Discontinuous Polyacrylamide Gel Electrophoresis Research Articles

Plasma Haptoglobin and Group-specific Component Phenotypic Variants Increase the Risk of Chronic Kidney Disease

ABSTRACT The researchers studied the correlation of Haptoglobin (HP) and Group-specific Component (GC) plasma protein biomarkers in chronic kidney disease (CKD) patients and non-CKD controls using discontinuous-polyacrylamide gel electrophoresis. The results showed a significant difference between the CKD and control clusters of 2-2 variants for HP (p=0.0036) and GC (p= 0.0033). The current research indicates 2-2 phenotype is an independent risk determinant for CKD, while risk analysis reports odds value >1 for both protein polymorphisms signifying an effective risk towards CKD. The multifactor dimensional reduction analysis also detailed HP and GC markers have a strengthening and stronger association and are causative for CKD development. Collectively, the findings signify a potential risk of plasma HP and GC polymorphisms being susceptible to CKD and their key role in the deterioration of kidney functions.

Heparin Binding Proteins of Black Bengal Buck Semen and Their Correlation with Sperm Characters and Freezability

This experiment was conducted to study the electrophoretic characters of heparin binding proteins (HBP) of Black Bengal buck semen and their correlation with sperm characters and cryo-survivability. Semen ejaculates (n=20/buck) were collected from nine bucks and in vitro sperm characters were evaluated at collection, after equilibration and after freeze - thawing. HBP were isolated through heparin column and discontinuous Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) was performed to assess molecular weight. Significant difference (plessthan0.01) were observed among the bucks in sperm characters and freezability. Eight protein bands of 17 to 180 kDa in seminal plasma and 7 bands in sperm were found. 180 -136 kDa HBP of seminal plasma and 134-101 kDa HBP of sperm had showed high correlation with in vitro sperm characters. Further studies on identification of these proteins and their correlation with in vivo pregnancy are needed to find their role as marker for buck selection.

The No-Nonsens SDS-PAGE.

The discontinuous polyacrylamide gel electrophoresis system devised by Laemmli (Nature 227:680-685, 1970) has not only been used in numerous laboratories but has also been modified in several ways since its birth. In our laboratories, we use a modified Laemmli SDS-PAGE system for following protein purification as well as for analysis of certain protein-protein interactions, mainly involving filametous actin.

Electrophoretic profile of boar seminal proteins

ABSTRACTThe objective of this study was to study the electrophoretic properties of Large White Yorkshire boar seminal plasma and sperm proteins. Semen was collected from eight sexually mature breeding boars by the gloved hand method; seminal plasma proteins were precipitated by the ice-cold ethanol method while sperm proteins were isolated through the Triton X detergent extraction method. Discontinuous sodium dodecyl sulphate polyacrylamide gel electrophoresis was performed to assess the molecular weight of seminal proteins. A total of 11 protein bands with the molecular weight ranging from 14 to 200 kDa were observed in the porcine seminal plasma. Protein bands of 116–200 kDa, 66–97 kDa, 55–66 kDa and 45–55 kDa were present in all the 8 boars (100%) while protein bands of molecular weight 97–116 kDa, 36–45 kDa, 29–36 kDa, 20–29 kDa and 14–20 kDa were observed only in 50%, 12.5%, 62.5%, 25% and 25% of the boars, respectively. In the case of sperm proteins, a total of 7 bands with molecular weight ranging from 29 to 200 kDa were detected. Protein bands of molecular weight 97–116 kDa, 36–45 kDa and 29–36 kDa were observed only in 62.5%, 25% and 62.5% of the boars, respectively. Seminal plasma protein fractions such as 20–29 kDa and 14–20 kDa were not detected in sperm. In conclusion, some of the seminal protein fractions in porcine semen are similar to those reported in other animal species and their role in boar fertility needs to be studied to use them as a marker for selection of breeding boars.

Separation of native allophycocyanin and R-phycocyanin from marine red macroalga Polysiphonia urceolata by the polyacrylamide gel electrophoresis performed in novel buffer systems.

