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Nancy C. Stellwagen - One of the best experts on this subject based on the ideXlab platform.
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Electrophoretic mobility is a reporter of hairpin structure in single-stranded DNA oligomers.
Biochemistry, 2007Co-Authors: Earle Stellwagen, Arian Abdulla, Qian Dong, Nancy C. StellwagenAbstract:The electrophoretic mobilities of 24 single-stranded DNA oligomers, each containing 26 nucleotide residues, have been measured in polyacrylamide gels and in free solution. The mobilities observed at 20 degrees C differed by approximately 20% in polyacrylamide gels and by approximately 10% in free solution, even though the oligomers contained the same number of bases. Increasing the temperature or adding urea to the solution equalized the mobilities of the oligomers, suggesting that the variable mobilities observed at 20 degrees C are due to the formation of stable secondary structures, most likely hairpins. Thermal melting profiles were measured for eight oligomers in 40 mM Tris Acetate buffer. The observed melting temperatures of most oligomers correlated roughly with the mobilities observed at 20 degrees C; however, one oligomer was much more stable than the others. The melting temperatures of four of the oligomers were close to the values predicted by DINAMelt [Markham, N. R., and Zuker, M. (2005) Nucleic Acids Res. 33, W577-W581]; melting temperatures of the other oligomers differed significantly from the predicted values. Thermal melting profiles were also measured for two oligomers as a function of the Tris Acetate buffer concentration. The salt concentration dependence of the melting temperatures suggests that 0.15 Tris+ ion per phosphate is released upon denaturation. Because the apparent number of Tris+ ions released is greater than that observed by others for the release of Na+ ions from similar hairpins, the results suggest that DNA hairpins (and, presumably, duplexes) bind more Tris+ ions than Na+ ions in solution.
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The free solution mobility of DNA in Tris-Acetate-EDTA buffers of different concentrations, with and without added NaCl.
Electrophoresis, 2002Co-Authors: Earle Stellwagen, Nancy C. StellwagenAbstract:Abstract The free solution mobility of a high-molecular-weight DNA, linear pUC19, and a 20-bp oligomer called dsA5 have been studied as a function of Tris-Acetate-EDTA (TAE) buffer concentration, with and without added NaCl. The two DNAs migrate as separate peaks during capillary electrophoresis, because the mobility of linear pUC19 is higher than that of the 20-bp oligomer. In TAE buffers ranging from 10-400 mM in concentration, the migration times and peak areas of the two DNAs are independent of whether they are electrophoresed separately or in mixtures, indicating that DNA-DNA and DNA-buffer interactions are absent in these solutions. The migration times of the two DNAs vary and the peak areas are not additive when the TAE buffer concentration is reduced to 5 mM or below, indicating that DNA-DNA and DNA-buffer interactions are occurring at very low TAE buffer concentrations. The mobilities of linear pUC19 and dsA5 decrease slowly with increasing conductivity or ionic strength when the conductivity is increased by increasing the TAE buffer concentration. When the Tris buffer concentration is held constant and the conductivity is increased by adding various concentrations of NaCl to the solution, the mobilities of linear pUC19 and dsA5 first increase slightly, then become independent of solution conductivity (or ionic strength), and finally decrease when the NaCl concentration is increased above approximately 50 mM. The mobility variations observed in the various TAE and TAE-NaCl solutions are described qualitatively by Manning's theory, although quantitative agreement is not achieved. The free solution mobilities of single-stranded pUC19 and two 20-base oligonucleotides have also been measured. The free solution mobility of single-stranded pUC19 is approximately 15% lower than that of native pUC19, in agreement with other results in the literature. Somewhat surprisingly, the mobilities of the single- and double-stranded 20-mers are equal to each other in TAE buffers with and without added NaCl.
