The Experts below are selected from a list of 231 Experts worldwide ranked by ideXlab platform
Harry W. Jarrett - One of the best experts on this subject based on the ideXlab platform.
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DNA Affinity Chromatography of Transcription Factors: The Oligonucleotide Trapping Approach
Cell Biology, 2006Co-Authors: Suchareeta Mitra, Robert A Moxley, Harry W. JarrettAbstract:Publisher Summary This chapter describes the current oligonucleotide trapping method practiced in laboratories. A column is prepared with the single-stranded oligonucleotide ACACACA CAC attached to CNBr-activated Sepharose through an aminoalkyl linker. Another DNA is prepared, which has a double-stranded region containing the element bound by a transcription factor and additionally has a GTGTGTGTGT single-stranded tail. The probe is mixed with a cell extract containing a transcription factor to be purified along with salt and various competitors and surfactants, which improve selectivity. Chromatography is either by simply using gravity flow with a fraction collector or using a Bio-Rad Biologic LP Chromatograph. They can also be annealed by boiling 50 ml water in a 100-ml beaker, removing it from the heat, and adding the sealed tube containing the DNA. After allowing the beaker to cool for 30 min, the tube is removed to an ice bath. By adding different dilutions of nuclear extract to a gel shift, performed using a known amount of DNA, usually a total shift of the DNA can be obtained.
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Effect of the detergent Tween-20 on the DNA Affinity Chromatography of Gal4, C/EBPalpha, and lac repressor with observations on column regeneration.
Journal of chromatography. A, 2004Co-Authors: F Darlene Robinson, Robert A Moxley, Harry W. JarrettAbstract:C/EBPalpha, Gal4, and lac repressor, representing three different transcription factor homology families, were expressed as fusion proteins and used to characterize the effects of column aging, Mg2+, the nonionic detergent Tween-20, column loading, and bovine serum albumin on DNA-Affinity Chromatography. When lac-repressor-beta-galactosidase fusion protein is loaded onto a new DNA-Sepharose column, less elutes from a new column than one that has been used two or more times. Higher amounts of lac repressor, the Green Fluorescent Protein fusions with CAAT enhancer binding protein (C/EBPalpha) and Gal4, elute from the columns when 0.1% Tween-20 is added to the mobile phase. The amount of improvement found depends upon the transcription factor studied and the amount of the protein loaded on the column; lac repressor and Gal4 are eluted in higher amounts over a large range of protein loads while C/EBP shows the greatest effect at low protein loads. This detergent effect is seen when either Sepharose or silica is used for the stationary phase. Including bovine serum albumin in the mobile phase gives a similar though lesser improvement to that observed with Tween-20. Mg2+ or EDTA in the mobile phase gave similar Chromatography for C/EBP; since EDTA protects columns from DNAses, its inclusion in the mobile phase is preferred. After extended use, the DNA Affinity columns no longer bind transcription factors and this is not due to losses of DNA from the columns. Two simple methods (sodium dodecylsulfate and KSCN) were developed to regenerate such worn out columns.
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Effect of the detergent Tween-20 on the DNA Affinity Chromatography of Gal4, C/EBPα, and lac repressor with observations on column regeneration
Journal of Chromatography A, 2003Co-Authors: F Darlene Robinson, Robert A Moxley, Harry W. JarrettAbstract:C/EBPalpha, Gal4, and lac repressor, representing three different transcription factor homology families, were expressed as fusion proteins and used to characterize the effects of column aging, Mg2+, the nonionic detergent Tween-20, column loading, and bovine serum albumin on DNA-Affinity Chromatography. When lac-repressor-beta-galactosidase fusion protein is loaded onto a new DNA-Sepharose column, less elutes from a new column than one that has been used two or more times. Higher amounts of lac repressor, the Green Fluorescent Protein fusions with CAAT enhancer binding protein (C/EBPalpha) and Gal4, elute from the columns when 0.1% Tween-20 is added to the mobile phase. The amount of improvement found depends upon the transcription factor studied and the amount of the protein loaded on the column; lac repressor and Gal4 are eluted in higher amounts over a large range of protein loads while C/EBP shows the greatest effect at low protein loads. This detergent effect is seen when either Sepharose or silica is used for the stationary phase. Including bovine serum albumin in the mobile phase gives a similar though lesser improvement to that observed with Tween-20. Mg2+ or EDTA in the mobile phase gave similar Chromatography for C/EBP; since EDTA protects columns from DNAses, its inclusion in the mobile phase is preferred. After extended use, the DNA Affinity columns no longer bind transcription factors and this is not due to losses of DNA from the columns. Two simple methods (sodium dodecylsulfate and KSCN) were developed to regenerate such worn out columns.
