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PubMed2004

An electrokinetic characterization of low charge density cross-linked polyacrylamide gels.

Hydrogels of polyacrylamide have found wide application in separation science and, more recently, in speciation techniques. These applications ideally require an uncharged, inert polymer matrix to act as a conduit for either diffusion or electrically driven migration. However, the electrical effects due to the structural charge of the gel itself are typically neglected. In order to address this issue, streaming potential and conductivity measurements were carried out on thin films of porous polymer gels of varying fixed charge density. The films consisted of gels of polyacrylamide-co-sodium acrylate, cross-linked with N,N'-methylenebisacrylamide. Conductivity data allowed the estimation of the Donnan potential difference between the bulk gel and electrolyte solution as a function of ionic strength. The data illustrated that even a gel with very modest fixed charge density can give rise to Donnan potentials of tens of millivolts in the submillimolar regime of electrolyte concentration. Streaming potential data were used to calculate zeta potentials using a form of the Smoluchowski equation expanded to account for conductance in the gel layers. The resulting zeta potentials are not consistent with the Donnan potentials obtained from the analysis of the conductivity data and demonstrate the need for a more complete electrokinetic model for soft interfaces.

Journal Article↗
PubMed2006

Spectroscopic investigation of the adsorption mechanisms of polyacrylamide polymers onto iron oxide particles.

The mechanisms of high-molecular-weight polyacrylamide nonionic homopolymer and 25 mol% anionic acrylate-substituted copolymer adsorption onto iron oxide particles were investigated via DRIFT and UV-vis spectroscopies at three pH values (6, 8.5, and 11). While electrostatic interactions play an important role in charged polymer adsorption, this information is not spectroscopically available. At pH values above and below pH 8.5 (the isoelectric point for the anionic polymer), bidentate chelation and hydrogen bonding were the main adsorption mechanisms. At the isoelectric point, monodentate chelation was observed to be the main mode of adsorption, along with hydrogen bonding. For the nonionic polymer, in all cases, hydrogen bonding through the carbonyl group was the main mode of adsorption. The adsorption of both polymers conformed well to the Freundlich model, suggesting that the adsorbed polymer amount increases with increasing polymer concentration up to 7500 g/t solid, rather than approaching monolayer coverage. Spectroscopic evidence was found to suggest that hydrolysis of nonionic polyacrylamide occurs at high pH.

Journal Article↗
PubMed2006

Monthly variations in ovine seminal plasma proteins analyzed by two-dimensional polyacrylamide gel electrophoresis.

