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Polyacrylamide Gels (polyacrylamide + gel)
Terms modified by Polyacrylamide Gels Selected AbstractsComplications from Injectable Polyacrylamide Gel, a New Nonbiodegradable Soft Tissue FillerDERMATOLOGIC SURGERY, Issue 12p2 2004Snehal P. Amin Background. Polyacrylamide gels, containing a hydrogel composed of polyacrylamide and water, are used for soft tissue augmentation and contour correction. There are no reports of significant complications after injection of this material into the face. Objective. We report an inflammatory reaction after injection of polyacrylamide gels for zygomatic facial augmentation. Methods. A retrospective chart review of single case is presented. Results. An inflammatory reaction at the sites of polyacrylamide gels injection was noted at 1 month after initial injection. Despite two ensuing courses of broad-spectrum antibiotics, the patient presented to us with persistent draining nodules. Intralesional steroid injections resulted in prompt resolution and no recurrence. Conclusion. Inflammatory reactions have been noted in patients receiving polyacrylamide gels for breast augmentation. Facial polyacrylamide gels injections may also be associated with an inflammatory reaction that responds to intralesional steroids. With increasing availability of a variety of soft tissue fillers, dermatologists should be aware of this delayed complication from polyacrylamide gels. [source] New Approaches to the Formation of a Stationary Phase in an Immunoadsorption WallARTIFICIAL ORGANS, Issue 2 2005Tsung-Hua Yang Abstract:, The concept of an immunoadsorption wall, which combines the principles of immunoisolation and immunoadsorption, was proposed in 1999 to remove certain toxins accumulated in patients' blood. However, realization of this concept is obviously handicapped by the inefficient use of immunoadsorbent. This study is intended to improve the use of immunoadsorbent and optimize the formation of a stationary phase in an immunoadsorption wall. Polyacrylamide gel, which has the advantages of being chemically inert, having minimal diffusion effect and reasonable cost, could be considered as the medium of choice for a stationary phase. In this study, new approaches aimed at effective allocation of immunoadsorbent utilizing polyacrylamide gel are attempted. The advantages and disadvantages of these new approaches are discussed according to the preparation, formation, and outcome of a stationary phase. It is hoped that these new approaches could serve as a first step toward building an immunoadsorption wall. [source] Complications from Injectable Polyacrylamide Gel, a New Nonbiodegradable Soft Tissue FillerDERMATOLOGIC SURGERY, Issue 12p2 2004Snehal P. Amin Background. Polyacrylamide gels, containing a hydrogel composed of polyacrylamide and water, are used for soft tissue augmentation and contour correction. There are no reports of significant complications after injection of this material into the face. Objective. We report an inflammatory reaction after injection of polyacrylamide gels for zygomatic facial augmentation. Methods. A retrospective chart review of single case is presented. Results. An inflammatory reaction at the sites of polyacrylamide gels injection was noted at 1 month after initial injection. Despite two ensuing courses of broad-spectrum antibiotics, the patient presented to us with persistent draining nodules. Intralesional steroid injections resulted in prompt resolution and no recurrence. Conclusion. Inflammatory reactions have been noted in patients receiving polyacrylamide gels for breast augmentation. Facial polyacrylamide gels injections may also be associated with an inflammatory reaction that responds to intralesional steroids. With increasing availability of a variety of soft tissue fillers, dermatologists should be aware of this delayed complication from polyacrylamide gels. [source] An optimal method of DNA silver staining in polyacrylamide gelsELECTROPHORESIS, Issue 6 2008Yan-Chuang Han Abstract DNA silver staining has widely been used to detect DNA fragments in polyacrylamide gels with high sensitivity. We developed an optimal method for DNA silver staining on polyacrylamide gels. The novel procedure can be completed within 10,min instead of over 20,min with the conventional methods. The sensitivity is significantly improved by the silver-ion sensitizer (Eriochrome black T (EBT)) and the minimum of 0.11 and 1.75,ng of DNA amount can be detected in denaturing and nondenaturing polyacrylamide gel, respectively. Compared with the conventional silver staining methods, the improved optimal method can save time and display high sensitivity, color uniformity, and long storage time of the staining gels. [source] An optimal method of DNA silver staining in polyacrylamide gelsELECTROPHORESIS, Issue 8 2007Yun-Tao Ji Abstract A silver staining technique has widely been used to detect DNA fragments with high sensitivity on polyacrylamide gels. The conventional procedure of the silver staining is tedious, which takes about 40,60,min and needs five or six kinds of chemicals and four kinds of solutions. Although our previous improved method reduced several steps, it still needed six kinds of