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PubMed2000

Optimized MR imaging for polyacrylamide gel dosimetry.

Polyacrylamide gels are a powerful tool to measure radiation dose by quantifying the NMR T2 relaxation times of the irradiated gel. The exploitation of these radiation sensitive gels in clinical radiotherapy requires accurate mapping of T2 values. This paper describes the optimization strategy used to identify accurate and practical methods of measuring the range of T2 values typical of gel dosimeters (140-700 ms). The MR imaging techniques used to measure T2 values and the choice of image acquisition parameters are described. Four sequences are compared and the results are analysed in terms of accuracy, signal-to-noise ratio and acquisition time. A multiple spin echo sequence was found to yield the most accurate results (98.9%). Single spin echo sequences, such as Hahn spin echo and EPI spin echo, were found to measure gel T2 values with an accuracy of 90.1%. This paper reports the importance of careful selection and optimization of the MR imaging sequences for accurate and reliable polyacrylamide gel dosimetry.

Echo-Planar Imaging↗

PubMed2000

The reproducibility of polyacrylamide gel dosimetry applied to stereotactic conformal radiotherapy.

The reproducibility of polyacrylamide gel (PAG) dosimetry has been evaluated when used to verify two radiotherapy treatment plans of increasing complexity. The plans investigated were a three-field coplanar arrangement, using the linac jaws for field shaping, and a four-field, conformal, non-coplanar plan using precision-cast lead alloy shielding blocks. Each treatment was performed three times using phantoms and calibration gels manufactured in-house. Two phantoms were specially designed for this work to aid accurate positioning of the gels for irradiation and imaging. All gels were imaged post-irradiation using a Siemens Vision 1.5T MR scanner. T2 relaxation images were calibrated to absorbed dose distributions using a number of smaller calibration vessels to produce distribution maps of relative dose. The relative dose distributions were found to be reproducible, with the standard deviation on the mean areas enclosed by the > or = 50% isodose lines measured in three orthogonal planes being 6.4% and 4.1% for the coplanar and non-coplanar plans respectively. The measured distributions were also consistent with those planned, with isodose lines generally agreeing to within a few millimetres. However, the measured absolute doses were on average 23.5% higher than those planned. Although the polyacrylamide gel dosimetry technique has some limitations, particularly when calibrating distributions to absolute dose, the ability to resolve sharp dose gradients in three dimensions with millimetre precision is invaluable when verifying complex conformal treatment plans, where avoidance of proximal, critical structures is a treatment criterion.

Acrylic Resins↗

PubMed2006

An NMR relaxometry and gravimetric study of gelatin-free aqueous polyacrylamide dosimeters.

In conformal radiation therapy, a high dose of radiation is given to a target volume to increase the probability of cure, and care is taken to minimize the dose to surrounding healthy tissue. The techniques used to achieve this are very complicated and the precise verification of the resulting three-dimensional (3D) dose distribution is required. Polyacrylamide gelatin (PAG) dosimeters with magnetic resonance imaging and optical computed tomography scanning provide the required 3D dosimetry with high spatial resolution. Many basic studies have characterized these chemical dosimeters that polymerize under irradiation. However, the investigation of the fundamental properties of the radiation-induced polymerization in PAG dosimeters is complicated by the presence of the background gelatin matrix. In this work, a gelatin-free model system for the study of the basic radiation-induced polymerization in PAG dosimeters has been developed. Experiments were performed on gelatin-free dosimeters, named aqueous polyacrylamide (APA) dosimeters, containing equal amounts of acrylamide and N,N'-methylene-bisacrylamide. The APA dosimeters were prepared with four different total monomer concentrations (2, 4, 6 and 8% by weight). Nuclear magnetic resonance (NMR) spin-spin and spin-lattice proton relaxation measurements at 20 MHz, and gravimetric analyses performed on all four dosimeters, show a continuous degree of polymerization over the dose range of 0-25 Gy. The developed NMR model explains the relationship observed between the relaxation data and the amount of crosslinked polymer formed at each dose. This model can be extended with gelatin relaxation data to provide a fundamental understanding of radiation-induced polymerization in the conventional PAG dosimeters.

