Use Of Polyacrylamide Gel Electrophoresis Research Articles

Probing of alpha, beta and kappa-caseins polymorphic variants in Gangatiri cow milk with the use of polyacrylamide gel electrophoresis and HRAMS.

This research communication aimed to probe the genetic polymorphisms of alpha, beta, and kappa caseins in Gangatiri cows (an indigenous Indian cattle). Detection of variants has received considerable research interest in the dairy industry and animal breeding in recent years as a source of good quality protein, but also of bioactive peptides that may be linked to health implications. The polymorphic nature of casein fractions and their association with milk production traits, composition, and quality also attracted several efforts in evaluating the allelic distribution of different casein locus as a potential dairy trait marker. Molecular techniques of polyacrylamide gel electrophoresis and high-resolution accurate mass-spectrometry have been applied to this probe. Sequence analysis of different casein types in the cows showed the presence of four specific variants.

Quantitative Application for SDS–PAGE in a Biochemistry Lab

A variety of laboratory experiments have been published in this Journal describing the use of polyacrylamide gel electrophoresis to investigate different properties of proteins, including determination of the subunit composition of individual proteins and molar mass. The experiment described extends the capabilities of this experimental technique and provides a mechanism to determine the concentration of a single protein from a heterogeneous mixture.

The First Identification of Rotavirus B from Children and Adults with Acute Diarrhoea in Kathmandu, Nepal

Rotavirus B (RVB) in the genus Rotavirus of the family Reoviridae is known to be a cause of acute gastroenteritis among children and adults in parts of Asia including China, India, Bangladesh and Myanmar. In a 15-month surveillance programme between March 2007 and May 2008, 3,080 stool specimens were collected from children and adults with acute gastroenteritis in an infectious disease hospital in Kathmandu, Nepal. In 25 (0.8%) specimens RVB was detected, for the first time in Nepal, by the use of polyacrylamide gel electrophoresis followed by confirmation with reverse-transcription PCR and sequencing. The strains detected in this study had very similar electropherotypes, and their VP7 sequences were almost identical and phylogenetically belonged to the Indo-Bangladeshi lineage which was distinct from the Chinese lineage. Thus, this study showed the circulation of RVB strains belonging to the Indo-Bangladeshi lineage in a broader region than previously documented, suggesting that this phylogenetic divide corresponded to the geographic divide created by the Himalayan Mountains. Further studies may be warranted to identify and characterize the RVB strains in northern Vietnam which is adjacent to southern China with a long and less mountainous border.

Isozyme evidence on the specific distinctness and phylogenetic position of Vicia incisa (Fabaceae)

Abstract Vicia incisa is a taxonomically controversial species that has been also treated as a subspecies of V. sativa because of a great morphological similarity. The phylogenetic position of V. incisa is uncertain because various DNA markers have provided contradictory results. Isozymes of V. incisa encoded by 15 loci and resolved with the use of polyacrylamide gel electrophoresis (PAGE) are described and compared with those of seven related species belonging to sections Vicia, Sepium, Lathyroides and Pseudolathyrus in order to get new evidence about its taxonomic rank and phylogenetic position. Phylogenetic relationships are analyzed with maximum parsimony and neighbour joining methods. Vicia incisa is shown to differ from all three subspecies of V. sativa including, sativa, cordata and nigra, by alternate variants of ten isozymes out of 15 analysed. Instead, V. incisa has much more similarity to V. grandiflora and V. sepium by sharing eight isozyme variants which differ from the subspecies of V. sativa. The most parsimony and neighbour joining analyses of the isozyme variation placed V. incisa as basally linked to the V. grandiflora and V. sepium species couple in the clade of section Sepium (= sect. Atossa), while the subspecies of V. sativa together with V. lathyroides formed a separate clade of section Vicia. The isozyme data provide further support to the species status of V. incisa.