chemicals. The objective of this study was to improve further the existing procedures and develop an optimal method for DNA silver staining on polyacrylamide gels. The novel procedure could be completed with only four chemicals and two solutions within 20,min. The steps of ethanol, acetic acid, and nitric acid precession before silver impregnation have been eliminated and the minimal AgNO3 dose has been used in this up-to-date method. The polyacrylamide gel of the DNA sliver staining displayed a golden yellow and transparent background with high sensitivity. The minimum 0.44 and 3.5,ng of DNA amount could be detected in denaturing and nondenaturing polyacrylamide gel, respectively. This result indicated that our optimal method can save time and cost, and still keep a high sensitivity for DNA staining in polyacrylamide gels. [source] Efficient and sensitive method of DNA silver staining in polyacrylamide gelsELECTROPHORESIS, Issue 1 2005Lujiang Qu Abstract DNA silver staining is widely used to detect DNA fragment in polyacrylamide gel with high sensitivity. Conventional procedures of the silver staining involve several steps, which take about 40 min to 2 h in total. To improve the efficiency of DNA silver staining, a more efficient protocol is developed in this study. The procedure comprises only four steps including impregnating, rinsing, developing, and stopping, and could be completed within 20 min. Nitric acid and ethanol in the silver-impregnation step of the new procedure eliminates the need for prior treatment of gels with a fixing solution and following rinse prior to impregnation with silver. The procedure has high sensitivity and long storage lifetime. The minimum detectable mass of DNA is 0.44 and 3.5 ng in denaturing and nondenaturing polyacrylamide gel, respectively. [source] Electronic gel protein transfer and identification using matrix-assisted laser desorption/ionization-mass spectrometryELECTROPHORESIS, Issue 9 2004Jonathan W. Cooper Abstract An electronic protein transfer technique is described for achieving the rapid and efficient recovery of sodium dodecyl sulfate (SDS)-protein complexes from polyacrylamide gels. This process involves the use of small-dimension capillaries in physical contact with a resolved protein band within the polyacrylamide gel, providing a large potential drop and high electric field strength at the capillary/gel interface. Several factors controlling the electronic protein transfer, including the applied electric field strength, the electrophoresis buffer concentration, and the capillary dimension, are studied to further enhance the use of field-amplification for sample stacking of extracted SDS-protein complexes. As a result of sample stacking, the extracted proteins from a 50 ng gel loading are present in a narrow (,80 nL) and highly concentrated (0.46 mg/mL or 3.3×10,5 M for cytochrome c) solution plug. Three model proteins with molecular mass ranging from 14 kDa (cytochrome c) to 116 kDa (,-galactosidase) are stained by Coomassie blue and electrophoretically extracted from gels with protein loadings as low as 50 ng. The capillary format of the electronic protein transfer technique allows direct deposition of extracted proteins onto a matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) target. Various matrices and solvent compositions are evaluated for the analysis of extracted and concentrated SDS-protein complexes using MALDI-MS. The electronic protein transfer technique, when operated under optimized conditions, is demonstrated for the effective (>70% recovery), speedy (less than 5 min), and sensitive MS identification of gel resolved proteins (as low as 50 ng). [source] Background-free, fast protein staining in sodium dodecyl sulfate polyacrylamide gel using counterion dyes, zincon and ethyl violetELECTROPHORESIS, Issue 24 2002Jung-Kap Choi Abstract A background-free, fast protein staining method in polyacrylamide gel electrophoresis using an acidic dye, zincon (ZC) and a basic dye, ethyl violet (EV) is described. It is based on the counterion dye staining technique that employs two oppositely charged dyes to form an ion-pair complex in staining solution. The selective binding of free dye molecules to proteins in acidic solution produces bluish violet-colored bands. It is a rapid and end-point staining procedure, involving only fixing and staining steps that are completed in 1,1.5 h. The detection limit of this method is 8,15 ng of protein that is comparable to the sensitivity of the colloidal Coomassie Brilliant Blue G (CBBG) stain. Due to its sensitivity and speed, this stain may be more practical than any other dye-based stains for routine laboratory purposes. [source] Purification, characterization and amino-acid sequence analysis of a thermostable, low molecular mass endo-,-1,4-glucanase from blue mussel, Mytilus edulisFEBS JOURNAL, Issue 16 2000Bingze Xu A cellulase (endo-,-1,4- d -glucanase, EC 3.2.1.4) from blue mussel (Mytilus edulis) was purified to homogeneity using a combination of acid precipitation, heat precipitation, immobilized metal ion affinity chromatography, size-exclusion chromatography and ion-exchange chromatography. Purity was analyzed by SDS/PAGE, IEF and RP-HPLC. The cellulase (endoglucanase) was