Acrylamides↗

PubMed1985

Purification of chick interferon by zinc chelate affinity chromatography and sodium dodecylsulfate-polyacrylamide gel electrophoresis.

An improved purification method for chick interferon from the allantoic fluid of embryonated chick eggs is described. Interferon prepurified by perchloric acid treatment, zinc acetate precipitation, and chromatography on SP-Sephadex C-25 was further enriched by column chromatography on zinc chelate. Analysis on sodium dodecylsulfate polyacrylamide gel electrophoresis of the interferon preparation with a specific activity of 8 X 10(5) units/mg protein shows that the major antiviral activity migrated in a broad band in the range of 20-29 kD molecular weight. Several protein bands were stainable with Coomassie blue and silver nitrate in this molecular weight range. Between 80 and 95% of the total protein charged to the gel could be removed from the interferon containing fractions by sodium dodecylsulfate polyacrylamide gel electrophoresis.

Allantois↗

PubMedDate not supplied

Polyacrylamide gel analysis of spiroplasmas proteins and its contribution to the taxonomy of spiroplasmas.

Representative strains of the known groups and serogroups of spiroplasmas have been compared on the basis of the results of one- and two-dimensional protein analysis on polyacrylamide gels. Each of the four subgroups of the Spiroplasma citri complex (S. citri and honeybee, corn stunt, and 277F spiroplasmas) has characteristic protein profiles and maps. Spiroplasma G1 isolated from flowers has a protein profile closely related to that of the honeybee spiroplasmas (KC3, BC3, AS576, B1707, B88, and B63). The flower spiroplasmas fall into two groups on the basis of the mole percentage (mol%) of guanine plus cytosine (G + C) of their deoxyribonucleic acid (DNA) 26 and 30 mol %, respectively) and their serologic properties. The three flower spiroplasmas (OBMG, BNR1, and 23-6) with 26 mol % of G + C have the same basic protein pattern. Although some of the flower spiroplasmas with 30 mol % of G + C have closely related protein patterns, others have more distant profiles. Spiroplasmas B13 and L89, isolated from honeybees and froghoppers, respectively, had DNA with 30 mol % of G + C and could be included with other flower spiroplasmas with similar DNA base composition on the basis of their protein profiles and maps. Individual proteins separated by two-dimensional analysis on polyacrylamide gels have been used for the production of monospecific immunoglobulins, which are useful in taxonomic studies.

Animals↗

PubMed1992

Effect of glycerol on the separation of nucleosomes and bent DNA in low ionic strength polyacrylamide gel electrophoresis.

We report that glycerol changes the separation characteristics of polyacrylamide nucleoprotein gels in which it is included as a stabilizing agent. Polyacrylamide gel electrophoresis fractionates DNA and nucleosomes according to net negative charge, mass and conformation. With glycerol included, fractionation seems to be largely based on particle mass and charge. The conformation factor in separation is progressively lost with increasing glycerol concentrations. Nucleosome positions on the same DNA fragment are no longer resolved, while the difference in electrophoretic mobility between core particles and nucleosomes carrying longer DNA becomes smaller and is eventually lost. The retardation of bent DNA is also much reduced. Using the differences in separation characteristics between glycerol-containing and regular nucleoprotein gels could be a new means to obtain information on macromolecules in solution.

Animals↗

PubMed1996

A fluorescence-labeling method for sequencing small RNA on polyacrylamide gel.

A practical fluorescence-labeling method for sequencing small RNAs by the traditional 'direct read out' on polyacrylamide gel electrophoresis was established. The 3' terminus of RNA was oxidized into dialdehyde by sodium periodate and then labeled with fluorescein-5-thiosemicarbazide through the condensation reaction between carbazide and aldehyde. The fluorescence-labeled RNA was partially degraded enzymatically and fractionated by polyacrylamide gel electrophoresis. The fluorescent bands were visualised by ultraviolet photography. A partial sequence of yeast 5S rRNA was determined. The result indicates that this method can be used in sequencing small RNAs rapidly, conveniently and safely.