Three buffer systems of Imidazole−Acetic acid, HEPES−Imidazole/Bis-tris and Bis-tris−HEPES−MES were designed based on the principle of discontinuous polyacrylamide gel electrophoresis (PAGE) for the native PAGE which could be performed in pH 7.0 and 6.5 in order to analyze and prepare the minor components of allophycocyanin (AP) and R-phycocyanin (R-PC) from marine red macroalga Polysiphonia urceolata. These AP and R-PC phycobiliproteins are easily denatured in alkaline environments. The obtained results demonstrated that the PAGE modes performed in the buffer systems of HEPES−Imidazole/Bis-tris and Bis-tris−HEPES−MES gave the satisfactory resolution and separation of AP and R-PC proteins. The absorption and fluorescence spectra of the AP and R-PC proteins which were prepared by the established PAGE modes proved that they maintained natural spectroscopic characteristics. The established PAGE modes may also provide useful references and selections for some other proteins that are sensitive to alkaline environments or are not effectively separated by the classical PAGE modes performed normally in alkaline buffer systems.

Emulsion liquid membrane extraction with D2EHPA mobile carrier Hirudinaria manillensis hirudin in experimental study

To study the extraction system of hirudin emulsion liquid membrane with the Poecilobdella manillensis as raw material, di-(2-ethylhexyl) phosphate (D2EHPA) as carrier, Span 80 as emulsifier, octane and D2EHPA mixed to constitute membrane solution, diluted HCl solutions as internal aqueous phase. Using the orthogonal experiment to optimize the extraction conditions of hirudin reference substance such as membrane phase, internal aqueous phase volume ratio (MIPVR), external aqueous phase pH, internal aqueous phase pH, mobile carrier concentration and so on, and then using hirudin crude extracts to do purifying experiment, and gaining experimental samples. The optimal conditions of hirudin extraction were as follows: MIPVR 10: 3, internal aqueous phase pH 2.6, external aqueous phase pH 3.4, the mass fraction of carrier D2EHPA 2%. In the optimal extraction conditions, when the initial concentration of hirudin was one anti-thrombin activity units (ATU) x mL(-1), ATU recovery rate of the reference substance was 83.06%. In the purifying experiment of crude extracts, ATU recovery rate was 82.99%, and the specific activity of sample was 3 289.48 the ATU x mg(-1). Discontinuous polyacrylamide gel electrophoresis and spectral scanning, the results showed that the purity and reference substance were considerable. The method of preparation hirudin was relatively simple, the purity of the experimental samples and ATU recovery were both high.

Extraction, purification and identification of surface adhesion ligand from <i>Lactobacillus</i>

The objective of this experiment was to purify and analyze adhesion protein from the surface of Lactobacillus. The surface adhesion ligand of Lactobacillus was isolated with of LiCl method and (NH4)2SO4 method, followed by sephadex chromatography, analyzed with discontinuous native polyacrylamide gel electrophoresis, and tested with a binding assay with intestinal epithelial cells. We get the adhesion matter by two methods: ammonium sulfate and LiCl extraction. Further purification with sephadex chromatography produced 3 components. The second component of eluted from chromatography had adhesion enhancing ability compared with the control. A single band was present in discontinuous native—poly-acrylamide gel electrophoresis analysis and molecular weight was determined to be 43-66 KDa. The purified adhesion ligand can improve the number of Lactobacillus adhering to the intestinal epithelial cells.

Partial characterization of hepatopancreatic and extracellular digestive proteinases of wild and cultivated Octopus maya

Proteinases from hepatopancreas (HP) and gastric juice (GJ) from wild and cultured red octopus (Octopus maya) were characterized. Hepatopancreas assays revealed optimal activity at pH 4, 9–10 and 10 for wild and pH 3, 8, and 9, for cultured octopuses, for total proteinases, trypsin and chymotrypsin, respectively. In the gastric juice, maximum activity was recorded at pH 6, 8, and 7 for total proteinases, trypsin, and chymotrypsin, respectively for both wild and cultured octopus. A reduction on enzyme activity of 70 and 20% was observed in HP and GJ extracts, respectively when protease inhibitor Pepstatin A was used. That result suggests that the main proteases in the HP were aspartic acid proteinases type (possibly Cathepsin D) and some of them were present in the GJ. Dissociating discontinuous polyacrylamide gel electrophoresis showed activity bands between 20 and 28, 30 and 34, 35 and 45, 60 and 70 kDa, and a last one between 75 and 100 kDa. We concluded that extracellular digestion of O. maya takes place in an acid environment, around pH 6. In contrast, intracellular digestion in the HP is developed at pHs between 3 and 4, where cathepsin D could be the most important enzyme for O. maya.