characterized with regard to enzymatic properties, isoelectric point, molecular mass and amino-acid sequence. It is a single polypeptide chain of 181 amino acids cross-linked with six disulfide bridges. Its molecular mass, as measured by MALDI-MS, is 19 702 Da; a value of 19 710.57 Da was calculated from amino-acid composition. The isoelectric point of the enzyme was estimated by isoelectric focusing in a polyacrylamide gel to a value of 7.6. According to amino-acid composition, the theoretical pI is 7.011. The effect of temperature on the endoglucanase activity, with carboxymethyl cellulose and amorphous cellulose as substrates, respectively, was studied at pH 5.5 and displayed an unusually broad optimum activity temperature range between 30 and 50 °C. Another unusual feature is that the enzyme retains 55,60% of its maximum activity at 0 °C. The enzyme readily degrades amorphous cellulose and carboxymethyl cellulose but displays no hydrolytic activity towards crystalline cellulose (Avicel) and shows no cross-specificity for xylan; there is no binding to Avicel. The enzyme can withstand 10 min at 100 °C without irreversible loss of enzymatic activity. Amino-acid sequence-based classification has revealed that the enzyme belongs to the glycoside hydrolase family 45, subfamily 2 (B. Henrissat, Centre de Recherches sur les Macromolecules Végétales, CNRS, Joseph Fourier Université, Grenoble, France, personal communication). [source] Sucrose phosphorylase of the rumen bacterium Pseudobutyrivibrio ruminis strain AJOURNAL OF APPLIED MICROBIOLOGY, Issue 3 2009A. Kasperowicz Abstract Aims:, To verify the taxonomic affiliation of bacterium Butyrivibrio fibrisolvens strain A from our collection and to characterize its enzyme(s) responsible for digestion of sucrose. Methods and Results:, Comparison of the 16S rRNA gene of the bacterium with GenBank showed over 99% sequence identity to the species Pseudobutyrivibrio ruminis. Molecular filtration, native electrophoresis on polyacrylamide gel, zymography and thin layer chromatography were used to identify and characterize the relevant enzyme. An intracellular sucrose phosphorylase with an approximate molecular mass of 52 kDa exhibiting maximum activity at pH 6·0 and temperature 45°C was identified. The enzyme was of inducible character and catalysed the reversible conversion of sucrose to fructose and glucose-1-P. The reaction required inorganic phosphate. The Km for glucose-1-P formation and fructose release were 3·88 × 10,3 and 5·56 × 10,3 mol l,1 sucrose, respectively , while the Vmax of the reactions were ,0·579 and 0·9 ,mol mg protein,1 min,1. The enzyme also released free glucose from glucose phosphate. Conclusion:,Pseudobutyrivibrio ruminis strain A utilized sucrose by phosphorolytic cleavage. Significance and Impact of the Study:, Bacterium P. ruminis strain A probably participates in the transfer of energy from dietetary sucrose to the host animal. [source] Infrared laser desorption and ionization of polypeptides from a polyacrylamide gelJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 3 2002Michelle Baltz-Knorr Abstract We observed direct desorption and ionization of angiotensin II and bovine insulin from a frozen polyacrylamide gel without the addition of an exogenous matrix, using picosecond pulses from a tunable, mid-infrared free-electron laser tuned to strong absorption bands of the gel. At 5.7, 5.9, 6.1 and 6.3 µm we were able to desorb and ionize both analyte molecules, with the strongest analyte signal generated at 5.9 µm. However, no analyte signal was observed at 5.5 µm. Consistent with a previous report, we did not observe ions of either polypeptide at 2.9 µm, in spite of strong overall absorption. We discuss the implications of this wavelength-dependent ionization, including possible ablation mechanisms and energy partitioning between competing vibrational modes. Copyright © 2002 John Wiley & Sons, Ltd. [source] In-gel deglycosylation of sodiumdodecyl sulfate polyacrylamide gel electrophoresis-separated glycoproteins for carbohydrate estimation by matrix-assisted laser desorption/ionization time-of-flight mass spectrometryJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 3 2002S. Kilz Abstract Mass determination by mass spectrometric methods (electrospray ionization mass spectrometry (ESI-MS), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS)) of sodiumdodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)-separated proteins is a well known procedure and reliable protocols are available. In our efforts to use the established methods to determine the molecular mass of the disulfide brigded, heterodimeric glycoprotein GP3 and to determine the carbohydrate content of each protein subunit we developed an in-gel chemical deglycosylation method. For this purpose we established experimental conditions that allow maximum extraction of the high molecular mass protein subunits and developed a routine method to apply the HF,pyridine deglycosylation protocol to proteins isolated from polyacrylamide gel pieces. The novel protocol and extraction procedure described can be used to analyze O -glycosylated proteins up to 150 kDa after SDS-PAGE separation. Copyright © 2002 John Wiley & Sons, Ltd. [source] Comparison