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apparent pore size of polyacrylamide gels comparison of gels cast and run in Tris Acetate edta and Tris borate edta buffers
Electrophoresis, 1998Co-Authors: Nancy C. StellwagenAbstract:The electrophoretic mobilities of DNA molecules in three different molecular weight ladders were measured in polyacrylamide gels containing different acrylamide concentrations (%T) and cross-linker ratios (%C), cast and run in TrisAcetate-EDTA (TAE) buffer. The apparent pore radius of each gel was estimated from Ferguson plots of the relative mobilities of each of the DNA molecules, using the mobility of the monomer fragment in each molecular weight ladder as the reference mobility. The effective size of each of the DNA molecules was estimated from its radius of gyration. The apparent gel pore radii calculated in this manner ranged from 21 nm in gels containing 10.5%T, 5%C to 200 nm in gels with 4.6%T, 2%C, similar to the values observed for polyacrylamide gels cast and run in Tris-borate-EDTA (TBE) buffer (Holmes and Stellwagen, Electrophoresis 1991, 12, 612-619). Hence, the effective pore size of polyacrylamide gels is essentially independent of whether the gels are cast and run in TAE or TBE buffer.
Jose Luis Rosa - One of the best experts on this subject based on the ideXlab platform.
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Tris-Acetate Polyacrylamide Gradient Gels for the Simultaneous Electrophoretic Analysis of Proteins of Very High and Low Molecular Mass.
Methods in molecular biology (Clifton N.J.), 2018Co-Authors: Monica Cubillos-rojas, Ramon Bartrons, Francesc Ventura, Fabiola Amair-pinedo, Irantzu Tato, Jose Luis RosaAbstract:Polyacrylamide gel electrophoresis (PAGE) is one of the most powerful tools used for protein analysis. We describe the use of Tris-Acetate buffer and 3-15% polyacrylamide gradient gels to simultaneously separate proteins in the mass range of 10-500 kDa. We show that this system is highly sensitive, it has good resolution and high reproducibility, and it can be used for general applications of PAGE such as Coomassie Brilliant Blue staining and immunoblotting. Moreover, we describe how to generate mini Tris-Acetate polyacrylamide gels to use them in miniprotein electrophoresis systems. These economical gels are easy to generate and to manipulate and allow a rapid analysis of proteins. All these features make the Tris-Acetate-PAGE system a very helpful tool for protein analysis.
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Tris-Acetate polyacrylamide gradient gel electrophoresis for the analysis of protein oligomerization
Analytical and Bioanalytical Chemistry, 2016Co-Authors: Monica Cubillos-rojas, Taiane Schneider, Susana Sánchez-tena, Ramon Bartrons, Francesc Ventura, Jose Luis RosaAbstract:Here we report a new approach for studying protein oligomerization in cells using a single electrophoresis gel. We combined the use of a crosslinking reagent for sample preparation, such as glutaraldehyde, with the analysis of oligomers by Tris-Acetate polyacrylamide gel electrophoresis. The use of a 3-15% Tris-Acetate polyacrylamide gradient gel allows for the simultaneous analysis of proteins of masses ranging from 10 to 500 kDa. We showed the usefulness of this method for analyzing endogenous p53 oligomerization with high resolution and sensitivity in human cells. Oligomerization analysis was dependent on the crosslinker concentration used. We also showed that this method could be used to study the regulation of oligomerization. In all experiments, Tris-Acetate polyacrylamide gel electrophoresis proved to be a robust, manageable, and cost- and time-efficient method that provided excellent results using a single gel. This approach can be easily extrapolated to the study of other oligomers. All of these features make this method a highly useful tool for the analysis of protein oligomerization.