This study was conducted to evaluate monthly changes in the ram seminal plasma protein profile using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) with a polyacrylamide linear gradient gel. Likewise, comparative analyses of the protein composition of ovine seminal plasma (SP) from ejaculates obtained along the year, and its relationship with sperm motility, viability and concentration of ejaculate were carried out. Western-blot analysis was performed to specifically detect P14, a ram SP protein postulated to be involved in sperm capacitation and gamete interaction [Barrios B, Fernández-Juan M, Muiño-Blanco T, Cebrián-Pérez JA. Immunocytochemical localization and biochemical characterization of two seminal plasma proteins which protect ram spermatozoa against cold-shock. J Androl 2005;26:539-49], and its variations along the year have also been established. The experiment was carried out from May 2003 to April 2004, with nine Rasa Aragonesa rams. Ejaculates obtained every 2 days were pooled and used for each assay, to avoid individual differences, and three two-dimensional SDS-PAGE gels were run for each month. The high resolution of the gradient gel allowed the image analysis software to detect around 252 protein spots, with pIs ranging from 4.2 to 7.6, and molecular weight (M(r)) from 12.5 to 83.9 kDa. Four protein spots (1, 2, 3 and 4) of low M(r) (15.1, 15.7, 15.9 and 21.0 kDa) and acidic pI (5.9, 5.3, 5.7 and 6.6), respectively, had the highest relative intensity in the SP map (11.2, 9.3, 4.7 and 7.7%, respectively). Spot 3 was more abundant (P<0.05) from May to December, and negatively correlated (P<0.05, r=-0.34) with sperm viability and concentration (P<0.05, r=0.36). Another 12 protein spots also had significant quantitative differences (P<0.05) along the year, and 17 protein spots, which correlated with some seminal quality parameter, did not show quantitative monthly changes. Western-blot analysis indicated that spots 1 and 2 reacted with the anti-P14 antibody, raised against the P14 band (approximate M(r) 14 kDa) of ram SP. This indicates that spots 1 and 2 are similar to RSP15 [Bergeron A, Villemure M, Lazure C, Manjunath P. Isolation and characterization of the major proteins of ram seminal plasma. Mol Reprod Dev 2005;71:461-70], bovine PDC-109 [Esch FS, Ling NC, Bohlen P, Ying S, Guillemin R. Primary structure of PDC-109, a major protein constituent of bovine seminal plasma. Biochem Biophys Res Commun 1983;113:861-7] (also called BSP A1/A2 [Manjunath P, Sairam MR. Purification and biochemical characterization of three major acidic proteins (BSP-A1, BSP-A2 and BSP-A3) from bovine seminal plasma. Biochem J 1987;241:685-92]) and goat GSP-14/15 kDa [Villemure M, Lazure C, Manjunath P. Isolation and characterization of gelatine-binding proteins from goat seminal plasma. Reprod Biol Endocrinol 2003;1:39], based on our previous results on the P14 amino acid sequence [Barrios B, Fernández-Juan M, Muiño-Blanco T, Cebrián-Pérez JA. Immunocytochemical localization and biochemical characterization of two seminal plasma proteins which protect ram spermatozoa against cold-shock. J Androl 2005;26:539-49].

Animals↗
PubMed2002

Laser capture microdissection and two-dimensional polyacrylamide gel electrophoresis: evaluation of tissue preparation and sample limitations.

Laser capture microdissection (LCM) is now well established as a tool for facilitating the enrichment of cells of interest from tissue sections, overcoming the problem of tissue heterogeneity. LCM has been used extensively in combination with analysis at the DNA and RNA levels, but only a small number of studies have employed LCM with subsequent protein analysis, albeit with promising results. This study focuses on the potential of LCM in combination with two-dimensional polyacrylamide gel electrophoresis. The effects of tissue section preparation and sample type were evaluated to fully determine the suitability of using LCM in global protein profiling. The effects of several histochemical stains (hematoxylin and eosin, methyl green and toluidine blue) and immunolabeling on subsequent two-dimensional polyacrylamide gel electrophoresis were investigated. Quantitative analysis was performed to establish the extent of changes in the relative intensity of protein species and their reproducibility. All staining protocols tested were found to be compatible with protein analysis although there was variation in protein recovery and the quality of the protein profiles obtained. LCM of renal and cervix samples indicated that protein yield after dissection was acceptable, although the extent of enrichment and dissection time was tissue-dependent, which may preclude the use of this approach with some tissue types. These results indicate that LCM has potential as a tool in proteomic research.

Electrophoresis, Gel, Two-Dimensional↗
PubMed1975

Observations on the staining of ceruloplasmin following disc-electrophoresis utilizing polyacrylamide gels.

In our hands the dianisidine technique for the staining of ceruloplasmin in polyacrylamide gels following disc electrophoresis has proven unsatisfactory for three reasons. First, staining of ceruloplasmin could not be achieved with physiological amounts of the glycoglobulin present in serum. Second, even when relatively massive quantities of pure ceruloplasmin permitted effective staining there was loss of stain intensity with increasing electrophoresis time. Third, the necessity of utilizing dianisidine in alcoholic solution produces undesirable shrinkage and distortion of the gels. However, a modification of the p-phenylenediamine technique for the staining of ceruloplasmin in agar gels has been found to stain ceruloplasmin very effectively in polyacrylamide gels following disc electrophoresis.