by restriction fragment differential display RT‐PCR of gene expression pattern in bovine oocytes matured in the presence or absence of fetal calf serumMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 1 2001S. Jacek Rzucidlo Abstract A novel restriction fragment differential display (RFDD) RT‐PCR has been used to compare patterns of mRNA expression in bovine oocytes matured in vitro in the presence (10%) or absence of fetal calf serum (FCS). Total RNA extracted from matured and denuded oocytes was processed using display Profile kit (Display System Biotech). RFDD RT‐PCR products were separated on 6% polyacrylamide gel and analyzed using a Storm 860 scanner. Selected bands representing potentially differentially expressed fragments were excised from the gel and re‐amplified. Re‐amplified fragments with size matched to the original fragment were cloned into the TA vector and sequenced. Initially, 10 and 15 differentially expressed fragments were isolated from oocytes matured in the presence and absence of FCS, respectively. Eight out of 10 and 10 out of 15 fragments were re‐amplified successfully as evidenced bysize similarity to the original fragments. Finally, the size of six inserts sequenced from each group matched the size of corresponding original as well as re‐amplified fragments. Sequence comparison search revealed similarity of some isolated fragments to 18s ribosomal RNA, bovine apolipoprotein A‐I, bovine mitochondrion DNA, human CGI‐79 mRNA, human Ab1‐interactor protein, and bovine satellite DNA. The other sequenced fragments may represent novel genes. We showed that RFDD RT‐PCR can be effectively applied to contrast gene expression pattern in bovine oocytes and that presence or absence of FCS during maturation interval affects gene expression pattern in matured bovine oocytes. Mol. Reprod. Dev. 59:90–96, 2001. © 2001 Wiley‐Liss, Inc. [source] Bifunctional indole-3-acetyl transferase catalyses synthesis and hydrolysis of indole-3-acetyl- myo -inositol in immature endosperm of Zea maysPHYSIOLOGIA PLANTARUM, Issue 2 2003Stanislaw Kowalczyk 1- O -(indole-3-acetyl)- , - d -glucose: myo -inositol indoleacetyl transferase (IA- myo -inositol synthase) is an important enzyme in IAA metabolism. This enzyme catalyses the transfer of the indole acetyl (IA) moiety from 1- O -(indole-3-acetyl)- , - d -glucose to myo -inositol to form IA- myo- inositol and glucose. IA- myo -inositol synthase was purified to an electrophoretically homogenous state from maize liquid endosperm by fractionation with ammonium sulphate, anion-exchange, adsorption on hydroxylapatite, affinity chromatography on ConA-Sepharose, preparative PAGE and isoelectric focusing. We thus obtained two enzyme preparations which differ in their Rf on 8% polyacrylamide gel. The preparation of Rf 0.36 contained a single 56.4 kDa polypeptide, whereas the preparation of Rf 0.39 consisted of two polypeptides of 56.4 and 53.5 kDa. Both purified preparations of IAInos synthase also exhibited the activity of an IAInos hydrolase, showing that the dual activity was associated with a single protein. Results of gel filtration and analytical SDS-PAGE suggest that the native enzyme exists as both a monomeric (65 kDa) and homo- or heterodimeric form (110,130 kDa). Analysis of peptide maps and amino acid sequences of two 21 amino-acid peptides showed that polypeptides of 56.4 and 53.5 kDa have the same primary structure and that the 3 kDa difference in molecular mass is probably caused by different glycosylation levels. Comparison of this partial and internal amino acid sequence with sequences of other plant acyltransferases indicated similarity to several proteins which belonged to the serine carboxypeptidase-like (SCPL) acyltransferase family. [source] Regulation of the catalytic behaviour of L-form starch phosphorylase from sweet potato roots by proteolysisPHYSIOLOGIA PLANTARUM, Issue 4 2002Han-Min Chen Starch phosphorylase (SP) is an enzyme used for the reversible phosphorolysis of the ,-glucan in plant cells. When compared to its isoform in an animal cell, glycogen phosphorylase, a peptide containing 78 amino acids (L78) is inserted in the centre of the low-affinity type starch phosphorylase (L-SP). We found that the amino acid sequence of L78 had several interesting features including the presence of a PEST region, which serves as a signal for rapid degradation. Indeed, most L-SP molecules isolated from mature sweet potato roots were nicked in the middle of a molecule, but still retained their tertiary or quaternary structures, as well as full catalytic activity. The nicking sites on the L78 were identified by amino acid sequencing of these peptides, which also enabled us to propose a proteolytic process for L-SP. Enzyme kinetic studies of L-SP in the direction of starch synthesis indicated that the Km decreased during the proteolytic process when starch was used as the limiting substrate, but the Km for the other substrate (Glc-1-P) increased. On the other hand, the maximum velocities (Vmax) increased for both substrates. Mobility of the nicked L-SP was retarded on a native polyacrylamide gel containing soluble starch, indicating the increased affinity for starch. Results in this study suggested that L78 