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PubMed1994

Sodium dodecyl sulfate polyacrylamide gel- and replaceable polymer-filled capillary electrophoresis for molecular mass determination of proteins of pharmaceutical interest.

Performance of commercially available replaceable SDS polymer-filled capillary electrophoresis (rPGCE) systems were evaluated for the determination of the molecular mass of proteins of pharmaceutical interest. The mass values obtained by these systems were compared with those obtained by SDS-PAGE and high-performance size exclusion chromatographic (HPSEC). The molecular masses of proteins determined by rPGCE were not significantly different and agreed closely with those obtained by SDS-PAGE and with the theoretical values of the proteins. HPSEC gave the least reliable data. Performance of the SDS crosslinked polyacrylamide gel-filled capillary electrophoresis system was superior to that of the commercially available rPGCE systems. Nevertheless, the rPGCE systems present significant advantages over the conventional SDS-PAGE and HPSEC methods for rapid analysis and improved precision for the determination of the molecular masses of proteins of pharmaceutical interest.

Electrophoresis, Polyacrylamide Gel↗
PubMedDate not supplied

Studies and observations on the use of isoelectric focusing in ultra-thin polyacrylamide gels as a method of typing human red cell phosphoglucomutase.

The technique of isoelectric focusing in ultra-thin polyacrylamide gels as a method of typing human red cell phosphoglucomutase (PGM1) has been studied. Typing was possible without the samples attaining true equilibrium focusing conditions. The isozyme patterns so obtained were clearly defined and free from distortion. The importance of assessing relative band intensities when interpreting the isozyme patterns is discussed. Our experience of using the technique to analyse casework material is described.

Acrylic Resins↗
PubMed1988

Analysis of human vaginal secretions by SDS-polyacrylamide gel electrophoresis.

Proteins in 68 vaginal secretions from 50 women were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Although there were considerable numbers of intermediate types, the secretions could be classified into the following groups based on the densitometric patterns. A relatively low concentration of 67K protein (albumin) and a high concentration of 52K protein (IgG) were the patterns common to pre- and post-menstrual phases and pregnant stadium. A relatively high concentration of the 67K protein and a moderate concentration of the 52K protein characterized the midcycle. A considerably high concentration of 67K protein compared with the 79K (transferrin), 58K (IgA) and 52K bands was the pattern for menstrual phase and postpartum stage.

Adolescent↗
PubMed1989

Non-equilibrium pH gradient electrophoresis (NEPHGE) on ultrathin polyacrylamide gels containing separators: improved erythrocyte phosphoglucomutase (PGM) and esterase D (EsD) diagnosis in red cell lysates and bloodstains.

Two rapid and reliable electrophoretic techniques for PGM1 and EsD typing on ultrathin polyacrylamide gels are described. They have been based on non-equilibrium pH gradient electrophoresis and on the addition of chemical spacers (EPPS for PGM1 and HEPES for EsD) to the gel mixture.

Blood Stains↗
PubMed1995

Use of nondenaturing silver-stained polyacrylamide gel analysis of polymerase chain reaction amplification products for the differential diagnosis of Leptospira interrogans infection.

A 285-bp DNA fragment was amplified using the polymerase chain reaction from 38 Leptospira serovars of six different genomic species. The fragments amplified exhibited differential mobilities on nondenaturing polyacrylamide gels resulting from sequence-dependent conformational alterations. Leptospira interrogans serovars could be distinguished from those of other species on this basis.

Bacterial Typing Techniques↗
PubMed1991

An improved pulsed-field polyacrylamide gel electrophoresis system for physical selection of linking clones: isolation of SfiI linking clones from a chromosome 21-specific library.

We had previously developed an efficient procedure for selective cloning of rare-cutter linking fragments that is based on physical separation of linking clone DNAs by pulsed-field polyacrylamide gel electrophoresis (PF-PAGE). An advantage of the physical selection procedure over the conventional cloning-based ones utilizing the insertion of selection marker or vector sequences into the rare-cutter sites is that it can be readily applied to the selection of linking fragments for rare-cutters, generating ambiguous cohesive end sequences such as SfiI (GGCCNNNN/NGGCC). In the present work, the physical separation procedure was improved by introducing a discontinuous buffer system into PF-PAGE, and its feasibility was exemplified by the selective isolation of SfiI linking clones from a human chromosome 21-specific library. This simple and efficient procedure will provide a useful tool for genome analysis.