Animals↗
PubMed1982

Migration of fresh and cryopreserved human spermatozoa in polyacrylamide gel.

The ability of freshly collected and frozen human spermatozoa to migrate in round capillary tubes containing specially formulated polyacrylamide gel was investigated, using 33 ejaculates from 27 donors. Each semen sample was divided; one portion was left undiluted, and the other portion was diluted to 50 x 10(6) sperm/ml. Glycerol was used as the cryoprotectant. The percentage of motile sperm cells was determined before and after freezing. Fresh semen contained a higher percentage of motile cells, which migrated farther than those of cryopreserved-thawed semen. Various correlations between the percentage of motile sperm and migration distance ranged from 0.57 to 0.62. There was a low positive correlation of migration distance with sperm cell concentration per milliliter, r = 0.25 to 0.34; and thus adjusting semen samples to a standard sperm concentration improved the accuracy of the test only slightly. The regression coefficient of migration distance on the percentage of motile sperm in fresh semen was 0.65, indicating that for each 10% increase in sperm motility, migration distance is predicted to increase 6.5 mm. Five batches of polyacrylamide gel gave uniform results, and the application of this stable gel to fertility investigations is discussed.

Acrylic Resins↗
PubMed1996

Two-dimensional polyacrylamide gel electrophoresis of bovine semen after cryopreservation in half-milliliter straws.

One of the problems encountered with two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is the streaking of proteins so that individual spot identification is compromised. This study was conducted to determine whether a low loading dose (50 microg) of protein would permit resolution of more discrete protein spots using megapixel camera technology, and if so, to present a nomenclature for future comparisons of the identified proteins. If the major proteins could be identified in a 50-microg sample we aimed to determine whether they could be identified in the supernatant (seminal plasma plus extender) of cryopreserved semen. Two ejaculates were obtained from each of 6 bulls and bovine seminal plasma (BSP) protein concentration was standardized to 50 microg/10 microl. Isoelectric points (pI) and molecular weights (MWt) of BSP proteins were determined by measuring spot mobility on 2-D PAGE (15% polyacrylamide). Three distinct protein spot constellations (a,b,c) could be readily seen by the naked eye and a faintly stained constellation "d" was identified by the megapixel camera. The image analysis software located 6 protein spots in both constellation "a" (MWt 26 kDa; pI 4.2 to 4.8) and "b" ( MWt 27 kDa; pI 6.6 to 8.0). Constellation "c" contained 13 protein spots distributed in a right-angled triangle with its base towards the acidic end of the gel (MWt 14.7 to 18.8 kDa; pI 5.3 to 7.4). Only spots c(2), c(3), c(5), c(8), and c(13) were present in all 12 samples. Streaking can be eliminated by using 50 microg protein for 2-D PAGE, and the major protein spots are readily identified by megapixel camera technology. Protein spots c(3), c(5), c(13) and constellation "a" appear to correspond with Manjunath's proteins (BSP-A(1), -A(2); -A(3); -30 kDa). Killian's 2 low fertility proteins may lie in the "c" constellation, and 1 of the high fertility proteins may lie in the "b" constellation. The 3 major BSP proteins can be visualized in the supernatant of cryopreserved semen. We believe that the technique may prove useful for retrospective analysis of processed semen batches that achieve less than satisfactory results in the field.

Journal Article↗
PubMed1994

Obstruction of aqueous humor outflow by cross-linked polyacrylamide microgels in bovine, monkey, and human eyes.