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Tris–Acetate Polyacrylamide Gradient Gels for the Simultaneous Electrophoretic Analysis of Proteins of Very High and Low Molecular Mass
Methods of Molecular Biology, 2012Co-Authors: Monica Cubillos-rojas, Ramon Bartrons, Francesc Ventura, Fabiola Amair-pinedo, Irantzu Tato, Jose Luis RosaAbstract:Polyacrylamide gel electrophoresis (PAGE) is one of the most powerful tools used for protein analysis. We describe the use of Tris-Acetate buffer and 3-15% polyacrylamide gradient gels to simultaneously separate proteins in the mass range of 10-500 kDa. We show that this system is highly sensitive, it has good resolution and high reproducibility, and that it can be used for general applications of PAGE such as Coomassie Brilliant Blue staining and immunoblotting. Moreover, we describe how to generate mini Tris-Acetate polyacrylamide gels to use them in miniprotein electrophoresis systems. These economical gels are easy to generate and to manipulate and allow a rapid analysis of proteins. All these features make the Tris-Acetate-PAGE system a very helpful tool for protein analysis.
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Simultaneous electrophoretic analysis of proteins of very high and low molecular mass using Tris-Acetate polyacrylamide gels.
Electrophoresis, 2010Co-Authors: Monica Cubillos-rojas, Ramon Bartrons, Francesc Ventura, Fabiola Amair-pinedo, Irantzu Tato, Jose Luis RosaAbstract:To separate and analyze giant and small proteins in the same electrophoresis gel, we have used a 3-15% polyacrylamide gradient gel containing 2.6% of the crosslinker bisacrylamide and 0.2 M of Tris-Acetate buffer (pH 7.0). Samples were prepared in a sample buffer containing lithium dodecyl sulphate and were run in the gel described above using Tris-Tricine-SDS-sodium bisulfite buffer, pH 8.2, as electrophoresis buffer. Here, we show that this system can be successfully used for general applications of SDS-PAGE such as CBB staining and immunoblot. Thus, by using Tris-Acetate 3-15% polyacrylamide gels, it is possible to simultaneously analyze proteins, in the mass range of 10-500 kDa, such as HERC1 (532 kDa), HERC2 (528 kDa), mTOR (289 kDa), Clathrin heavy chain (192 kDa), RSK (90 kDa), S6K (70 kDa), beta-actin (42 kDa), Ran (24 kDa) and LC3 (18 kDa). This system is highly sensitive since it allows detection from as low as 10 microg of total protein per lane. Moreover, it has a good resolution, low cost, high reproducibility and allows for analysis of proteins in a wide range of weights within a short period of time. All these features together with the use of a standard electrophoresis apparatus make the Tris-Acetate-PAGE system a very helpful tool for protein analysis.
Claudia G. Benítez-cardoza - One of the best experts on this subject based on the ideXlab platform.
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Self-Association of Enolase from Trichomonas vaginalis. Monomers, Dimers, and Octamers Coexist in Solution
ACS Omega, 2018Co-Authors: Elibeth Mirasol-meléndez, Enrique Lima, Victor Lara, Luis G. Brieba, Samuel Lara-gonzález, Claudia G. Benítez-cardozaAbstract:We used small-angle X-ray scattering to study the self-association of enolase from Trichomonas vaginalis as a function of the protein concentration and cosolute type. We observed coexisting monomers, dimers, and octamers in variable relative populations, depending on whether Tris–Acetate, Tris–HCl, or potassium phosphate buffers were used. Phosphate ions hindered the formation of dimers and octamers. In contrast, the populations of dimers and octamers increased in Tris–Acetate or Tris–HCl buffers and additionally increased by augmenting protein concentration or adding magnesium. Single oligomeric species could not be isolated in any of the experimental conditions tested. Furthermore, the secondary and tertiary structures, as well as the temperature-induced denaturation of the mixtures of species, were investigated. The acquired species lost enzymatic activity, but they were prone to interact with plasminogen, as judged from changes in the secondary and tertiary structures upon complex formation.