Esterase isoenzymes are linked to embryogenic structures induction in cotton cell suspension cultures

Esterase activity and isoenzymes pattern of two cultivars of cotton (Gossypium hirsutum L.), Coker 312 an embryogenic cultivar and ISA 205N a non embryogenic cultivar, were studied and compared during cell suspension cultures. The use of polyacrylamide gel electrophoresis allowed the identification of isoenzymes that number increased with the successive stages of cell culture of the two cultivars. At the stage of embryogenic structures induction which occurs only in Coker 312 cell suspension, we noted the presence of two isoenzymes (y and z) identified as aryl esterase, while one isoenzyme (x) identified as choline esterase wasexclusively found in the cell suspensions of the non embryogenic cultivar ISA 205N. Esterase activity increased in cells of Coker 312 whereas it’s remained constant in ISA 205N. These results suggested a great implication of esterase enzyme in the induction of embryogenic structures during cotton cell suspension cultures. Key words: Gossypium hirsutum L., cultivar, cell suspensions, embryogenic structures, Esterase; isoenzyme.

Changes in protein and nutrient composition of milk throughout lactation in dogs.

To evaluate changes in protein and nutrient composition of milk throughout lactation in dogs. Milk samples collected from 10 lactating Beagles. Milk samples were collected on days 1, 3, 7, 14, 21, 28, 35, and 42 after parturition and analyzed to determine concentrations of nitrogen, nonprotein nitrogen, casein, whey proteins, amino acids, lipids, lactose, citrate, minerals, and trace elements. Optimum conditions for separating casein from whey proteins and distribution of milk proteins throughout lactation were assessed by use of polyacrylamide gel electrophoresis. Protein concentration was high in samples collected on day 1 (143 g/L), decreased through day 21 (68.4 g/L), and increased thereafter. Concentration of nonprotein nitrogen did not change throughout lactation (5.7 to 9.9% of total nitrogen content). Casein-to-whey ratio was approximately 70:30 and remained constant throughout lactation. Lactose concentration increased from 16.6 g/L on day 1 to 34.0 to 40.2 g/L on days 7 to 42. Lipid concentration ranged from 112.5 to 1372 g/L. Citrate concentration increased from day 1 (4.8 mM) to day 7 (6.6 mM), then gradually decreased until day 42 (3.9 mM). Iron, zinc, copper, and magnesium concentrations decreased during lactation, whereas calcium and phosphorus concentrations increased. Calcium-to-phosphorus ratio remained constant throughout lactation (approx 1.6:1). Energy content of milk ranged from 1,444 to 1,831 kcal/L. Protein and nutrient composition of milk changes throughout lactation in dogs. These data can provide valuable information for use in establishing nutrient requirements of puppies during the suckling period.

Von Willebrand disease phenotype and von Willebrand factor marker genotype in Doberman Pinschers.

To define the relationship between clinical expression of a type-1 von Willebrand disease phenotype and genotype at 2 von Willebrand factor marker loci in Doberman Pinschers. 102 client-owned Doberman Pinschers. Dogs were recruited on the basis of plasma von Willebrand factor concentration, clinical history, and pedigree. Blood samples and response to a history questionnaire were obtained for each dog. Plasma von Willebrand factor concentration was measured by use of an ELISA, and genotyping was performed via polymerase chain reaction for 1 intragenic and 1 extragenic von Willebrand factor marker. Amplification product size was determined by use of polyacrylamide gel electrophoresis (intragenic marker) or automated sequence analysis (extragenic marker). Western blots were prepared from a subset of dogs with low plasma von Willebrand factor concentration to evaluate multimer distribution. Strong associations were detected between plasma von Willebrand factor concentration and von Willebrand factor marker genotype. Twenty-five dogs had substantial reduction in plasma von Willebrand factor concentration and multiple hemorrhagic events. All were homozygous for a 157-base-pair intragenic marker allele and homozygous or compound heterozygous for 1 of 4 extragenic marker alleles. These marker genotypes were exclusively detected in dogs with low plasma von Willebrand factor concentration, although some dogs with these genotypes did not have abnormal bleeding. Type-1 von Willebrand disease in Doberman Pinschers is associated with the von Willebrand factor gene locus; however, the expression pattern in this breed appears more complex than that of a simple recessive trait.