Electrophoresis, Polyacrylamide Gel↗

PubMed1999

Synthesis of polyacrylamides N-substituted with PNA-like oligonucleotide mimics for molecular diagnostic applications.

Two types of oligonucleotide mimics relative to peptide nucleic acids (PNAs) were tested as probes in nucleic acid hybridisation assays based on polyacrylamide technology. One type of mimic oligomers represented a chimera constructed of PNA and phosphono-PNA (pPNA) monomers, and the other one contained pPNA residues alternating with PNA-like monomers on the base of trans -4-hydroxy-L-proline (HypNA). A chemistry providing efficient and specific covalent attachment of these DNA mimics to acrylamide polymers using a convenient approach based on the co-polymerisation of acrylamide and some reactive acrylic acid derivatives with oligomers bearing 5'- or 3'-terminal acrylamide groups has been developed. A comparative study of polyacrylamide conjugates with oligonucleotides and mimic oligomers demonstrated the suitability and high potential of PNA-pPNA and HypNA-pPNA chimeras as sequence-specific probes in capture and detection of target nucleic acid fragments to serve current forms of DNA arrays.

Acrylamide↗

PubMed1981

Identification and suppression of secondary structures formed from deoxy-oligonucleotides during electrophoresis in denaturing polyacrylamide-gels.

Nature and stability of secondary structures formed from DNA-fragments during electrophoresis in urea containing polyacrylamide-gels were investigated. Duplices and especially hairpin loops were found to be surprisingly stable. Polyacrylamide-gels containing high concentrations of formamide effectively melt these structures. Such gels will improve nucleotide sequence analysis in regions of dyad symmetry that often cause 'band compressions' under standard electrophoresis conditions.

Electrophoresis, Polyacrylamide Gel↗

PubMed1981

Adenosine dimethylation of 16S ribosomal RNA: effect of the methylgroups on local conformational stability as deduced from electrophoretic mobility of RNA fragments in denaturing polyacrylamide gels.

The electrophoretic mobility of RNA fragments derived from the 3'-end of 16S rRNA on slabs of polyacrylamide gel in the presence of urea is strongly influenced by dimethylation of the N6-aminogroup of two adjacent adenosines. This is not due to the presence of the methylgroups per se, but must be ascribed to an effect of methylation on long range intramolecular interactions at these denaturing conditions. When it is assumed that the electrophoretic mobilities of the RNA fragments in the polyacrylamide matrix are determined by the conformational state(s) of the fragments, dimethylation of the adenosines leads in the smaller fragments to a less compact average conformation and in the larger fragments to a more compact average conformation. An effort is made to comprehend the effects of adenosine dimethylation in terms of secondary structure based on nucleotide sequence.

Adenosine↗

PubMed1999

Rapid and inexpensive recovery method of DNA fragments from agarose and polyacrylamide gels by a cotton-wool column tube.

We developed a rapid, convenient, simple, and inexpensive method for isolating pure DNA from agarose and polyacrylamide gels using cotton wool tubes. DNA fragments ranging in size from 193-23,130 bp can be easily recovered within 2 hours by centrifugation through cotton wool from gel slices. The recovery rate of this method is 35% to 50%, when estimated for isolation of lambda DNA-HindIII fragments. We have also recovered 700-bp polymerase chain reaction (PCR) products using cotton wool tubes from electrophoresis on both a 0.8% agarose gel and a 6% polyacrylamide gel, in which satisfactory yields of more than 50% were obtained. The DNA thus recovered in this way is biologically active and can be used as a substrate for further experimental procedures without additional purification steps.

Bacteriophage lambda↗

PubMed1987

Real-time monitoring of polyacrylamide gel electrophoresis by schlieren optics.

