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PubMed2005

Enantiomeric purity determination of tamsulosin by capillary electrophoresis using cyclodextrins and a polyacrylamide-coated capillary.

The chiral separation of racemic tamsulosin hydrochloride (TH) was carried out using cyclodextrin (CD)-mediated capillary electrophoresis (CE) with DAD at 200 nm. The best separation of enantiomers of the studied compound was achieved at 20 kV with 30 cm x 50 microm I.D. polyacrylamide (PAA)-coated fused-silica capillary (effective length 20 cm) and running buffer with sulfated-beta-CD (S-beta-CD) as chiral selector. Other selected native or derivatized CDs were also tested: beta-CD (5, 15 mmol l(-1)), carboxymethyl-beta-CD (5, 30 mmol l(-1)), dimethyl-beta-CD (15 mmol l(-1)) and hydroxypropyl-beta-CD (5, 30 mmol l(-1)). Several parameters such as capillary pretreatment, buffer type and concentration, pH of background electrolyte, methanol content, separation temperature and voltage, were optimized. The excellent baseline separation of chiral TH was successfully achieved within 12 min using 100 mmol l(-1) phosphate buffer with pH 2.5 containing 1.7 mmol l(-1) S-beta-CD. Rectilinear calibration range was 50.0-500.0 mumol l(-1) of each enantiomer (r = 0.9993-0.9996). The method was applied to the assay of R-TH in Omnic, capsules (nominal content 0.4 mg per capsule) with R.S.D. 2.75% (n = 6), recovery 99.3-101.7% and it was suitable for the chiral purity control of the active enantiomer in the pharmaceutical.

Acrylic Resins↗
PubMed2006

Encapsulation efficiency and controlled release characteristics of crosslinked polyacrylamide particles.

Polyacrylamide (pAAm) particles crosslinked with N,N-methylenebis-acrylamide/ethylene glycol dimethacrylate (NNMBA/EGDMA) have been prepared in water-methanol medium by the dispersion polymerization using poly(vinyl pyrrolidone), PVP as a steric stabilizer. 5-fluorouracil an anticancer drug, has been loaded in situ into the crosslinked pAAm particles. Plain as well as drug loaded microparticles have been characterized by differential scanning calorimetry (DSC) and X-ray diffraction studies (XRD) and scanning electron microscopy (SEM). DSC and XRD studies have indicated a molecular level dispersion of the drug in pAAm particles during in situ loading and SEM pictures have shown the formation of spherical and oval-shaped particles. In vitro release of 5-fluorouracil from the crosslinked pAAm particles has been carried out in 7.4 pH buffer medium. Both encapsulation efficiency and release patterns are found to depend on the nature of the crosslinking agent, amount of crosslinking agent used and the amount of drug loaded. In vitro release studies indicated the controlled release of 5-fluorouracil up to 12 h.

Acrylamides↗
PubMed2004

Staining of proteins on SDS polyacrylamide gels and on nitrocellulose membranes by Alta, a colour used as a cosmetic.

We describe here the use of Alta, a pre-existing scarlet-red stain of cosmetic use, for staining proteins on sodium dodecyl sulfate (SDS) polyacrylamide gels, as well as for a single step staining of gels and nitrocellulose membranes during Western blot analysis. This stain, which is composed of 0.8% Crocein scarlet (brilliant crocein) and 0.2% Rhodamine B, is inexpensive, easy to use and nearly as sensitive as Coomassie Brilliant Blue (CBB) R-250. The gels can be stained in 10% Alta (2 h) and can be destained effectively only with 7% acetic acid as opposed to the conventional destainer (methanol/acetic acid/water) required for CBB-stained gels. In an alternative procedure, the proteins can be stained on the gel while electrophoresis by simply using 5% Alta in the top tank buffer and the stain can be viewed under UV-transilluminator. This procedure can also be used for Western blot analysis, as a single step procedure for staining of proteins on the gel as well as on the nitrocellulose membrane, as the stain is retained on the membrane after protein transfer. Thus, this staining procedure allows monitoring of proteins after each step in the Western blot, thereby eliminating the need to run separate gels for staining and Western blot analysis, and also the need for Ponceau Red S staining of the nitrocellulose membrane during Western blot analysis.