The use of polyacrylamide gel electrophoresis in animal systematics, phylogeny, and ecophysiology.

In the 1980s, the use of polyacrylamide gel electrophoresis (PAGE) was popular. But more recent developments in other electrophoretic techniques have resulted in this method being less widely used. However, it is adequate for the needs of naturalists for issues which do not require high-performance methods, such as in systematics, phylogeny, studies of intraspecific or clinal variability, ecology and ecophysiology. We illustrate the application of PAGE by the results of a research program on marine and terrestrial invertebrates which was conducted from 1979 to 1992, but which is still used to initiate graduate students to the research. Thus, issues faced by the zoologist can be clarified by this method, not yet obsolete and still useful in natural history despite considerable advances in other electrophoretic methods.

ポリアクリルアミドゲル電気泳動法による蛍光標識糖分析法（ＦＡＣＥ）

FACE (fluorophore-assisted carbohydrate electrophoresis) is a method for the separation of fluorophore-labeled saccharides by the use of polyacrylamide gel electrophoresis. The method is characterized by high resolution, high sensitivity, and ease of use without expensive giant-sized machine such as mass spectrometry. It has been found to be useful for the analysis of oligosaccharides profiling of N-linked or O-linked glycans after releasing saccharides from glycoproteins and for sequencing oligosaccharides component. Judging from the importance of oligosaccharides in glycoproteins and the changes in oligosaccharides of cancer cell surface, the method described here is hopeful as a unique analysis tool in the field of basic medical science.

Biodegradable polyalkylcyanoacrylate nanoparticles for the delivery of oligonucleotides

Antisense oligonucleotides with base sequences complementary to a specific RNA can, after binding to intracellular mRNA, selectively modulate the expression of a gene. However, these molecules are poorly stable in biological fluids and are characterized by a low intracellular penetration. In view of using oligonucleotides as active molecules, the development of polymeric particulate carriers was considered. Oligonucleotides were associated with biodegradable polyalkylcyanoacrylate nanoparticles through the formation of ion pairs between the negatively charged oligonucleotides and hydrophobic cations. Oligonucleotides bound to these nanoparticles were found to be protected from nuclease attack in cell culture media and their cellular uptake was increased as the result of the capture of nanoparticles by an endocytotic/phagocytotic pathway. The in vivo pharmacokinetic profile of oligonucleotides free or associated with nanoparticles has been investigated after intravenous administration to mice and the stability of these molecules has been evaluated by original methodology based on the use of polyacrylamide gel electrophoresis (PAGE) followed by multichannel radioactivity counting. Stability in vivo in the plasma and in the liver was shown to be improved when the oligonucleotides were adsorbed onto the nanoparticles. These results obtained both in vitro and in vivo open exciting perspectives for the specific delivery of oligonucleotides to the liver, thus considering this approach for the treatment of liver diseases (e.g. liver metastasis or hepatitis).

Isoenzyme diversity and phylogenetic affinities in Vicia subgenus Vicia (Fabaceae)

Isoenzyme composition of ten enzymes and variability of their genetically heterologous isoenzymes (heterozymes) encoded by 17 loci has been studied with the use of polyacrylamide gel electrophoresis in 21 vetch species of the type subgenus of the genus Vicia in comparison with V. pisiformis of the subgenus Vicilla. In total, 118 electromorphs (putative allozymes) of different frequency and variability pattern within and between species were recorded. Cladistic analysis of the allozyme data revealed in the subgenus two basic monophyletic groups with two monophyletic subclades in both. One subclade of the first group includes species related to sections Vicia, Sepium, Pseudolathyrus and Lathyroides. The second subclade of the first group consists of species of the section Peregrinae. The species of the section Narbonensis, which are considered to be closest wild relatives of the cultivated faba bean by morphological similarities, form a subclade in a second monophyletic group, with V. hybrida, V. pannonica, V. anatolica, and V. melanops of the section Hyperchusa as a sister subclade. Vicia hyrcanica and V. lutea of the same section are placed on the cladogram as basally paraphyletic in the second monophyletic group. Vicia faba is placed either as basally paraphyletic to both monophyletic groups or in the Peregrinae subclade, depending on the use of evolutionarily less variable isoenzymes alone or together with polymorphic ones in the data matrixes. The placement of cultivated V. faba and its morphologically closest wild relatives of the section Narbonensis in different monophyletic clades on the allozyme phylograms shows that they belong to different phylogenetic branches within the subgenus and indicates more remote relationship between them than previously assumed from morphological similarity. UPGMA phenograms of Manhattan distances characterizing the extent of allozymic divergence between species are presented.