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Self-Association of Enolase from Trichomonas vaginalis. Monomers, Dimers, and Octamers Coexist in Solution
2018Co-Authors: Elibeth Mirasol-meléndez, Enrique Lima, Victor Lara, Luis G. Brieba, Samuel Lara-gonzález, Claudia G. Benítez-cardozaAbstract:We used small-angle X-ray scattering to study the self-association of enolase from Trichomonas vaginalis as a function of the protein concentration and cosolute type. We observed coexisting monomers, dimers, and octamers in variable relative populations, depending on whether Tris–Acetate, Tris–HCl, or potassium phosphate buffers were used. Phosphate ions hindered the formation of dimers and octamers. In contrast, the populations of dimers and octamers increased in Tris–Acetate or Tris–HCl buffers and additionally increased by augmenting protein concentration or adding magnesium. Single oligomeric species could not be isolated in any of the experimental conditions tested. Furthermore, the secondary and tertiary structures, as well as the temperature-induced denaturation of the mixtures of species, were investigated. The acquired species lost enzymatic activity, but they were prone to interact with plasminogen, as judged from changes in the secondary and tertiary structures upon complex formation
Alexander N Glazer - One of the best experts on this subject based on the ideXlab platform.
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fluorometric assay using dimeric dyes for double and single stranded dna and rna with picogram sensitivity
Analytical Biochemistry, 1993Co-Authors: J M Dabora, Mark A Quesada, Richard A Mathies, Alexander N GlazerAbstract:Thiazole orange homodimer (TOTO; 1,1′-(4,4,7,7-tetramethyl-4,7-diazaundecamethylene)-bis-4-[3-methyl-2,3-dihydro-(benzo-1,3-thiazole)-2-methylidene]-quinolinium tetraiodide) and oxazole yellow homodimer (YOYO; an analogue of TOTO with a benzo-1,3-oxazole in place of the benzo-1,3-thiazole) bind with very high affinity to nucleic acids with more than a 1000-fold fluorescence enhancement upon binding. A linear dependence of fluorescence intensity on DNA concentration over a range from 0.5 to 100 ng/ml in the presence of 2 × 10−7 M TOTO or YOYO in 4 mM Tris-Acetate/0.1 mM EDTA/50 mM NaCl, pH 8.2 allows sensitive quantitation of double-stranded DNA in a conventional fluorometer. With nucleic acid-dye mixtures in an array of 25-μl wells in a block of low autofluorescence plastic and detection with a laser-excited confocal fluorescence scanner, as little as 20 pg of double-stranded DNA can be detected per well. The array scanning method is rapid, has high throughput, and requires small amounts of sample. It also allows quantitation of single-stranded DNA and RNA.
Monica Cubillos-rojas - One of the best experts on this subject based on the ideXlab platform.
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Tris-Acetate Polyacrylamide Gradient Gels for the Simultaneous Electrophoretic Analysis of Proteins of Very High and Low Molecular Mass.
Methods in molecular biology (Clifton N.J.), 2018Co-Authors: Monica Cubillos-rojas, Ramon Bartrons, Francesc Ventura, Fabiola Amair-pinedo, Irantzu Tato, Jose Luis RosaAbstract:Polyacrylamide gel electrophoresis (PAGE) is one of the most powerful tools used for protein analysis. We describe the use of Tris-Acetate buffer and 3-15% polyacrylamide gradient gels to simultaneously separate proteins in the mass range of 10-500 kDa. We show that this system is highly sensitive, it has good resolution and high reproducibility, and it can be used for general applications of PAGE such as Coomassie Brilliant Blue staining and immunoblotting. Moreover, we describe how to generate mini Tris-Acetate polyacrylamide gels to use them in miniprotein electrophoresis systems. These economical gels are easy to generate and to manipulate and allow a rapid analysis of proteins. All these features make the Tris-Acetate-PAGE system a very helpful tool for protein analysis.