Base Sequence↗
PubMed1990

Genomic variation of herpes simplex virus type 2 isolates analysed by hybridization after electroblotting from polyacrylamide gels.

Genomic variation of herpes simplex virus type 2 (HSV-2) strains was analysed by polyacrylamide gradient gel electrophoresis and subsequent hybridization to cloned HSV-2 sequences. Two probes were used, one from the L-segment and one from the S-segment of the HSV-2 genome. The probes did not contain a-repeat sequences. Hybridization to the specific sequences in individual DNA fragments obtained by use of the frequently cleaving restriction endonuclease Alu I revealed variations in the genome not detectable by analysing the fragment size only. The use of 35S-labelled deoxynucleotide in the radioactive labelling of the probe further improved the resolution of the method.

DNA, Viral↗
PubMed1995

Nucleoside phosphatase activities on pig pancreas zymogen granule membranes analyzed by non-denaturing polyacrylamide gel electrophoresis.

The membrane of the pancreatic zymogen granule plays an important part in the sequence of storage, transport and exocytosis of digestive enzymes. While much is known on stimulus-secretion coupling, very little is understood about how the storage organelles move in the cytoplasm to the luminal plasma membrane and why and how they fuse with it to release the contents. It is assumed that nucleoside phosphatases are involved in these energy consuming processes. Pancreatic zymogen granule membranes contain one major glycoprotein, GP-2, and a few minor proteins all with unknown functions. In order to identify functions we have purified zymogen granule membranes from pig pancreas, solubilized the proteins under non-denaturing conditions with the detergent CHAPS and characterized the extracted proteins by polyacrylamide gel electrophoresis, histochemistry and lectins. Three major protein bands, often fused in one broad band, revealed enzymatic activity for adenosine-, cytidine-, inositol- and guanidine- di- and triphosphates by the precipitation of liberated phosphate by Pb(NO3)2. This activity was sensitive to known ATP diphosphohydrolase inhibitors. The band with activity arises from a 92 kDa glycoprotein. A different narrow band showed monophosphatase activity for AMP, GMP, IMP and CMP. Some of the activities were inhibited by different lectins, indicating glycosyl groups near the active site. Electron microscopical cytochemistry confirmed a nucleoside phosphatase activity on granule membranes. Our results show for the first time that the nucleoside phosphatase activity of the zymogen granule membranes is carried by a 92 kDa glycoprotein, probably the known self-associating form of GP-2. The hydrolysis of tri- and diphosphate nucleotides could provide the energy required by exocytosis.

Animals↗
PubMed2003

Electrophoretic mobility of Alzheimer's amyloid-beta peptides in urea-sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

The 43-amino acid Alzheimer's amyloid-beta peptide (Abeta peptide) retains a predominantly alpha-helix and beta-strand structure in sodium dodecyl sulfate (SDS) solution. This conformer has a high tendency to aggregate during conventional SDS-polyacrylamide gel electrophoresis (PAGE). Both the secondary structure and the proclivity for aggregation are obviated by the use of urea-SDS-PAGE: In 8M urea-with or without SDS-the Abeta peptide becomes 100% random coil and remains monomeric. However, during electrophoresis in this medium, the peptide and its truncated variants do not obey the law of mass/mobility relationship that most proteins-including Abeta peptides-follow in conventional SDS-PAGE. Rather, the smaller carboxy-terminally truncated peptides migrate slower than the larger full-length peptide, while the amino terminally truncated peptide does migrate faster than the full-length Abeta peptide. Thus, despite their small size (2-4kDa) and minor differences between their lengths, the Abeta peptides display a wide separation in this low-porosity (12% acrylamide) gel. We found that this unusual electrophoretic mobility in 8M urea is due to the fact that the quantity of [35S]SDS bound to the Abeta peptides, instead of being proportional to the total number of amino acids, is rather proportional to the sum of the hydrophobicity consensus indices of the constituent amino acids. It is then their hydrophobicity and, hence, the net negative charges contributed by the peptide-bound SDS that plays a major role in determining the mobility of Abeta peptides in 8M urea-SDS-PAGE. The high selectivity of the 8M urea-SDS-PAGE method allowed us to detect the presence of hitherto unknown Abeta peptide variants that were secreted in the conditioned medium by cultured HeLa cells.