and its proteolytic modifications might play a regulatory role on the catalytic behaviour of L-SP in starch biosynthesis. [source] Granule-bound starch synthase (GBSS) diversity of ancient wheat and related speciesPLANT BREEDING, Issue 6 2008L. Caballero Abstract Granule-bound starch synthase of ancient wheat and related species was examined by sodium dodecyl sulphate polyacrylamide gel. A total of 13 different alleles were revealed in a collection of three accessions of diploid wheat, six accessions of tetraploid wheat, 49 accessions of spelt wheat, nine accessions of Sitopsis and two accessions of Aegilops tauschii. A new allele named Wx-A1a, appeared in four spelt wheat accessions. The tetraploid wheat accessions evaluated did not show any polymorphism; nevertheless the tetraploid accessions of Sitopsis section revealed three novel alleles. The novel allele Wx-Ddn1g was found in two accessions of A. ventricosa and the Wx-Ddcm1h and Wx-Ddcm1i in two accessions of A. crassa. A novel allele named Wx-Au1g was found in Triticum urartu, which is different from the also new Wx-Am1h allele of T. monococcum. The diploid-related species accessions revealed two novel alleles named Wx-Bsl1h and Wx-Bs1g found, respectively, in A. longissima and A. speltoides. The amylose content was measured for the different alleles found in all evaluated species and no significant effects of the allele composition on the amylose content were detected. [source] Proteomic analysis of human bronchoalveolar lavage fluid: expression profiling of surfactant-associated protein A isomers derived from human pulmonary alveolar proteinosis using immunoaffinity detectionPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 1 2003Chang He Abstract Human bronchoalveolar lavage fluid (BALF) proteins from pulmonary alveolar proteinosis (PAP) obtained by washing the epithelial lining of the lung with phosphate-buffered saline, were separated using high resolution two-dimensional gel electrophoresis (2-DE) under denaturing and reducing conditions. By Western blotting, the proteins were transferred from polyacrylamide gel onto a chemical resilient membrane. The surfactant-associated protein A (SP-A) isomers were then identified with enhanced chemiluminescence detection (ECL) using antibody-antigen reaction. Some of the gels were treated with silver staining after 2-DE. The molecular masses of SP-A isomers in BALF from PAP ranged from 20.5 to 26, 26 to 32, and 32 to 42 kDa, respectively; and isoelectric points (pI) were in pH range of 4.5,5.4 under denaturing and reducing conditions. In the mass range of 20.5,26 kDa and pI of 4.5,5.4, there were five isomers, and in mass range of 26,32 kDa and pI of 4.5 to 5.4, there were at least eight isomers on the ECL detection film. However, in the mass range of 32,42 kDa and pI of 4.5,5.4, there were three isomers separated one from another but there was also a cluster of overlapping spots on the ECL detection film. Thus, this communication describes a characteristic 2-DE pattern of SP-A isomers in BALF from PAP as follows. (1) The five isomers of mass 20.5,26 kDa and pI of 4.5,5.4; (2) the eight isomers of mass 26,32 kDa and pI of 4.5,5.4; and (3) the three isomers of mass 32,42 kDa and pI of 4.5,5.4. [source] New Approaches to the Formation of a Stationary Phase in an Immunoadsorption WallARTIFICIAL ORGANS, Issue 2 2005Tsung-Hua Yang Abstract:, The concept of an immunoadsorption wall, which combines the principles of immunoisolation and immunoadsorption, was proposed in 1999 to remove certain toxins accumulated in patients' blood. However, realization of this concept is obviously handicapped by the inefficient use of immunoadsorbent. This study is intended to improve the use of immunoadsorbent and optimize the formation of a stationary phase in an immunoadsorption wall. Polyacrylamide gel, which has the advantages of being chemically inert, having minimal diffusion effect and reasonable cost, could be considered as the medium of choice for a stationary phase. In this study, new approaches aimed at effective allocation of immunoadsorbent utilizing polyacrylamide gel are attempted. The advantages and disadvantages of these new approaches are discussed according to the preparation, formation, and outcome of a stationary phase. It is hoped that these new approaches could serve as a first step toward building an immunoadsorption wall. [source] Neutrophils display biphasic relationship between migration and substrate stiffnessCYTOSKELETON, Issue 6 2009Kimberly M. Stroka Abstract Neutrophils are one type of migrating cell in the body's innate immune system and are the first line of defense against inflammation or infection. While extensive work exists on the effect of adhesive proteins on neutrophil motility, little is known about how neutrophil motility is affected by the mechanical properties of their physical environment. This study investigated the effects of substrate stiffness on the morphology, random motility coefficient, track speed (v), spreading area, and distribution of turning angles of neutrophils during chemokinesis. Human neutrophils were plated onto polyacrylamide gels of varying stiffness, ranging from 3 to 13 kPa, and coated with the extracellular matrix protein