PURPOSE: Orcolon, a synthetic viscoelastic, may have contributed to refractory intraocular pressure (IOP) elevation after intracameral injection in some patients. Cross-linked polyacrylamide (microgels), an altered form of the polymer, was investigated as an etiologic candidate. METHODS: Four adult rhesus monkeys underwent anterior chamber exchange with mock aqueous humor containing microgels in one eye and a vehicle in the other. Outflow facility (perfusion) and IOP (applanation) were determined before and at various times thereafter. Facility also was determined before and after microgel or vehicle infusion into organ-cultured individual human (n = 9) and paired calf (n = 6) anterior segments. Representative monkey and human eyes were examined by light and electron microscopy. RESULTS: In the microgel-infused monkey eyes, IOP was consistently higher, by approximately 5 mmHg for approximately 1 month. In all three species, microgel infusion acutely decreased facility by approximately 50% to 80%. In the living monkeys where longer-term observation and retesting were possible, a facility reduction of approximately 40% to 50% persisted for at least 1 to 2 months, and rechallenge again produced an acute 80% facility decrease and subsequent 10-mmHg IOP rise. Results of electron microscopic examination in human and monkey eyes showed accumulation of microgels in the cribriform meshwork and beneath the inner wall of Schlemm's canal, with no cellular alterations or inflammatory infiltrate. CONCLUSIONS: Cross-linked polyacrylamide microgels can produce an acute and longstanding obstruction of trabecular drainage experimentally, and might therefore do so clinically.

Acrylic Resins↗
PubMed1998

Biology of giant proteins of Plasmodium: resolution on polyacrylamide-agarose composite gels.

The malaria parasite Plasmodium falciparum that infects humans encodes several extremely large proteins with molecular masses in the hundreds of thousands to megadalton range. Studies on the structure, function and antigenicity of these ;giant proteins' are hindered by the inability to resolve them effectively in conventional polyacrylamide gels. In this report, Jochen Wiesner, Denise Mattei, Artur Scherf and Michael Lanzer describe a convenient gel system, based on a composite polyacrylamide-agarose matrix, which facilitates analysis of giant proteins.

Journal Article↗
PubMed2006

Identification of trichloroethanol visualized proteins from two-dimensional polyacrylamide gels by mass spectrometry.

Proteins visualized by 2,2,2-trichloroethanol (TCE) on two-dimensional electrophoresis gels are efficiently identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and MS/MS. In a previous study, a method was developed that placed TCE in the polyacrylamide gel so that protein bands can be visualized without staining in less than 5 min. A visible fluorophore is generated by reaction of TCE with tryptophan that allows for protein visualization. In this study, MALDI-TOF MS and LC-MS/MS are used to identify randomly selected Escherichia coli proteins. The identification of TCE visualized proteins is compared to the identification of Coomassie brilliant blue (CBB) stained proteins from two-dimensional gel electrophoresis of E. coli proteins. This study demonstrated that TCE visualized proteins are compatible with protein identification by MALDI-TOF peptide mass fingerprinting. For 10 randomly selected spots, TCE visualization lead to statistically significant identification of 5 proteins and CBB visualization lead to identification of 6 proteins. TCE visualized proteins are also shown to be well suited for protein identification using LC-MS/MS. In 16 spots selected for MS/MS analysis, TCE samples lead to the identification of 79 peptides; while CBB samples lead to the identification of 65 peptides. TCE samples also supported the identification of more proteins. The low stoichiometry of labeling of tryptophan residues does not require inclusion of this modification for database searches. In addition to being a rapid visualization technique compatible with MS, TCE visualization utilizes rapid washing conditions for sample preparation of proteins spots excised from polyacrylamide gels.

Amino Acid Sequence↗
PubMed2001

A simple apparatus for generating stretched polyacrylamide gels, yielding uniform alignment of proteins and detergent micelles.

Compressed and stretched polyacrylamide hydrogels previously have been shown to offer a robust method for aligning proteins. A simple, funnel-like apparatus is described for generating uniformly stretched hydrogels. For prolate-shaped proteins, gels stretched in the direction of the magnetic field yield two-fold larger alignment than gels compressed to the same aspect ratio in this direction. Empirically, protein alignment is found to be proportional to (c-2.3)2 [(d(o/dN)3-1], where do and dN are the diameters of the cylindrical gels before and after stretching, respectively, and c is the polyacrylamide weight fraction in percent. Low gel densities, in the 4-7% range, are found to have minimal effects on macromolecular rotational correlation times, tauc, and no effect of the compression ratio on tauc could be discerned over the range studied (do/dN < or = 1.4). Application is demonstrated for a sample containing the first Ig-binding domain of protein G, and for a detergent-solubilized peptide.