A modernized schlieren optics was applied to follow protein bands visually during polyacrylamide gel electrophoresis. A band containing as little as 0.3 microgram of a protein could be detected. Besides the protein bands, usually overlooked phenomena such as boundary migration of the buffer components could be visualized. As an example, electrophoretic patterns thus obtained for SDS-polyacrylamide gel electrophoresis with a discontinuous buffer system are presented. The use of a split-type colored filter was found to be useful for coloring a particular location on the gel red or blue depending on the sign of the refractive index gradient. This means of detection also seems useful in that, firstly, it makes electrophoretic mobility measurement more quantitative and, secondly, it allows localization of a protein band and sampling in the intact state.

Computer Systems↗

PubMed1975

Retarding effect of dodecyl alcohol on polyacrylamide gel electrophoresis of SDS micelles and SDS-protein polypeptide complexes.

Micelles of sodium dodecyl sulfate (SDS) are significantly retarded by the addition of a small amount of dodecyl alcohol to a sample solution in SDS-polyacrylamide gel electrophoresis. The phenomenon can be ascribed to the decrease in charge density due to the incorporation of dodecyl alcohol into SDS micelles. The effect is extended to SDS-protein polypeptide complexes when the amount of SDS micelles is insufficient to accomodate the dodecyl alcohol. A similar effect is likely to occur when SDS-polyacrylamide gel electrophoresis is applied to a sample containing lipophilic materials.

Alcohols↗

PubMed1986

Transferrin variants in sheep: separation and characterization by polyacrylamide gel electrophoresis and isoelectric focusing.

Isoelectric focusing with carrier ampholytes in ultrathin polyacrylamide gels and polyacrylamide gel electrophoresis in a discontinuous buffer system were used for the separation of sheep transferrin variants. For identification of the different iron-binding sites of transferrin a stepwise urea gradient, different degrees of iron saturation and double one-dimensional electrophoresis were used. Isoelectric focusing results in an increased resolution of the Fe0-transferrin, Fe1-transferrin and Fe2-transferrin region. At the level of Fe0-transferrin and Fe1-transferrin the variants I, A, G, B, C, D, M, E, Q, P can be identified. The method is especially suitable for genetic studies. For screening purposes up to 108 samples can be separated within one run in an ultrathin gel.

Animals↗

PubMed1984

Immunological detection of Plasmodium knowlesi antigens after electrophoretic transfer from SDS-polyacrylamide gels to diazophenylthioether paper.

Freeze-thawed extracts of mature schizonts of Plasmodium knowlesi (strain W1) were electrophoresed in sodium dodecylsulphate (SDS) polyacrylamide gels, then transferred electrophoretically to diazophenylthioether paper. The transferred antigen was probed using a purified polyvalent, polyclonal immune serum pool isolated from rhesus monkeys (Macaca mulatta). This method for identifying parasite antigens was compared with immunoabsorption, using parasites which had been metabolically labelled in vitro with 35S-methionine. The methods showed general correspondence but some antigens failed to react after transfer because of loss of antigenicity during SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Some antigens were observed preferentially after the transfer procedure, possibly because of their failure to elute from the immunoabsorbant or because they may have a low methionine content. The transfer method failed to reveal the antigen reactivity of three out of four monoclonal antibodies tested, apparently because the epitopes involved were inactivated by SDS. During P. knowlesi infection in the resistant kra monkey (Macaca fascicularis) the transfer method showed the presence by day 10 of antibodies against two components having an apparent molecular weight by SDS-PAGE similar to those of putative protective antigens described in other studies using monoclonal antibodies.

Animals↗

PubMed1982

Role of sialic acid in the mobility of membrane proteins containing O-linked oligosaccharides on polyacrylamide gel electrophoresis in sodium dodecyl sulfate.