Azo Compounds↗
PubMed2005

Blue native polyacrylamide gel electrophoresis and the monitoring of malate- and oxaloacetate-producing enzymes.

We demonstrate a facile blue native polyacrylamide gel electrophoresis (BN-PAGE) technique to detect two malate-generating enzymes, namely fumarase (FUM), malate synthase (MS) and four oxaloacetate-forming enzymes, namely pyruvate carboxylase (PC), phosphoenolpyruvate carboxykinase (PEPCK), citrate lyase (CL) and aspartate aminotransferase (AST). Malate dehydrogenase (MDH) was utilized as a coupling enzyme to detect either malate or oxaloacetate in the presence of their respective substrates and cofactors. The latter four oxaloacetate-forming enzymes were identified by 2,6-dichloroindophenol (DCIP) and p-iodonitrotetrazolium (INT) while the former two malate-producing enzymes were visualized by INT and phenazine methosulfate (PMS) in the reaction mixtures, respectively. The band formed at the site of enzymatic activity was easily quantified, while Coomassie staining provided information on the protein concentration. Hence, the expression and the activity of these enzymes can be readily evaluated. A two-dimensional (2D) BN-PAGE or SDS-PAGE enabled the rapid purification of the enzyme of interest. This technique also provides a quick and inexpensive means of quantifying these enzymatic activities in normal and stressed biological systems.

Aspartate Aminotransferases↗
PubMed2006

Electroelution of lipoprotein(a) [Lp(a)] from native polyacrylamide gels: a new, simple method to purify Lp(a).

Lipoprotein(a), Lp(a), is an atherogenic lipoprotein consisting of an LDL like core particle and a covalently linked glycoprotein of variable size. Lp(a), isolated from serum always contains LDL and HDL(2) as contaminants since Lp(a) floats in the density range 1.05-1.12 g/ml which overlaps that of LDL and HDL(2). Purified Lp(a) is increasingly needed as a standard to overcome various problems in the standardization of Lp(a) measurements and for in vitro biological studies. Problems inherent to the purification of Lp(a) include the aggregation of Lp(a) with LDL, overlapping size distribution and the inability of some fractions to bind to affinity columns. Here, we describe the development of a new method to purify Lp(a) from contaminating LDL and HDL(2) particles. Lp(a) was isolated from serum by sequential ultracentrifugation, resolved by native polyacrylamide gel electrophoresis and the gel segments were electroeluted to obtain pure Lp(a). l-Proline was added to the sample to a final concentration of 0.1 M to prevent the aggregation of Lp(a) with LDL.

Acrylic Resins↗
PubMed2005

Amperometric biosensor for the detection of hydrogen peroxide using catalase modified electrodes in polyacrylamide.

A simple biosensor for the detection of hydrogen peroxide in organic solvents has been developed and coupled to a flow injection analysis (FIA) system. Catalase was entrapped in polyacrylamide gel and placed on the surface of platinum (working electrode) fixed in a Teflon holder with Ag-wire (auxiliary electrode), followed by addition of filter paper soaked in KCl. The entrapped catalase gel was held on the electrode using membranes. The effects of cellulose and polytetrafluroethylene (PTFE) membranes on the electrode response towards hydrogen peroxide have been studied. The modified electrode has been used to study the detection of hydrogen peroxide in solvents like water, dimethyl sulfoxide (DMSO), and 1,4-dioxane using amperometric techniques like cyclic voltammetry (CV) and FIA. The CV of modified catalase electrode showed a broad oxidation peak at -150 mV and a clear reduction peak at -212 mV in the presence of hydrogen peroxide. Comparison of CV with hydrogen peroxide in various solvents has been carried out. The electrode showed an irreversible kinetics with DMSO as the solvent. A flow cell has been designed in order to carry on FIA studies to obtain calibration plots for hydrogen peroxide with the modified electrode. The calibration plots in several solvents such as water, dimethyl sulfoxide, 1,4-dioxane have been obtained. The throughput of the enzyme electrode was 10 injections per hour. Due to the presence of membrane the response time of the electrode is concentration dependent.