Acrylic Resins↗
PubMedDate not supplied

Penis invalidating cicatricial outcomes in an enlargement phalloplasty case with polyacrylamide gel (Formacryl).

The present article reports the case of a patient subjected to polyacrylamide polymers-composed gel cutaneous infiltration in the penis for cosmetic purposes, resulting in severe invalidating outcomes. A significant tissue reaction to the subcutaneous injection of polyacrylamide gel for the penis enlargement purpose resulted in permanent and invalidating scars both on the esthetic and functional levels. Such a result must be simply taken into account both singly and in the light of the international literature to exclude this method as standard uro-andrologic activity.

Acrylic Resins↗
PubMed1998

Reduction of Cr, Mo, Se and U by Desulfovibrio desulfuricans immobilized in polyacrylamide gels.

Intact cells of Desulfovibrio desulfuricans, immobilized in polyacrylamide gel, removed Cr, Mo, Se and U from solution by enzymatic-mediated reduction reactions. Lactate or H2 served as the electron donor and the oxidized Cr(VI), Mo(VI), Se(VI) and U(VI) served as electron acceptors. Reduction of the oxidized metal species resulted in the precipitation of solid phases of the metals. Metal removal efficiencies of 86-96% were achieved for initial concentrations of 1 mM Mo, Se, and U and 0.5 mM Cr. Insoluble metal phases accumulated on both the surface and the interior of the polyacrylamide gel. In column tests conducted for U removal, effluent concentrations less than 20 micrograms L(-1) were achieved with initial concentrations of 5 mg L(-1) and 20 mg L(-1) U and residence times from 25-37 h. The enzymatic reduction of Cr, Mo, Se, and U by immobilized cells of D. desulfuricans may be a practical method for removing these metals from solution in a biological reactor.

Acrylic Resins↗
PubMed1968

A study by polyacrylamide-gel electrophoresis of the effect of proteolysis on Micrococcus lysodeikticus polynucleotide phosphorylase.

1. Trypsin digestion of Micrococcus lysodeikticus polynucleotide phosphorylase (nucleoside diphosphate-polynucleotide nucleotidyltransferase) causes a progressive increase in electrophoretic mobility in polyacrylamide gels of the single active degradation product. 2. A marked increase in primer requirement for CDP polymerization occurs before a more mobile product is formed. 3. alpha-Chymotrypsin digestion yields a product that separates into several active species on polyacrylamide-gel electrophoretograms. 4. No separation of ADP-and CDP-polymerization activities occurs during electrophoresis after either trypsin or alpha-chymotrypsin treatment.

Adenine Nucleotides↗
PubMed1971

Molecular-sieve chromatography and electrophoresis in polyacrylamide gels.

1. The absolute electrophoretic mobilities of eight proteins have been measured at pH8.76, I 0.05, in polyacrylamide gels of 20 different compositions at 10 degrees C. 2. The partition coefficients of these proteins have been determined chromatographically under the same conditions by using columns of granulated polyacrylamide gel prepared simultaneously. 3. The electrophoretic mobilities are an exponential function of the gel concentrations when the latter are corrected for water uptake. The constants of this function have been determined by curvefitting methods. They have been shown to be related to the free solution mobility and to the mean molecular radius respectively. 4. The reduced mobilities have been shown to be a linear function of the partition coefficients by statistical analyses. 5. The physical significance of the relation between electrophoretic mobility and chromatographic phase distribution in gel media is discussed in the context of these results.

Acrylamides↗
PubMed2003

Characterization of protein variants and post-translational modifications: ESI-MSn analyses of intact proteins eluted from polyacrylamide gels.