Amyloid beta-Peptides↗
PubMed2006

Suppression of sodium dodecyl sulfate-polyacrylamide gel electrophoresis sample preparation artifacts for analysis of IgG4 half-antibody.

Human IgG4 subtype antibodies have often been reported to have a significant portion (5-50%) of a heavy chain-light chain dimer ("half-antibody") on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), in which the heavy chain is not covalently linked through the hinge disulfides to another heavy chain. We demonstrate here that there can be artifactual sources of half-antibody. One occurred during SDS-PAGE sample preparation where rapid disulfide scrambling was initiated by preexisting free sulfhydryls in the monoclonal antibody (mAb) and by free sulfhydryl produced by destruction of disulfide bonds during heating. Inclusion of N-ethylmaleimide in the sample buffer prevented the disulfide scrambling. Presumably, cyclization of the flexible IgG4 hinge during this disulfide scrambling leads to the preferential separation of heavy chains. A second condition producing half-antibody was reoxidation after exposure to reductant, where 46% of the antibody was trapped in the intrachain disulfide form. The amount of half-antibody was reduced to 4% by reoxidation in the presence of a mixture of oxidized and reduced glutathione. When the improved sample preparation conditions were used, IgG4 mAb freshly isolated from cells contained 4.5-15% half-antibody, indicating that equilibration of the interchain and intrachain hinge disulfide pairing was not always attained in cells.

Antibodies, Monoclonal↗
PubMed2006

In-gel activity staining of oxidized nicotinamide adenine dinucleotide kinase by blue native polyacrylamide gel electrophoresis.

Oxidized nicotinamide adenine dinucleotide (NAD(+)) kinase (NADK, E.C. 2.7.1.23) plays an instrumental role in cellular metabolism. Here we report on a blue native polyacrylamide gel electrophoretic technique that allows the facile detection of this enzyme. The product, oxidized nicotinamide adenine dinucleotide phosphate (NADP(+)), formed following the reaction of NADK with NAD(+) and adenosine 5'-triphosphate was detected with the aid of glucose-6-phosphate dehydrogenase or NADP(+)-isocitrate dehydrogenase, iodonitrotetrazolium chloride, and phenazine methosulfate. The bands at the respective activity sites were excised and subjected to native and denaturing two-dimensional electrophoresis for the determination of protein levels. Hence this novel electrophoretic method allows the easy detection of NADK, a critical enzyme involved in pyridine homeostasis. Furthermore, this technique allowed the monitoring of the activity and expression of this kinase in various biological systems.

Electrophoresis, Polyacrylamide Gel↗
PubMed2004

Implication of the differential roles of metallothionein 1 and 2 isoforms in the liver of rats as determined by polyacrylamide-coated capillary zone electrophoresis.