fibronectin, and timelapse images were taken with phase contrast microscopy. Our results show a biphasic behavior between neutrophil motility and substrate stiffness, with the optimum stiffness for motility depending on the concentration of fibronectin on the surface of the gel. On 100 ,g/mL fibronectin, the optimum stiffness is 4 kPa (v = 6.9 ± 0.6 ,m/min) while on 10 ,g/mL fibronectin, the optimum stiffness increases to 7 kPa (v = 4.5 ± 2.0 ,m/min). This biphasic behavior most likely arises because neutrophils on soft gels are less adherent, preventing production of traction forces, while neutrophils on stiff gels adhere strongly, resulting in decreased migration. At intermediate stiffness, however, neutrophils can attain optimal motility as a function of extracellular matrix coating. Cell Motil. Cytoskeleton 2009. © 2009 Wiley-Liss, Inc. [source] Sensitivity of alveolar macrophages to substrate mechanical and adhesive propertiesCYTOSKELETON, Issue 6 2006Sophie Féréol Abstract In order to understand the sensitivity of alveolar macrophages (AMs) to substrate properties, we have developed a new model of macrophages cultured on substrates of increasing Young's modulus: (i) a monolayer of alveolar epithelial cells representing the supple (,0.1 kPa) physiological substrate, (ii) polyacrylamide gels with two concentrations of bis-acrylamide representing low and high intermediate stiffness (respectively 40 kPa and 160 kPa) and, (iii) a highly rigid surface of plastic or glass (respectively 3 MPa and 70 MPa), the two latter being or not functionalized with type I-collagen. The macrophage response was studied through their shape (characterized by 3D-reconstructions of F-actin structure) and their cytoskeletal stiffness (estimated by transient twisting of magnetic RGD-coated beads and corrected for actual bead immersion). Macrophage shape dramatically changed from rounded to flattened as substrate stiffness increased from soft ((i) and (ii)) to rigid (iii) substrates, indicating a net sensitivity of alveolar macrophages to substrate stiffness but without generating F-actin stress fibers. Macrophage stiffness was also increased by large substrate stiffness increase but this increase was not due to an increase in internal tension assessed by the negligible effect of a F-actin depolymerizing drug (cytochalasine D) on bead twisting. The mechanical sensitivity of AMs could be partly explained by an idealized numerical model describing how low cell height enhances the substrate-stiffness-dependence of the apparent (measured) AM stiffness. Altogether, these results suggest that macrophages are able to probe their physical environment but the mechanosensitive mechanism behind appears quite different from tissue cells, since it occurs at no significant cell-scale prestress, shape changes through minimal actin remodeling and finally an AMs stiffness not affected by the loss in F-actin integrity. Cell Motil. Cytoskeleton 2006. © 2006 Wiley-Liss, Inc. [source] Complications from Injectable Polyacrylamide Gel, a New Nonbiodegradable Soft Tissue FillerDERMATOLOGIC SURGERY, Issue 12p2 2004Snehal P. Amin Background. Polyacrylamide gels, containing a hydrogel composed of polyacrylamide and water, are used for soft tissue augmentation and contour correction. There are no reports of significant complications after injection of this material into the face. Objective. We report an inflammatory reaction after injection of polyacrylamide gels for zygomatic facial augmentation. Methods. A retrospective chart review of single case is presented. Results. An inflammatory reaction at the sites of polyacrylamide gels injection was noted at 1 month after initial injection. Despite two ensuing courses of broad-spectrum antibiotics, the patient presented to us with persistent draining nodules. Intralesional steroid injections resulted in prompt resolution and no recurrence. Conclusion. Inflammatory reactions have been noted in patients receiving polyacrylamide gels for breast augmentation. Facial polyacrylamide gels injections may also be associated with an inflammatory reaction that responds to intralesional steroids. With increasing availability of a variety of soft tissue fillers, dermatologists should be aware of this delayed complication from polyacrylamide gels. [source] Determination of DNA methylation by COBRA: A comparative study of CGE with LIF detection and conventional gel electrophoresisELECTROPHORESIS, Issue 17 2009Simon Goedecke Abstract DNA methylation as an epigenetic modification of the human genome is under emphatic investigation. Several studies have demonstrated a role of DNA methylation in oncogenesis. In conjunction with histone modifications, DNA methylation may cause the formation of heterochromatin and thus mediate the inactivation of gene transcription. It is important to develop methods that allow for an accurate quantification of the amount of DNA methylation in particular DNA regions, to gain information concerning the threshold of methylation levels necessary for gene inactivation. In this article, a CGE method with on-column LIF detection using SYBR Green is compared with a conventional slab-gel electrophoresis. We thus investigate the validity to analyze DNA methylation in the