Acrylic Resins↗
PubMed2005

Lead removal by polyacrylamide-bentonite and zeolite composites: effect of phytic acid immobilization.

Polyacrylamide (PAA)-bentonite (B) and zeolite (Z) composites were prepared by direct polymerization of PAA in suspensions of B and Z. Phytate (Phy) immobilized onto the composites to obtain their Phy modifications. FT-IR and XRD were employed to characterize their structures. The sorption of Pb(2+) from various adsorption media onto the composites was then investigated. The reusability and storage characteristics of the adsorbents with and without Phy were also considered for Pb(2+) adsorption. All isotherms were L and H type of the Giles classification. The Phy adsorption capacities (X(M)) of PAA-B and Z lower than those of bare B and Z due to their surface coverage by PAA. The Phy modification decreased the Pb(2+) adsorption capacities of B and PAA-B but ascended those of Z and PAA-Z. However, Phy significantly increased the Langmuir equilibrium constants (K(L)) for all adsorbents. Ionic strength of the adsorption media was favorable for the adsorption capacity of PAA-B while not for that of PAA-Z. The chemical structure of the composites was not changed after five reuses and the storage foregoing. It is foreseen that the use of PAA-B and PAA-Z and their Phy modifications will provide practicality and effectiveness for Pb(2+) separation and removal procedures. The X(M) (mol kg(-1)) and K(L) (L mol(-1)) values for Pb(2+) adsorption from a model solution were 0.16 and 3625 for PAA-B, 0.18 and 5.1 x 10(5) for PAA-B-Phy, 0.28 and 13210 for PAA-Z and 1.65 and 2.0 x 10(5) for PAA-Z-Phy.

Acrylic Resins↗
PubMed2005

Adsorption of cationic polyacrylamide onto sepiolite.

The adsorption of PAM onto sepiolite from aqueous solutions has been investigated systematically as a function of some parameters such as calcination temperature of sepiolite, pH, ionic strength and temperature. The adsorption of cationic polyacrylamide (PAM) increases with pH from 5.50 to 11.00, temperature from 25 to 55 degrees C and ionic strength from 0 to 0.1molL(-1). The sepiolite sample calcined at 200 degrees C has a higher adsorption capacity than the other calcined samples. Adsorption isotherms of PAM onto sepiolite have been determined and correlated with common isotherm equations such as Langmuir and Freundlich isotherm models. The Langmuir isotherm model appeared to fit the isotherm data better than the Freundlich isotherm model. The physical properties of this adsorbent are consistent with the parameters obtained from the isotherm equations. The zeta potentials of sepiolite suspensions have been measured in aqueous solutions of NaCl and different PAM concentrations and pH. From the experimental results: (i) pH strongly alters the zeta potential of sepiolite, (ii) sepiolite has an isoelectric point at about pH 6.6 in water and about pH 8 in 250mgL(-1) PAM concentration, (iii) PAM changes the interface charge from negative to positive for sepiolite. Effect of temperature on adsorption has been quantified by calculating various thermodynamic parameters such as Gibbs free energy, enthalpy and entropy. The dimensionless separation factor (R(L)) has shown that sepiolite can be used for adsorption of PAM from aqueous solutions.

Acrylic Resins↗
PubMed2003

Use of acridine orange staining for the detection of rotavirus RNA in polyacrylamide gels.