ISOENZYME DIFFERENCES BETWEEN THE WILD DIPLOID AND TETRAPLOID WHEATS

Variability of acid phosphatase (ACP), esterase (EST), and superoxide dismutase (SOD) isoenzymes among 660 accessions of wild diploid wheats Triticum boeoticum (475) and T. urartu (185) and among 35 accessions of wild tetraploid wheats T. araraticum (21) and T. dicoccoides (14) was studied with the use of polyacrylamide gel electrophoresis. The wild diploids are differentiated by the electrophoretic variants of ACP-B, ACP-C, EST-A, EST-B, EST-C, EST-D, and SOD-B. Triticum urartu matches the A genome of wild tetraploid wheats T. araraticum and T. dicoccoides by allozymes of ACP-B, ACP-C and SOD-B, but differs distinctly by the monomorphic allozyme EST-A66. Triticum boeoticum fits the A genome of polyploid wheats by allozymes of EST-A and EST-B, but differs in allozymes of ACP-B and SOD-B. Thus, none of the two contemporary diploid wheats corresponds exactly to the A genome of wild tetraploid wheats by all diagnostic isoenzymes. Triticum boeoticum and T. arararicum share EST-C46 and EST-D71, whereas T. urartu and T. dicoccoides are fixed for silent EST-C0 and share EST-D73, suggesting independent origin of the two tetraploid wheats with a contribution from ancestral forms of both diploids.

Use of polyacrylamide gel electrophoresis to detect structural variations in kilobase-sized DNAs.

The electrophoresis of linear, kilobase-sized DNA molecules with permuted sequences has been studied in polyacrylamide and agarose gels. Plasmid pBR322, bacteriophage phi X174, and the SV40 minichromosome were each digested with a series of single-cut restriction enzymes. The linearized, permuted isomers of all three DNAs exhibit different mobilities in large-pore polyacrylamide gels, suggesting that all three DNAs contain sites of anisotropic, sequence-dependent curvature. Various experimental parameters such as acrylamide concentration, crosslinker ratio and buffer composition affect the magnitude of the observed differential mobilities. Band sharpness appears to be optimal in polyacrylamide gels containing 6.9-8.1%T and 0.5-1%C. Only small mobility differences are observed for the linearized, permuted sequence isomers in agarose gels.

Differential accumulation of β-1,3-glucanase and chitinase isoforms in pepper stems infected by compatible and incompatible isolates of Phytophthora capsici

Phytophthora capsici infection induced the synthesis and accumulation of β-1,3-glucanases and chitinases in the stem tissues of pepper plants. The hydrolase increase started soon after inoculation with P. capsici . After the appearance of symptoms on the pepper stems, the accumulation of β-1,3-glucanases became much pronounced in the incompatible interaction. However, induction of chitinases by P. capsici infection was very similar in both compatible and incompatible interactions, except that the enzyme activity was somewhat higher in the incompatible interaction at 4 days after inoculation. The use of polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing gels could detect and quantitate the isoforms of the two hydrolases in pepper plants. The direct detection of β-1,3-glucanases on PAGE gels revealed two acidic isoforms Ga 1 and Ga 2. The pI 4·8 isoform Ga 2 may be associated with the expression of resistance to P. capsici , because its high activity per unit protein amount was detected only in the incompatible interaction 3–4 days after inoculation. Most basic isoforms of β-1,3-glucanases were induced and accumulated in both the compatible and incompatible interactions, suggesting a possible involvement of the hydrolases in the infection process rather than in the resistance expression. The pI 8·8 isoform of β-1,3-glucanase was constitutively expressed in pepper stems, irrespective of P. capsici infection or mercuric chloride-treatment. No significant differences in electrophoretic patterns of chitinase isoforms were found between the compatible and incompatible interactions. A 32 kDa chitinase was identified on SDS-PAGE gels after its renaturation by treatment with deionized Triton X-100.