samples of a combined bisulfite restriction analysis. It is demonstrated that CGE is superior to gel electrophoresis in means of linearity, precision, accuracy, automatization (high throughput), and sample consumption. However, gel electrophoresis is easier to perform (simple devices, no PC usage), and the running costs are comparatively low. A further advantage of CGE is the sparse use of toxic compounds (MeOH and SYBR Green), whereas gel electrophoresis is performed in polyacrylamide gels with ethidium bromide staining. [source] An optimal method of DNA silver staining in polyacrylamide gelsELECTROPHORESIS, Issue 6 2008Yan-Chuang Han Abstract DNA silver staining has widely been used to detect DNA fragments in polyacrylamide gels with high sensitivity. We developed an optimal method for DNA silver staining on polyacrylamide gels. The novel procedure can be completed within 10,min instead of over 20,min with the conventional methods. The sensitivity is significantly improved by the silver-ion sensitizer (Eriochrome black T (EBT)) and the minimum of 0.11 and 1.75,ng of DNA amount can be detected in denaturing and nondenaturing polyacrylamide gel, respectively. Compared with the conventional silver staining methods, the improved optimal method can save time and display high sensitivity, color uniformity, and long storage time of the staining gels. [source] Sensitive fluorescence detection of polyphosphate in polyacrylamide gels using 4,,6-diamidino-2-phenylindolELECTROPHORESIS, Issue 19 2007Stephanie A. Smith Dr. Abstract PAGE is commonly used to identify and resolve inorganic polyphosphates (polyP). We now report highly sensitive and specific staining methods for polyP in polyacrylamide gels based on the fluorescent dye, 4,,6-diamidino-2-phenylindol (DAPI). DAPI bound to polyP in gels fluoresced yellow while DAPI bound to nucleic acids or glycosaminoglycans fluoresced blue. Inclusion of EDTA prevented staining of glycosaminoglycans by DAPI. We also identified conditions under which DAPI that was bound to polyP (but not nucleic acids or other anionic polymers) rapidly photobleached. This allowed us to develop an even more sensitive and specific negative staining method that distinguishes polyP from nucleic acids and glycosaminoglycans. The lower LOD using DAPI negative staining was 4,pmol (0.3,ng) phosphate per band, compared to conventional toluidine blue staining with a lower LOD of 250,pmol per band. [source] An optimal method of DNA silver staining in polyacrylamide gelsELECTROPHORESIS, Issue 8 2007Yun-Tao Ji Abstract A silver staining technique has widely been used to detect DNA fragments with high sensitivity on polyacrylamide gels. The conventional procedure of the silver staining is tedious, which takes about 40,60,min and needs five or six kinds of chemicals and four kinds of solutions. Although our previous improved method reduced several steps, it still needed six kinds of chemicals. The objective of this study was to improve further the existing procedures and develop an optimal method for DNA silver staining on polyacrylamide gels. The novel procedure could be completed with only four chemicals and two solutions within 20,min. The steps of ethanol, acetic acid, and nitric acid precession before silver impregnation have been eliminated and the minimal AgNO3 dose has been used in this up-to-date method. The polyacrylamide gel of the DNA sliver staining displayed a golden yellow and transparent background with high sensitivity. The minimum 0.44 and 3.5,ng of DNA amount could be detected in denaturing and nondenaturing polyacrylamide gel, respectively. This result indicated that our optimal method can save time and cost, and still keep a high sensitivity for DNA staining in polyacrylamide gels. [source] High-efficiency protein extraction from polyacrylamide gels for molecular mass measurement by matrix-assisted laser desorption/ionization-time of flight-mass spectrometryELECTROPHORESIS, Issue 6 2005Ya Jin Abstract A simple and fast method of protein extraction from Coomassie Brilliant Blue (CBB)-stained polyacrylamide gels suited for molecular mass measurement of proteins by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) is reported. Proteins in CBB-stained gel pieces were extracted by a 10-min soaking in 0.1,M NaOH at 25°C. The recovery of this one-step extraction method was 34,73% for proteins <67,kDa. CBB adduction to proteins during mass spectrometric analysis was avoided by a destaining step before the alkaline extraction. The molecular mass values of the extracted proteins coincided with those of purified proteins within ±0.01,0.10% deviation for all the proteins <36,kDa. Because of the high extraction recovery, mass measurement was possible for the proteins extracted from CBB-stained gels with loaded protein quantities as little as 34,ng for cytochrome,c, ,-lactalbumin, myoglobin, ,-lactoglobulin, trypsinogen, and carbonic anhydrase (12.4,29.0,kDa), 340,ng for glyceraldehyde-3-phosphate dehydrogenase (35.6,kDa) and albumin (66.3,kDa). This method provides a highly efficient approach to utilize CBB-stained one- or two-dimensional gels for whole protein analysis using MALDI-TOF-MS. [source] Efficient and sensitive method of DNA silver staining in polyacrylamide