An Electrophoretic Study of LDH-Isozyme Distribution in Developing Embryonic and Adult Tissues of the Toad Bufo arabicus

Lactate (LDH) dehydrogenases were studied by electrophoresis to investigate the possibility of isozymic differences in different stages and organs of B. arabicus embryos, tadpoles and adults. The expression pattern of lactate dehydrogenase (LDH) isozymes was analysed using nondenaturing discontinuous polyacrylamide gel electrophoresis (PAGE). Five LDH isozymes were observed in five different adult tissues. The most cathodal isozyme LDH-5 (A4) showed the strongest activity. All embryonic stages tested displayed the anodic isozymes first. Four LDH isozymes were present in the stages 1-16 (fertilized egg-tail bud stage). The first appearance of the fifth isozyme was detected in stage 17 when the muscular response started. This isozyme showed gradual increase in intensity from stage 17 to stage 43.

Purification and characterization of antitoxic proteins from the serum of Agkistrodon halys Pallas

In this study, the authors report the purification and characterization of antitoxic proteins from the serum of Agkistrodon halys Pallas. Two antitoxic proteins have been successfully isolated by the methods of (NH4) 2SO 4 fractional precipitation, chromatography and preparative discontinuous polyacrylamide gel electrophoresis (PAGE). We have measured their molecular weights by Sephadex G-150 chromatography and 0.1% SDS–Tris–HCl discontinue PAGE respectively. Antitoxin I was about 138,000±40 Da and antitoxin II was about 76,000±40 Da, they are all single-chain peptides. We have measured their capacity to neutralize the toxicity of agkistrodotoxin (ATX), and their capacity to inhibit the PLA 2 activity of ATX. The results showed that antitoxin I could increase LD 50 of ATX from 0.25±0.05 to 0.445±0.13 mg/kg, decrease its PLA 2 activity from 2.36 to 1.72 μm/mg min, and antitoxin II could increase LD 50 of ATX from 0.25±0.05 to 0.56±0.12 mg/kg, decrease Phospholipase A 2 (PLA 2) activity from 2.36 to 1.2 μm/mg min. When the natural antitoxins were mixed with different amounts of ATX and inoculated intraperitonially into eight mice, it was found that 0.5 mg antitoxin I could neutralize the toxicity of 0.4 mg ATX and 0.5 mg antitoxin II could neutralize the toxicity of 0.5 mg ATX completely. These antitoxic proteins could neutralize the toxicity of ATX completely and inhibit ATX's PLA 2 activity partially.

Partial amino acid sequence and physiological effects of a 27 kDa parasitism-specific protein present in the plasma of parasitized Lacanobia oleracea (Noctuidae)

Abstract. Parasitization of larvae of the tomato moth, Lacanonbia oleracea, by the ectoparasitic wasp, Eulophus pennicornis, results in the appearance of a 27 kDa parasitism-specific protein (PSP) in the plasma of the host. After isolation of this protein by native discontinuous polyacrylamide gel electrophoresis, whole gel elution and electroblotting, the N-terminal sequence of the 27 kDa PSP is determined by Edman degradation. The 20 amino acid residues obtained reveal 70% identity with a female-specific fat body protein from the moths Antheraea pernyi and Antheraea yamamai, 60% identity with a glutathione S-transferase (GST) isolated from Orthosia gothica, and a low level of identity with the N-termini of proteins belonging to the GST superfamily. Injection of the 27 kDa PSP into L. oleracea larvae has no significant effect on their ability to gain weight or the time at which they pupate. Furthermore, assays performed in vitro demonstrate that the 27 kDa PSP does not affect the ability of L. oleracea haemocytes to form aggregates. The precise source of the 27 kDa PSP remains unclear, although the current results suggest that it is most likely synthesized by host larvae in response to parasitism. The possible role(s) of the 27 kDa PSP are discussed with regard to the physiological effects of parasitism on the host.

