the total activity present in pig brain homogenates as measured under these conditions. The purified enzyme appeared to be homogeneous by the criterion of polyacryamide gel electrophoresis in the presence of sodium dodecyl sulphate. The subunit molecular weight and amino acid composition of the purified enzyme are presented and studies of the specificities of the soluble and membrane-bound enzymes indicated that they are both specific glutaminases.

Acquired dysfibrinogenaemia in acute and chronic liver disease.

Plasma from patients with both acute and chronic liver disease has been examined for evidence of acquired dysfibrinogenaemia, using electrophoretic methods and coagulation tests. An examination of isolated fibrins upon SDS polyacryamide gel electrophoresis failed to demonstrate any molecular or structural defect associated with the polypeptide chains of the patients' fibrinogen or fibrinogen derivatives produced by thrombin or plasmin. However, purified fibrin monomers isolated from plasma using both Reptilase and thrombin exhibited delayed polymerization rates and the occurrence of acquired dysfibrinogenaemia in liver disease is therefore confirmed.

Changes in Egg White During Incubation of the Fertile Egg

Fertile, Single Comb White Leghorn hatching eggs were obtained from the K.S.U. poultry farm and incubated at 37.8 ± 0.3° C. and 55–60% relative humidity. Eggs were taken from the incubator daily, candled to insure embryonic development, and quick frozen. The frozen eggs were sectioned and egg white collected carefully to avoid contamination with yolk material, embryonic tissue or blood.Albumen from incubated eggs was examined for changes in pH, free amino acid content, glucose, and sulfhydryl content. Albumen samples were subjected to polyacryamide gel electrophoresis and tested for lysozyme and conalbumin activity.Egg white from incubated eggs increased in free amino acids. Egg white pH at first increased, then decreased with incubation time. No change in sulfhydryl content was observed. Glucose content decreased rapidly after 3 days’ incubation. Electrophoretic analysis indicated severe alterations in egg white proteins during embryogenesis. Assays for lysozyme indicated a complete loss of enzymatic activity by the seventeenth day of incubation. Conalbumin, as determined by chromogenic iron-binding, decreased gradually and was nearly absent after seventeen days.

GENETICS: Normal, Duarte Variant, and galactosemic alleles code for immunologically identical gal-I-P uridyl transferase enzyme protein

Human galactose-l-phosphate uridyl transferase was purified from post-mortem liver to a preparation having a single band in polyacryamide gel electrophoresis. This preparation was used successfully to produce a rabbit antibody that precipitates transferase activity from solution and that forms a precipitin band in double immunodiffusion. Hemoglobulin-free erythrocyte preparations from homozygous normal (Gt+/Gt+), Durate Variant (GtD/GtD), and galactosemic (GtG/GtG) individuals show immunoprecipitin bands in double immunodiffusion against this antibody that are identical with that of the purified transferase preparation. The results indicate that the three alleles code for immunologically similar enzyme proteins suggesting that the functionally less active Duarte Variant and inactive galactosemic enzyme proteins have resulted from “point” mutations.