Acridine orange is a metachromatic intercalator used extensively in histochemistry to differentiate double- from single-stranded (ds, ss) nucleic acid by the emission of green and red fluorescence, respectively, under ultraviolet light. In the present study we standardised a protocol in order to use acridine orange to detect rotavirus ds RNA in polyacrylamide gels and compared it to silver and ethidium bromide staining. We demonstrated that the simplest and best condition was attained when gels containing rotavirus ds RNA bands, stained in green, were treated with 4.3 microM acridine orange after electrophoresis and destained with distilled water pH 6 at 37 degrees C. Under this protocol, rotavirus RNA concentration was calculated and the mean minimum amounts of nucleic acid detected by acridine orange, ethidium bromide, and silver staining were 26.0 +/- 4.29, 15.6 +/- 1.48 and 1.06 +/- 0.11 ng, respectively. The comparison of acridine orange sensitivity with ethidium bromide and silver staining, for 25 field strains of rotavirus and one cell-adapted strain (SA11), demonstrated concurrent results in 80% of the specimens. Red colour emission resulting from the interaction of acridine orange with ss nucleic acid was also shown by testing denatured 0.5 kb HindIII digest of lambda phage DNA. Furthermore, it was demonstrated that rotavirus ds RNA could be used for reverse transcription activity, followed by PCR amplification, after acridine orange staining. In conclusion, although acridine orange is less sensitive than ethidium bromide and silver staining, its practicality, low cost, metachromatic properties, and its non-interference on RT-PCR should be considered. It is suggested the use of acridine orange as an appropriate stain for various purposes in virology, as well as for the molecular biology of nucleic acid.

Acridine Orange↗
PubMed2005

Sensitive detection of transglycosylating activity of xyloglucan endotransglycosylase/hydrolase (XTH) after isoelectric focusing in polyacrylamide gels.

The paper describes a sensitive and rapid zymogram technique for detection of transglycosylating activity (XET) of xyloglucan endotransglycosylase/hydrolase (XTH; EC 2.4.1.207) in polyacrylamide isoelectric focusing gels. After the electrophoresis, the separating gel was overlaid and incubated with an agarose detection gel containing XET substrates: tamarind-seed xyloglucan as the glycosyl donor and sulphorhodamine-labeled xyloglucan-derived oligosaccharides (XGO-SRs) as the glycosyl acceptors. The transglycosylation catalyzed by XTH caused incorporation of the fluorescent label into the high-M(r) polysaccharide. Selective removal of unreacted XGO-SRs from the agarose replicas by washing with organic solvents revealed the zones corresponding to XET activity as bright pink fluorescent spots under UV-light. The method appears suitable for a number of purposes such as analysis of the isoenzyme composition of XTHs with XET activity in crude extracts from various plants and plant organs, monitoring the enzyme expression at various stages of plant development and/or for checking enzyme purity in the course of its isolation procedure.

Electrophoresis, Polyacrylamide Gel↗
PubMed2002

Microanalysis of hyaluronan oligosaccharides by polyacrylamide gel electrophoresis and its application to assay of hyaluronidase activity.

We established a rapid, sensitive polyacrylamide gel electrophoresis (PAGE) method for the analysis of hyaluronan (HA) oligosaccharides. Using mini-slab gels, but not large-slab gels so far reported, HA oligosaccharides of 5 to more than 50 repeating disaccharide units could be separated into discrete ladder-like bands in a short electrophoresis time of 45 min. Using a combined Alcian blue and silver staining protocol, the detection limit was less than 1 ng per band for 11 repeating disaccharide units, indicating 50 times higher sensitivity than that of an earlier-described sensitivity-enhanced PAGE method. Our PAGE method was applicable to the assay of hyaluronidase activity. When a total of multiple band intensities for 18-24 repeating disaccharide units was used as a measure of activity, as little as 3 x 10(-4) NFU of bovine testicular hyaluronidase was detectable on a 1-h incubation. This sensitivity permitted rapid measurements of human and rabbit serum hyaluronidases, the latter of which having never been detected even by a sensitive enzyme-linked immunosorbent assay (ELISA). Since this PAGE assay does not require specialized reagents and instruments and since it provides information on both the activity and the enzymatic HA degradation pattern, there may be many potential applications.