The use of polyacrylamide gel electrophoresis for the analysis of acidic glycans labeled with the fluorophore 2-aminoacridone.

A method is described for the electrophoretic analysis of fluorophore-labeled acidic glycans. Various glycoproteins were treated with the enzyme, peptide N-glycosidase F, to release mixtures of asparagine-linked glycans that were then labeled with the fluorophore 2-aminoacridone, and the resulting derivatives electrophoresed in high-density polyacrylamide gels. The acidic glycans were separated with high resolution and distinct, well-resolved fluorescent band patterns were produced. Neutral saccharides did not electrophorese in the system. Information on the structure of the glycans was revealed by the changes in the band patterns observed after the mixtures of the glycan fluorophore derivatives were treated with either of the enzymes neuraminidase or beta-galactosidase. Quantities of glycoprotein as little as 10 micrograms were analysed without difficulty. The electrofluorograms were viewed using a digital imaging system based on a cooled charge-coupled device. The method was also demonstrated using purified acidic glycans of known structure. The method was easy to use, enabled the sensitive analysis of multiple samples in parallel and should be a useful addition to the range of glycan analytical techniques.

DNA amplification fingerprinting using arbitrary oligonucleotide primers.

DNA amplification fingerprinting (DAF) is a strategy for genetic typing and mapping that uses one or more very short (> or = 5 nt) arbitrary oligonucleotides to direct the enzymatic amplification of discrete portions of a DNA template resulting in a spectrum of products characteristic of the DNA starting material. Polymorphisms from simple banding patterns are useful as genetic markers while more complex and informative patterns are suitable for DNA fingerprinting. The use of polyacrylamide gel electrophoresis and silver staining can adequately resolve the spectrum of DAF products into detailed and reproducible patterns.

Electrophoresis and detection of tin-labeled DNAs on open-faced gels.

Alternative protocols are necessary for the use of polyacrylamide gel electrophoresis in genome scale sequencing and mapping studies. The use of radioisotopes and manual gel reading will have to be replaced with a flexible labeling system that can be detected at levels similar or to better than radioisotopes but allows automated, high-speed detection. Labeling with stable isotopes is such an alternative. These nondecaying isotopes have the potential to be detected in sub-attomole quantities, despite being surrounded by the gel matrix, due to the high selectivity and sensitivity of resonance-ionization spectroscopy coupled with a mass spectrometer. In this study the detection limits of sputter-initiated resonance ionization spectroscopy (SIRIS) are investigated using thin, open-faced polyacrylamide gels supported by plastic. This system allows reproducibility and flexibility in the choice of gel size and buffer system since the gel can be cast, washed free of polymerization by-products, dried, and stored until use. Various concentrations of an Sn-labeled oligomer were run on these gels and loads of 5 femtomoles/mm could be detected on a 240 microns thick gel. Gels as thin as 60 microns lower the detectable concentration loads to 1 femtomole/mm. The limiting factor is tin contamination in the gel which, if reduced, will further increase detection. Polymerase chain reaction (PCR) products can also be labeled and detected using Sn isotopes, which could prove useful in mapping studies. Also presented are techniques which will facilitate resolution of these PCR products on open-faced gels by employing discontinuous buffers systems and DNA mobility modifiers.