Metallothioneins (MTs), determined by polyacrylamide-coated capillary zone electrophoresis (CZE), coincided well with those described by enzyme-linked immunosorbent assay. By using CZE, MT isoforms 1 (MT-1) and 2 (MT-2) were well separated and determined in the liver cytosol of LEC rats and Wistar rats administered CdCl(2). The total concentrations of MTs in the liver cytosol of LEC rats increased age-dependently as 1.0, 2.1, and 7.2mg/g wet weight of the liver at the age of 5, 10, and 15 weeks, respectively, and those of Wistar rats that had received daily CdCl(2) also increased with time of CdCl(2) as 0.5 and 1.2mg/g wet weight of the liver for 3 and 6 consecutive administration days, respectively. The MT-1/MT-2 ratio in the liver cytosol of LEC rats decreased age-dependently as 1.75, 1.49, and 0.76 at the age of 5, 10, and 15 weeks, respectively. In contrast, that of Wistar rats increased with time of exposure to the metal ion CdCl(2) as 1.1 and 1.6 for 3 and 6 administration days, respectively. Copper accumulation in the liver of LEC rats has already been reported. The present results indicated that the mechanism of the induction of MT synthesis differs between LEC rats, who lack ATP7B, and Wistar rats, who were given a toxic metal ion. On the basis of these results, we propose that MT-1 is related to the metabolism or detoxification of toxic metals such as Cd, and in contrast, MT-2 is responsible for the homeostasis of essential metals such as Cu.

Acrylic Resins↗
PubMed2006

Promotion of opsonization by antibodies and phagocytosis of Gram-positive bacteria by a bifunctional polyacrylamide.

This paper describes the application of a bifunctional polyacrylamide (pA-V-F) presenting both vancomycin and fluorescein groups, to modify the surfaces of multiple species of Gram-positive bacteria (Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, and Enterococcus faecalis) to control molecular recognition of these surfaces. The vancomycin groups allowed the specific recognition of a structural component of the bacterial cell wall: peptides terminated in D-Ala-D-Ala. The fluorescein groups allowed the imaging of binding of polymer to the surfaces of bacteria by fluorescence, and are representative, low molecular weight haptens; their recognition by anti-fluorescein antibodies provides proof-of-principle that bifunctional polymers can be used to introduce haptens onto the surface of the bacteria. Flow cytometry revealed that polymer-labeled S. aureus and S. pneumoniae were opsonized by anti-fluorescein antibodies approximately 20-fold more than were untreated bacteria; nearly all ( approximately 92%) polymer-labeled S. aureus, and a large (76%) fraction of polymer-labeled S. pneumoniae were opsonized. The bound antibodies then promoted phagocytosis of the bacteria by cultured J774 macrophage-like cells. Flow cytometry revealed that macrophages ingested S. aureus decorated with the polymer-antibody complexes approximately 2-fold more efficiently than S. aureus in control groups, in spite of the high background (caused by efficient antibody-independent ingestion of S. aureus by macrophages). This paper, thus, demonstrates the ability of a bifunctional polymer to carry out three distinct functions based on polyvalent molecular recognition: (i) recognition of the surface of Gram-positive bacteria, (ii) modification of this surface to generate specific binding sites recognized by an antibody, and (iii) promotion of phagocytosis of the opsonized bacteria.

Acrylic Resins↗
PubMed2005

Use of Cassia javahikai seed gum and gum-g-polyacrylamide as coagulant aid for the decolorization of textile dye solutions.

Investigations were carried out for possible exploitation of Cassia javahikai seeds as potential source of commercial gum for the textile wastewater treatment. Graft copolymerization with acrylamide was done to modify the seed gum for the favorable properties. C. javahikai seed gum, and its copolymer grafted with acrylamide were synthesized in the presence of oxygen using potassium persulphate/ascorbic acid redox system. Both C. javahikai seed gum (CJ) and its grafted-polyacrylamide (CJG), were found to be good working substitutes as coagulant aids in conjunction with PAC, for the decolorization of all the dyes in varying ratios. CJ and CJG alone could effectively decolorize direct dyes (DBR and DO) and in conjunction with a very low dose of PAC could decolorize all the dyes (DBR, DO, ASR, and PBB) to more than 70%. Grafting also increased the decolorizing ability of CJ gum.

Acrylic Resins↗
PubMed2005

Triton-polyacrylamide gel electrophoresis and leucine aminopeptidase activity staining detect Triton-slowed bands including high-molecular-mass aminopeptidase N (CD13) isoform in cholestatic patient sera.