Animals↗
PubMed1992

Quantitation of proteins separated in N, N'-1,2-dihydroxyethylenebisacrylamide-crosslinked polyacrylamide gels.

A simple and rapid method for the quantitation of proteins separated either by sodium dodecyl sulfate-electrophoresis or by isoelectric focusing in slab gels is presented. The method is based on the solubility of polyacrylamide gels crosslinked with N, N'-1, 2-dihydroxyethylenebisacrylamide (DHEBA) in periodic acid. After electrophoretic separation proteins are stained with Coomassie brilliant blue G-250. DHEBA gels show considerable swelling during the staining and destaining process but can be shrunk to their normal size in a 10% (w/v) solution of ammonium sulfate. Stained bands are cut from the gel and solubilized in periodic acid. During dissolution the dye decolorizes. Protein concentration in the solution is determined by a modified Coomassie dye-binding assay. Quantitation is linear in the range of 100 ng to 5 micrograms and not disturbed by dissolved gel. Separations in N, N'-1, 2-dihydroxyethylenebisacrylamide-crosslinked gels show qualities similar to those in normal crosslinked gels.

Acrylamides↗
PubMed1999

Modification of liposomes with N-substituted polyacrylamides: identification of proteins adsorbed from plasma.

Liposomes prepared from DMPC (80%) and cholesterol (20%) were modified with a series of hydrophobically modified N-substituted polyacrylamides, namely, poly[N-isopropylacrylamide] (PNIPAM), poly[N,N-bis(2-methoxyethyl) acrylamide] (PMEAM), and poly[(3-methoxypropyl)acrylamide] (PMPAM). The hydrophobic group, N-[4-(1-pyrenylbutyl)-N-n-octadecylamine was attached to one end of the polymer chains to serve as an anchor for incorporation into the liposome bilayer. Liposome-polymer interactions were confirmed using fluorescence spectroscopy and chemical analysis. Microscopy revealed differences in aggregation tendency between unmodified and polymer-modified liposomes. Proteins adsorbed to liposome surfaces during exposure to human plasma were identified by immunoblot analysis. It was found that both unmodified and polymer-modified liposomes adsorb a wide variety of plasma proteins. Contact phase coagulation proteins, complement proteins, cell-adhesive proteins, serine protease inhibitors, plasminogen, antithrombin III, prothrombin, transferrin, alpha(2)-microglobulin, hemoglobin, haptoglobin and beta-lipoprotein as well as the major plasma proteins were all detected. Some differences were found between the unmodified and polymer-modified liposomes. The unmodified liposomes adsorbed plasminogen mainly as the intact protein, whereas on the modified liposomes plasminogen was present in degraded form. Also, the liposomes modified with PNIPAM in its extended conformation (below the lower critical solution temperature) appeared to adsorb less protein than those containing the 'collapsed' form of PNIPAM (above the LCST).

Acrylamides↗
PubMed1986

Detection of a novel lymphocyte protein-tyrosine kinase by renaturation in polyacrylamide gels.

Protein kinase activity, including activity specific for the phosphorylation of tyrosine residues, can be detected among particulate fraction proteins of T cell lymphomas after separation by SDS-polyacrylamide gel electrophoresis. Putative protein kinases are detected by renaturation of enzyme activity directly within the gel following removal of detergent. LSTRA, a cell line that exhibits elevated levels of protein-tyrosine kinase activity, was found to express a predominant protein-tyrosine kinase of molecular weight 30,000. This same enzyme was present in T lymphocytes and other T lymphoid cell lines. Studies involving rapid preparation of protein fractions, limited proteolysis and one-dimensional peptide mapping did not demonstrate a direct relationship between the phosphorylated 30,000 dalton protein and the predominant 56,000 dalton phosphotyrosine containing protein that is observed following phosphorylation of LSTRA cell particulate fractions in vitro.