We have developed a strategy to characterize protein isoforms, resulting from single-point mutations and post-translational modifications. This strategy is based on polyacrylamide gel electrophoresis separation of protein isoforms, mass spectrometry (MS) and MSn analyses of intact proteins, and tandem MS analyses of proteolytic peptides. We extracted protein isoforms from polyacrylamide gels by passive elution using SDS, followed by nanoscale hydrophilic phase chromatography for SDS removal. We performed electrospray ionization MS analyses of the intact proteins to determine their molecular mass, allowing us to draw hypotheses on the nature of the modification. In the case of labile post-translational modifications, like phosphorylations and glycosylations, we conducted electrospray ionization MSn analyses of the intact proteins to confirm their presence. Finally, after digestion of the proteins in solution, we performed tandem MS analyses of the modified peptides to locate the modifications. Using this strategy, we have determined the molecular mass of 5-10 pmol of a protein up to circa 50 kDa loaded on a gel with a 0.01% mass accuracy. The efficiency of this approach for the characterization of protein variants and post-translational modifications is illustrated with the study of a mixture of kappa-casein isoforms, for which we were able to identify the two major variants and their phosphorylation site and glycosylation motif. We believe that this strategy, which combines two-dimensional gel electrophoresis and mass spectrometric analyses of gel-eluted intact proteins using a benchtop ion trap mass spectrometer, represents a promising approach in proteomics.

Amino Acid Sequence↗
PubMed1981

Isolation of human growth hormone isohormones D and E in milligram amounts (II), using isoelectric focusing on polyacrylamide gel.

Human growth hormone (hGH) isohormones D and E were prepared from a partial enzymatic hydrolyzate catalyzed by plasmin, using isoelectric focusing on polyacrylamide gel to fractionate the digest. Separation between the two isohormones species was optimized by employing a maximally flattened pH gradient between pH's 4.3 and 5.6. Gel slices containing isohormones D and E were extracted and concentrated by Steady-State Stacking on polyacrylamide gel. The extract was purified by gel filtration and lyophilized. Overall yields of lyophilized isohormones D and E were 10.9 mg and represented a recovery of 51% relative to the densitometrically estimated isohormone concentrations in the hGH digest. Purity of the products, based on Lowry analysis, U.V. absorbance and radioimmunoassay (RIA) was 60-70%. The isolated isohormone species were active in the rat tibia-line assay and in the specific RIA for hGH.

Acrylic Resins↗
PubMed2001

A computational study into the use of polyacrylamide gel and A-150 plastic as brain tissue substitutes for boron neutron capture therapy.

A precise evaluation of the dosimetric performance of epithermal neutron beams designed for boron neutron capture theory of brain tumours requires the use of a phantom material that closely matches brain tissue. The aim of this study was to investigate how well polyacrylamide gel (or PAG) and A- 150 plastic performed as substitutes for brain tissue compared with standard phantom materials such as water and polymethyl-methacrylate (or PMMA). Thermal neutron fluence, photon dose and epithermal neutron dose distributions were calculated for the epithermal neutron beam available at the University of Birmingham. The results presented in this paper show that the PAG provides a good simulation of radiation transport in the brain with differences from the real brain of +9.4%, - 10.8% and +5.1% at a depth of 50 mm for thermal neutron fluence, gamma dose and epithermal neutron dose distributions respectively. The polyacrylamide gel presented is therefore a promising substitute for brain tissue that can, as a dosimeter, provide a three-dimensional map of the absorbed dose delivered by the epithermal neutron beam. However, this study does not investigate the agreement between doses derived from magnetic resonance and physical doses for such gels. A- 150 plastic was shown to be a better substitute for brain tissue than PMMA, with differences from brain of -1.9%, -12.4% and - 13.2% at a depth of 50 mm for thermal neutron fluence, gamma dose and epithermal neutron dose distributions respectively, against +21.1%, -16.2% and +19.2% for PMMA. A-150 plastic should therefore be the material of choice for solid phantoms.

Acrylic Resins↗