BACKGROUND: Western blotting of aminopeptidase N (APN) detects a high-molecular-mass isoform (260 kDa) [M. Kawai, Y. Otake, Y. Hara High-molecular-mass isoform of aminopeptidase N/CD13 in serum from cholestatic patients. Clin Chim Acta 330 (2003) 141-149] in cholestatic patient serum but is time-consuming. METHODS: Human sera were electrophoresed on polyacrylamide gel containing Triton-X100 (Triton-PAGE) and stained with leucine-B-naphthylamide (LAP-staining). The stained bands were eluted from the gel, treated with N- and O-glycosidase if necessary, and analyzed by Western blotting [M. Kawai, Y. Otake, Y. Hara High-molecular-mass isoform of aminopeptidase N/CD13 in serum from cholestatic patients. Clin Chim Acta 330 (2003) 141-149]. RESULTS: Triton-PAGE and LAP-staining clearly detected fast bands in all the sera examined. Almost parallel with leucine aminopeptidase activity, slow bands were strongly stained in all 11 cholestatic patients but clearly stained in 3 out of 14 patients with hepatobiliary diseases other than cholestasis. PAGE with various concentrations of Triton showed that Triton slows down slow bands but not fast bands. Western blotting showed that Triton-PAGE-slow bands of cholestasis contained 140 and 260-kDa APN and that fast bands were slightly smaller than monomer-size slow bands after glycosidase treatment. CONCLUSIONS: Less time-consuming than Western blotting, Triton-PAGE and LAP-staining detect novel APN bands slowed by Triton and partly composed of the high-molecular-mass isoform in cholestasis. The slow bands seem to be homodimers of APN with transmembrane anchors. The polypeptide of the fast band seems to be processed differently from that of the slow band.

Blotting, Western↗
PubMed2004

Resveratrol-induced cell growth inhibition and apoptosis is associated with modulation of phosphoglycerate mutase B in human prostate cancer cells: two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry evaluation.

Several studies provide evidence for the anti-carcinogenic activity of resveratrol, a phytoalexin present in grapes and berries, but the precise mechanisms involved in the modulation of prostate carcinogenesis by resveratrol remain to be elucidated. The inhibitory effects induced by resveratrol in human prostate cancer cells impact diverse cellular mechanisms associated with tumor initiation, promotion, and progression. In our earlier studies with prostate cancer cells using cDNA microarray analysis, we indicated the importance of p53-mediated molecular targets of resveratrol. The present study based on two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (2D-SDS-PAGE) followed by mass spectrometry analysis of human prostate cells that have been treated with resveratrol clearly identifies the role of phosphoglycerate mutase B. For the first time, we report on phosphoglycerate mutase B in the resveratrol-treated prostate cancer cells LNCaP, DU145, and PC-3 at the transcription level. Our observations raise the possibility of its effect on metabolic enzymes in cancer cells without affecting the normal cells.

Antineoplastic Agents, Phytogenic↗
PubMed2005

Polyacrylamide sorption opportunity on interlayer and external pore surfaces of contaminant barrier clays.

Physico-chemical interactions among polymer molecules in aqueous solution and clay mineralogical/textural characteristics influence the sorption of polymer molecules on clay barrier minerals. Amendment of potentially unstable barrier clays with aqueous polymers can improve barrier material resistance to environmental stresses during service. In this research, the ability of molecular coils of polyacrylamide (PAM) to overlap in solution and to enter interlayer space in Na-montmorillonite (specific surface=31.82+/-0.22 m2 g(-1)) and kaolinite (specific surface=18+/-2 m2 g(-1)) were analyzed theoretically and experimentally, using solution viscosity measurements, and X-ray diffractometry. Experimental data on two theoretical indices: relative size ratio (RSR); and molecular availability (Ma) that are formulated to scale polymer molecular sorption on clay interlayer, indicate that the sorption of PAM A (Mw=4000000) and PAM B (Mw=7000000) does not produce any significant change in the d-spacing of both clay minerals. Although the negative Ma values of -3.51 g l(-1) for PAM A and -3.88 g l(-1) for PAM B indicate high levels of entanglement of polymer molecular coils in solution, sorption data confirm that the entangled coils are still able to sorb onto Na-montmorillonite highly and kaolinite to a lesser extent.

Acrylic Resins↗