gelsELECTROPHORESIS, Issue 1 2005Lujiang Qu Abstract DNA silver staining is widely used to detect DNA fragment in polyacrylamide gel with high sensitivity. Conventional procedures of the silver staining involve several steps, which take about 40 min to 2 h in total. To improve the efficiency of DNA silver staining, a more efficient protocol is developed in this study. The procedure comprises only four steps including impregnating, rinsing, developing, and stopping, and could be completed within 20 min. Nitric acid and ethanol in the silver-impregnation step of the new procedure eliminates the need for prior treatment of gels with a fixing solution and following rinse prior to impregnation with silver. The procedure has high sensitivity and long storage lifetime. The minimum detectable mass of DNA is 0.44 and 3.5 ng in denaturing and nondenaturing polyacrylamide gel, respectively. [source] Electronic gel protein transfer and identification using matrix-assisted laser desorption/ionization-mass spectrometryELECTROPHORESIS, Issue 9 2004Jonathan W. Cooper Abstract An electronic protein transfer technique is described for achieving the rapid and efficient recovery of sodium dodecyl sulfate (SDS)-protein complexes from polyacrylamide gels. This process involves the use of small-dimension capillaries in physical contact with a resolved protein band within the polyacrylamide gel, providing a large potential drop and high electric field strength at the capillary/gel interface. Several factors controlling the electronic protein transfer, including the applied electric field strength, the electrophoresis buffer concentration, and the capillary dimension, are studied to further enhance the use of field-amplification for sample stacking of extracted SDS-protein complexes. As a result of sample stacking, the extracted proteins from a 50 ng gel loading are present in a narrow (,80 nL) and highly concentrated (0.46 mg/mL or 3.3×10,5 M for cytochrome c) solution plug. Three model proteins with molecular mass ranging from 14 kDa (cytochrome c) to 116 kDa (,-galactosidase) are stained by Coomassie blue and electrophoretically extracted from gels with protein loadings as low as 50 ng. The capillary format of the electronic protein transfer technique allows direct deposition of extracted proteins onto a matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) target. Various matrices and solvent compositions are evaluated for the analysis of extracted and concentrated SDS-protein complexes using MALDI-MS. The electronic protein transfer technique, when operated under optimized conditions, is demonstrated for the effective (>70% recovery), speedy (less than 5 min), and sensitive MS identification of gel resolved proteins (as low as 50 ng). [source] A simple polyacrylamide gel electrophoresis procedure for separation of polyamidoamine dendrimersELECTROPHORESIS, Issue 16 2003Ajit Sharma Abstract A simple, inexpensive, and rapid electrophoresis technique was developed for use as a routine tool for evaluating purity of polyamidoamine (PAMAM) dendrimers. A variety of factors influencing migration of generations 0,7 dendrimers on nongradient polyacrylamide gels were evaluated. The low generation dendrimers were found to be very sensitive to diffusion during or after electrophoresis. The proposed method incorporates steps that minimize diffusion, in order to obtain improved resolution and sensitivity, especially for the lower-molecular-weight dendrimers. This was accomplished by inclusion of a dendrimer fixation step with glutaraldehyde and performing the electrophoresis separation, fixation, staining, and destaining at 4°C. PAMAM dendrimer separation was studied under basic and acidic conditions. Electrophoresis under acidic conditions gave increased resolution and sensitivity over separation at alkaline pH. Oligomers and trailing generations could be clearly separated and visualized under these conditions. The smallest PAMAM dendrimer, generation 0, was visible at 1.5 ,g under the optimized acidic conditions. With slight modifications, this technique should be applicable to separation of other water-soluble dendrimers. [source] Visualization of Differential Gene Expression , Using Fluorescence-Based cDNA-AFLPENGINEERING IN LIFE SCIENCES (ELECTRONIC), Issue 1 2004S. Gigliotti Abstract cDNA-AFLP is one of the techniques developed to study differentially expressed genes. This recent technique is advantageous because it does not need prior sequence knowledge and is reliable due to highly stringent PCR conditions. The traditional cDNA-AFLP method uses radioactively labelled products and is characterised by high sensitivity and resolution. Here, the use of Cy5-labelled primers to detect products on polyacrylamide gels is reported. This non-radioactive method, based on fluorescence, is shown to be faster and the recovery of interesting bands is easier. The study of the differential gene expression of the interaction between potato and Phytophthora infestans was used for the valuation of this method. Different gene expression profiles , such as up-regulation, down-regulation or point expression , were obtained. Moreover, this technique was shown to be highly reproducible. [source] | ||||||||||||||||