The use of polyacrylamide-gel electrophoresis for the high-resolution separation of reducing saccharides labelled with the fluorophore 8-aminonaphthalene-1,3,6-trisulphonic acid. Detection of picomolar quantities by an imaging system based on a cooled charge-coupled device

Various monosaccharides, oligosaccharides and small polysaccharides were labelled covalently at their reducing end groups with the fluorophore 8-aminonaphthalene-1,3,6-trisulphonic acid (ANTS), and the resulting fluorescent derivatives were separated by high-resolution PAGE. The electrophoretic mobilities of the labelled saccharides are related largely to the compounds' Mr values, but they are also influenced by the individual chemical structures of the saccharides. Various positional isomers and some epimers, for instance galactose and glucose, were resolved. Oligosaccharide and small polysaccharide derivatives, prepared from an enzymic digest of starch, each differing in size by a single hexose residue and with a range of degrees of polymerization from 2 to 26, were all resolved in a single gel. The method was relatively rapid and simple to perform. It enabled multiple samples to be analysed in parallel with high sensitivity. The fluorescent-labelling procedure was virtually quantitative. As little as 1 pmol of ANTS-labelled saccharide was detected photographically when the gels were illuminated by u.v. light. When the gels were viewed using an imaging system based on a cooled charge-coupled device, as little as 0.2 pmol was detected. The method may be useful for the structural analysis of the carbohydrate moieties of glycoconjugates and other naturally occurring oligosaccharides.

Interpretation of Western blots of specimens from children infected with human immunodeficiency virus type 1: Implications for prognosis and diagnosis

The current method of diagnosing human immunodeficiency virus type 1 infection in adults is based on serologic detection of HIV-1 antibodies by means of an enzymelinked immunosorbent assay, with subsequent confirmation by Western blot immunoassay. The Western blot qualitatively measures the presence of antibody to a number of virus-associated proteins, including major group-specific antigen proteins, envelope proteins, and polymerase gene products. In the Western blot assay, proteins from a lysate of HIV-1 are separated according to size with the use of polyacrylamide gel electrophoresis. The electrophoresed proteins are transferred to nitrocellulose paper and reacted with patient serum. The HIV-1 antibody from the patient's serum is then detected by an antihuman IgG antibody conjugated with an enzyme. When the nitrocellulose paper is exposed to substrate, a colored band will appear on the paper where serum antibody was bound to viral protein. Western blot immunoassays have recognized limitations: 32% of adults without antibody detected by ELISA, who are at low risk for HIV-1 infection, may have Western blot reactivity, resulting in an indeterminate interpretation. 1 Indeterminate Western blots also are obtained late in the course of adult HIV1 infection, when there is a loss of antibodies to core HIV-1 antigens, z, 3 In infants, the serologic diagnosis of HIV-1 infection is confused by the presence of HIV-1 antibodies transplacentally acquired from the mother. Of those infants who are offspring of H1V-1 seropositive mothers, but who are not

Identification of microbial isolates by DNA fingerprinting: analysis of ATCC Zymomonas strains

Nine Zymomonas strains available from the American Type Culture Collection were analyzed with a technique of restriction endonucleases fingerprinting of total DNA based on the combined use of polyacrylamide gel electrophoresis and silver staining. The pattern analysis was performed with a densitometer and a personal computer. Strains isolated independently showed very different fingerprints while strains obtained by mutagenesis had patterns identical to the parent. However, in one case it was possible to distinguish a mutant from the parent by the absence of a band in its fingerprint. The analysis of Zymomonas total protein extracts by polyacrylamide gel electrophoresis could not reveal any remarkable difference. The DNA fingerprinting similarity coefficients fitted very well with a previous taxonomical study based on classical tests, and confirmed the nomenclature problems regarding the Zymomonas CP4 strain pointed out in a recent paper (Yablonsky et al., 1988, J. Biotechnol. 9, 71–80). Furthermore a Zymomonas strain isolated in Argentina was compared with the ATCC strains showing a geographical correlation with other strains isolated in South America. On the basis of fingerprinting analysis it was possible to refer this strain to the subspecies mobilis.