Purification and characterization of lignin peroxidases from Penicillium decumbens P6

Peroxidases are essential enzymes in biodegradation of lignin and lignite which have been investigated intensively in the white-rot fungi. This is the first report of purification and characterization of lignin peroxidase from Penicillium sp. P6 as lignite degradation fungus. The results indicated that the lignin peroxidase of Penicillium decumbens P6 had physical and chemical properties and a N-terminal amino acid sequence different from the lignin peroxidases of white-rot fungi. The lignin peroxidase was isolated from a liquid culture of P. decumbens P6. This enzyme had a molecular weight of 46.3 KDa in SDS-PAGE and exhibited greater activity, temperature stability and wider pH range than those previously reported. The isolation procedure involved (NH4)2SO4 precipitation, ion-exchange chromatography on DEAE-cellulose and CM-cellulose, gel filtration on Sephadex G-100, and non-denaturing, discontinuous polyacrylamide gel electrophoresis. The K m and V max values of this enzyme using veratryl alcohol as substrate were 0.565 mmol L −1 and 0.088 mmol (mg protein) −1 min −1 respectively. The optimum pH of P6 lignin peroxidase was 4.0, and 70.6 of the relative activity was remained at pH 9.0. The optimum temperature of the enzyme was 45 °C.

Characterization of proteases from the digestive tract of sea cucumber ( Stichopus japonicus): High alkaline protease activity

Proteases from the digestive tract of sea cucumber ( Stichopus japonicus) have been characterized by biochemical and electrophoretic techniques. Casein digestion assay revealed acidic proteases with optimum activity at pH 2.0 and pH 5.0, and alkaline proteases with optimal activity at pH 8.0 and 13.5, respectively. The influence of temperature and pH on the activity of these proteases was studied. Specific protease inhibitors and the effect of various metal ions were also studied. Copper, Ca 2+ and Mg 2+ renatured the ethylenediamine tetraacetate (EDTA)-denatured protease at optimal pH range of 12.0–14.0. Non-dissociating discontinuous polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulphate-polycrylamide gel electrophoresis (SDS-PAGE) using casein as substrate with or without specific protease inhibitors confirmed the existence of at least three proteases, whose molecular weights were defined as 20.6, 39.1 and 114.1 kDa, respectively. Furthermore the 20.6 kDa protease with good pH and thermal stability was confirmed to be a metallo-protease containing Cu 2+ and the 39.1 kDa protease, a serine protease, probably a collagenase.

Rapid microsatellite analysis using discontinuous polyacrylamide gel electrophoresis.

A method for screening large numbers of samples for microsatellites using discontinuous, non-denaturing polyacrylamide gels and rapid fluorescent gel staining is described. Disc electrophoresis on slab gels provides high-resolution of PCR products. It is useful for collecting population data once microsatellite loci have been characterized.

Simultaneous typing of the STR loci, vWF and HumTPO, using discontinuous polyacrylamide gel electrophoresis

Analyses of short tandem repeat (STR) loci are very useful for improving the accuracy of paternity determination. Combined use of several STRs makes this test even more accurate. We have devised a simple but effective method which consists of the co-amplification of two STR loci ( vWF and HumTPO) in a single test tube, separation of the PCR products using discontinuous polyacrylamide gel electrophoresis, and detection using sensitive silver staining. An appropriate combination of PCR primer pairs gave a high resolution and good separation of the two STR bands in the same lane on the same gel. Many combinations of STR loci have potential usefulness in paternity and individualization testing or population genetic studies in forensic practice.