Cell Line↗
PubMed2001

Polyacrylamides as hydrophilic selectors in non-aqueous capillary electrophoresis.

Polyacrylamides (PAAms) were investigated as hydrophilic selectors in non-aqueous capillary electrophoresis (CE). Separation of 10 substituted benzoates and unsubstituted benzoate as model samples was greatly improved by the addition of PAAms in acetonitrile-CE. The migration behavior indicates that the carbonyl moiety of PAAms works as a good hydrogen-accepting site toward hydrogen-donating analytes such as 4-hydroxybenzoate anion (4OH-BA) in acetonitrile. PAAms also serve as electron-accepting agents with its amide proton interacting with the dissociated carboxyl groups of the benzoates. The ion-dipole interaction is useful to control the migration behavior of benzoates without hydrogen-donating substituents. The overall mode of the interaction is similar to that of polyethylene glycol (PEG) 20000 reported previously, but the complex formation constant of poly(N-tert.-butyl)acrylamide (PBAAm) with 40H-BA estimated here was 130-fold larger than that of PEG 20000. This would be ascribed to the strong basicity of the carbonyl oxygen atoms of PBAAm as compared with the ether oxygen atoms of PEG. Furthermore, a copolymer of (N-tert.-butyl)acrylamide-acrylamide [70:30 (in feed)] exhibited a complex formation constant of about fourfold larger toward 4OH-BA than PBAAm, most probably due to decrease in steric hindrance from the tert.-butyl groups. Adrenaline and its six precursors have been separated successfully using the PAAms.

Acetonitriles↗
PubMed1978

Globin chain separation by SDS polyacrylamide gel electrophoresis. Simple screening method for elongated hemoglobin chains.

A simple method for the separation of hemoglobin chains from hemolysate or globin, by sodium dodecyl sulfate polyacrylamide gel electrophoresis, is described. The alpha, beta, and gamma chains can be clearly separated from each other. The alpha chain has the highest mobility, the beta chain has a slower mobility than the gamma chain, while the delta chain has about the same mobility as the beta chain. Hemoglobins with elongated chains can easily be detected by this method. Tak-beta, elongated by 11 residues, moves much more slowly than betaA but is much faster than alpha Constant Spring which is elongated by 31 residues. Screening of several individuals with slow-moving hemoglobins using this method led to the finding of a case with Hb Tak-beta thalassemia and other carriers of Hb Tak.

Electrophoresis, Polyacrylamide Gel↗
PubMed1989

Improved method for amino acid analysis of stained collagen bands from polyacrylamide gels using high-performance liquid chromatography.

A procedure is described for the determination of the amino acid composition of stained collagen bands separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis using high-performance liquid chromatography and post-labelling with o-phthalaldehyde. Six alpha-chain bands from collagens I, III and V were analysed directly after gel electrophoresis, Coomassie blue staining and destaining. It was shown that good accuracy and reproducibility can be attained using this method, which makes it possible to determine simultaneously nineteen amino acids: Asx, Thr, Ser, Glx, Pro, Cys, Gly, Ala, Val, Met, Ile, Leu, Tyr, Phe, His, Lys and Arg together with 4-Hyp and Hyl, specific amino acids found in collagen.

Amino Acid Sequence↗
PubMed1988

Several intermediate forms in the processing of rat lutropin subunits as shown by sodium dodecyl sulphate polyacrylamide gel electrophoresis.

To study the biosynthesis in situ of lutropin (LH) subunits, anterior pituitary cells in culture were employed. The cells were incubated in the presence of [35S]methionine. Labelled polypeptides, immunologically related to alpha and LH beta subunits, were isolated by specific immunoprecipitation, then analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and revealed by fluorography. Electrophoretic analysis of immunoprecipitates is a crucial step in this methodology. It permits simultaneous purification, characterization and accurate quantitation of the radioactivity specifically incorporated into LH subunits. Also, using SDS-PAGE, it is possible to isolate and identify the different processed forms of LH subunits.

Animals↗