The major sialoglycoprotein of the human red-cell membrane, glycophorin A, contains 15 O-glycosidically linked oligosaccharides and one N-glycosidic oligosaccharide. The protein shows a decreased mobility on polyacrylamide gel electrophoresis in sodium dodecyl sulfate after neuraminidase treatment of the non-denatured protein. The molecular mechanism behind this phenomenon has been elucidated. Neuraminidase treatment of glycophorin A in intact cells or after solubilization in buffers containing Triton X-100 resulted in conversion of the predominant tetrasaccharide N-acetylneuraminosyl alpha 2-3galactosyl beta 1-3(N-acetylneuraminosyl alpha 2-6)-N-acetylgalactosamine to the trisaccharide galactosyl beta 1-3(N-acetylneuraminosyl alpha 2-6)-N-acetylgalactosamine and the disaccharide galactosyl beta 1-3-N-acetylgalactosamine. After denaturation with sodium dodecyl sulfate, Vibrio cholerae neuraminidase also liberated the N-acetylgalactosamine-bound sialic acids. Such treatment resulted in increased electrophoretic mobility. The results show that distal sialic acids linked to galactose are readily available to neuraminidase, and that their negative charge gives an increased electrophoretic mobility in polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. In contrast, most of the N-acetylgalactosamine-linked sialic acids of glycophorin A are not liberated by neuraminidase without denaturation of the substrate. Like sialic acids of complex-type oligosaccharides the decreased electrophoretic mobility caused by them is exclusively due to their mass while no significant contribution by the charge was seen.

Chemical Phenomena↗

PubMed1985

Identification of neuron-specific enolase and nonneuronal enolase in human and rat brain on two-dimensional polyacrylamide gels.

The location of the enzymes neuron-specific enolase and nonneuronal enolase on two-dimensional gels generated from tissue samples obtained from fresh human and rat cortex has been identified. This identification is based upon the following criteria: comigration on polyacrylamide gels with the appropriate purified protein and staining on nitrocellulose protein blots of human and rat cortex using antibodies specific for each protein. The results show that our preparation of neuron-specific enolase from rat and human brain is highly pure, as only one spot is obtained on two-dimensional gels. Further, the antiserum to neuron-specific enolase is highly specific, as it reacts only with neuron-specific enolase on nitrocellulose blots derived from two-dimensional gels of cortical tissue. The location of these proteins is of interest because it positively identifies two major brain proteins on two-dimensional polyacrylamide gels of fresh cortical tissue. This information will be useful in a variety of future studies aimed at both identifying specific proteins on two-dimensional gels and observing the effects of experimental manipulations on brain and other neuronal proteins.

Animals↗

PubMed2004

Migration of the activation peptide of thrombin-activatable fibrinolysis inhibitor (TAFI) during SDS-polyacrylamide gel electrophoresis.

Thrombin-activatable fibrinolysis inhibitor (TAFI) is a plasma zymogen, which upon activation is capable of delaying fibrinolysis. We investigated the migration and detection of the activation peptide of TAFI during SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Purified TAFI before and after activation by thrombin/thrombomodulin was electrophoresed on 4-20% polyacrylamide gels and stained with Coomassie blue as well as Western blotting. Before activation, Coomassie blue staining resulted in one main band of TAFI. After activation, a sharp band corresponding to TAFIa was observed. No distinct activation peptide was detected, in agreement with the literature. Western blotting using a polyclonal anti-TAFI antibody, on the other hand, showed one additional broad band with an Mr of about 33 000 after TAFI activation. N-terminal sequence analysis confirmed that this band represented the activation peptide of TAFI. In addition, we tested the reactivity of two anti-TAFI monoclonal antibodies (MA-T3D8 and MA-T18A8) towards TAFI before and after activation by Western blotting. Both monoclonal antibodies recognized TAFI. After activation of TAFI, MA-T3D8 reacted with TAFIa, while MA-T18A8 reacted with the activation peptide. We identify the 33 000 band as the activation peptide of TAFI and exemplify the use of this information for the characterization of monoclonal antibodies against TAFI.

Amino Acid Sequence↗