Purification of a toxic factor from Arabian Gulf catfish epidermal secretions

The Arabian Gulf catfish, Arius bilineatus (Valenciennes) secretes a proteinaceous epidermal secretion when threatened or injured. A toxic factor has been isolated and purified from the crude extract (crude skin toxin) of these secretions by a combination of gel filtration on Sephacryl S-300 and preparative discontinuous polyacrylamide gel electrophoresis. The purified skin toxin has a molecular weight of 39 000 Da and an isoelectric point (p I) of 5.45. Injection of the purified skin toxin into rabbits i.v. and determination of the ld 50 indicated that the protein had been purified approximately 30 fold by these procedures. Injection of the purified skin toxin into rabbits caused agitation, convulsions and death within 5 min. Analysis of plasma levels of lactate dehydrogenase, glutamate-oxaloacetate transaminase and glutamate–pyruvate transaminase in injected rabbits indicated that the skin toxin caused cardiac and liver damage to the animals.

Study on Antineurotoxic Factor from the Serum of Bungarus Multicinctus

AbstractAn antineurotoxic factor which can partially neutralize the toxicity of β bungarotoxin (β-BuTx) was purified to homogeneity from the serum of B. multicinctus by ammonium sulfate precipitation, DEAE-32 cellulose chromatography and preparative discontinuous polyacrylamide gel electrophoresis (PAGE). The purified antineurotoxic factor shows a single band in PAGE and SDS-PAGE, whose electrophoretic mobility is in the region of α2- or β-globulin. The molecular weight of the antineurotoxic factor is 75-85 KD estimated by SDS-PAGE and Sephadex G-150 gel filtration. The band of antineurotoxic factor in PAGE was stained pink with the PAS (periodic acid-Schiff) procedure. 0.12 g/kg antineurotoxic fraction can increase die LD50 of β-BuTx from 0.05 mg/kg mouse i.p. to 0.102 mg/kg mouse i.p. The antineurotoxic factor also possesses β-BuTx PLA2 enzymatic inhibition activity, which can decrease β-BuTx PLA2 enzymatic activity from 6.5 IU/mg to 5 IU/mg. Sephadex G-150 gel filtration shows no formation of a stable ...

Allele frequency distributions of D1S80 in the Polish population

The polymorphism of the D1S80 locus has been analyzed in a population sample of 208 unrelated individuals in the Southeast Poland and 103 mother/child pairs. PCR amplified alleles were separated by a vertical discontinuous polyacrylamide gel electrophoresis system. Nineteen different alleles and 52 phenotypes could be distinguished. The alleles 18 ( f = 0.267) and 24 ( f = 0.300) were most common in Poland. D1S80 genotype frequencies of Poland population do not deviate from Hardy-Weinberg equilibrium. All mother/child pairs shared at least one D1S80 allele.

Multiplex Amplification and Typing Procedure for the Loci D1S80 and Amelogenin

A method has been developed that enables multiplex amplification and simultaneous typing of the loci D1S80 and amelogenin using discontinuous polyacrylamide gel electrophoresis and silver staining. The protocol is sensitive, simple, rapid, and relatively inexpensive. The results of the multiplex analysis of the D1S80 and amelogenin loci were comparable to those obtained when each locus was analyzed individually. A small validation study was undertaken to evaluate the forensic applicability of this multiplex system. The data demonstrate that DNA exposed to a variety of environmental insults yields reliable multiplex typing results.

Enzymes from Pseudomonas sp. strain NCIB 11097 participating in biotransformation of acetaldehyde and glycine to threonine isomers.

Enzyme activities involved in L-threonine bioconversions present in cells of Pseudomonas sp. strain NCIB 11097 were separated by phenyl-Sepharose hydrophobic chromatography. The separation of the two main activity components was monitored by discontinuous polyacrylamide gel electrophoresis. Threonine aldolase catalyzed the conversion of glycine and acetaldehyde to a mixture of isomers, L-threonine and L-allothreonine, in a biotransformation reaction having pH and temperature optima of 7.5 and 25-30 degrees C, respectively. The fraction containing serine hydroxymethyltransferase converted acetaldehyde and glycine specifically to L-allothreonine in a biotransformation reaction having pH and temperature optima of 7.4 and 37 degrees C, respectively.