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Tris-Acetate polyacrylamide gradient gel electrophoresis for the analysis of protein oligomerization
Analytical and Bioanalytical Chemistry, 2016Co-Authors: Monica Cubillos-rojas, Taiane Schneider, Susana Sánchez-tena, Ramon Bartrons, Francesc Ventura, Jose Luis RosaAbstract:Here we report a new approach for studying protein oligomerization in cells using a single electrophoresis gel. We combined the use of a crosslinking reagent for sample preparation, such as glutaraldehyde, with the analysis of oligomers by Tris-Acetate polyacrylamide gel electrophoresis. The use of a 3-15% Tris-Acetate polyacrylamide gradient gel allows for the simultaneous analysis of proteins of masses ranging from 10 to 500 kDa. We showed the usefulness of this method for analyzing endogenous p53 oligomerization with high resolution and sensitivity in human cells. Oligomerization analysis was dependent on the crosslinker concentration used. We also showed that this method could be used to study the regulation of oligomerization. In all experiments, Tris-Acetate polyacrylamide gel electrophoresis proved to be a robust, manageable, and cost- and time-efficient method that provided excellent results using a single gel. This approach can be easily extrapolated to the study of other oligomers. All of these features make this method a highly useful tool for the analysis of protein oligomerization.
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Tris–Acetate Polyacrylamide Gradient Gels for the Simultaneous Electrophoretic Analysis of Proteins of Very High and Low Molecular Mass
Methods of Molecular Biology, 2012Co-Authors: Monica Cubillos-rojas, Ramon Bartrons, Francesc Ventura, Fabiola Amair-pinedo, Irantzu Tato, Jose Luis RosaAbstract:Polyacrylamide gel electrophoresis (PAGE) is one of the most powerful tools used for protein analysis. We describe the use of Tris-Acetate buffer and 3-15% polyacrylamide gradient gels to simultaneously separate proteins in the mass range of 10-500 kDa. We show that this system is highly sensitive, it has good resolution and high reproducibility, and that it can be used for general applications of PAGE such as Coomassie Brilliant Blue staining and immunoblotting. Moreover, we describe how to generate mini Tris-Acetate polyacrylamide gels to use them in miniprotein electrophoresis systems. These economical gels are easy to generate and to manipulate and allow a rapid analysis of proteins. All these features make the Tris-Acetate-PAGE system a very helpful tool for protein analysis.
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Simultaneous electrophoretic analysis of proteins of very high and low molecular mass using Tris-Acetate polyacrylamide gels.
Electrophoresis, 2010Co-Authors: Monica Cubillos-rojas, Ramon Bartrons, Francesc Ventura, Fabiola Amair-pinedo, Irantzu Tato, Jose Luis RosaAbstract:To separate and analyze giant and small proteins in the same electrophoresis gel, we have used a 3-15% polyacrylamide gradient gel containing 2.6% of the crosslinker bisacrylamide and 0.2 M of Tris-Acetate buffer (pH 7.0). Samples were prepared in a sample buffer containing lithium dodecyl sulphate and were run in the gel described above using Tris-Tricine-SDS-sodium bisulfite buffer, pH 8.2, as electrophoresis buffer. Here, we show that this system can be successfully used for general applications of SDS-PAGE such as CBB staining and immunoblot. Thus, by using Tris-Acetate 3-15% polyacrylamide gels, it is possible to simultaneously analyze proteins, in the mass range of 10-500 kDa, such as HERC1 (532 kDa), HERC2 (528 kDa), mTOR (289 kDa), Clathrin heavy chain (192 kDa), RSK (90 kDa), S6K (70 kDa), beta-actin (42 kDa), Ran (24 kDa) and LC3 (18 kDa). This system is highly sensitive since it allows detection from as low as 10 microg of total protein per lane. Moreover, it has a good resolution, low cost, high reproducibility and allows for analysis of proteins in a wide range of weights within a short period of time. All these features together with the use of a standard electrophoresis apparatus make the Tris-Acetate-PAGE system a very helpful tool for protein analysis.