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DNA Affinity Chromatography
Molecular Biotechnology, 2001Co-Authors: Priya Sethu Chockalingam, Luis A. Jurado, Harry W. JarrettAbstract:DNA-Affinity Chromatography has been used for the purification of DNA-binding proteins that control various cellular processes. There have been improvements in coupling methods and choice of supports over the years. The procedure for coupling 5′-aminoethyl-(dT)_18 to silica activated with N-hydroxysuccinimide and a carbodiimide has been described. Also, the cyanogen bromide mediated coupling of aminoethyl-(dT)_18 to Sepharose is described. Determination of (dT)_18-coupling to silica and Sepharose is by 5′ end-labeling an oligonucleotide containing a (dA)_18 stretch of sequence and determining how much hybridizes with the (dT)_18 support. Enzymatic synthesis of a double-stranded DNA-silica or Sepharose prevents modification of nucleotide bases. We have explained the use of DNA and RNA templates for template-directed enzymatic synthesis of Affinity columns. DNA-Affinity Chromatography is a powerful method with broad applicability and we are currently extending this technology for purifying transcription factors, polymerases, and nucleases.
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Temperature dependence of DNA Affinity Chromatography of transcription factors.
Analytical biochemistry, 2000Co-Authors: Harry W. JarrettAbstract:Abstract Oligonucleotides bound by the CAAT enhancer binding protein (C/EBP), the lactose repressor, and Gal4 were chemically coupled to cyanogen bromide-activated Sepharose and the temperature dependence of transcription factor Chromatography was characterized. Each transcription factor was applied to the appropriate column and eluted using a salt gradient at several temperatures. Each transcription factor showed a unique behavior. As temperature was increases, less salt was required to elute C/EBP, more salt was required to elute lac repressor, while Gal4 showed a biphasic dependency with the amount of salt first decreasing between 4 and 19°C and then increasing above 19°C. This temperature dependence is not due to protein or DNA unfolding but rather is a property of complex formation. By loading a column, washing it at a permissive temperature, and then rapidly changing the column temperature, highly selective elution can be obtained. The thermodynamics of this temperature effect are different for the binding of specific and nonspecific DNA sequences, making Chromatography at different temperatures a potentially important way of purifying transcription factors.
Eleanor N. Fish - One of the best experts on this subject based on the ideXlab platform.
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Genomic DNA Affinity Chromatography
Methods in molecular medicine, 2005Co-Authors: Jyothi Kumaran, Eleanor N. FishAbstract:Cytokines elicit responses in target cells by inducing changes in gene expression. For interferons (IFNs), this involves receptor-mediated activation of specific transcription factors, which then translocate into the nucleus to bind to cognate gene elements in the promoters of IFN-inducible genes. The prototypic IFN-inducible transcription factors are the signal transducer and activator of transcription (STAT) proteins. IFN-receptor interactions invoke Janus kinase activation via phosphorylation events, which in turn leads to the recruitment and phosphorylation of STAT proteins on tyrosine residues. Activated STATs then dimerize to form STAT complexes. IFNs-alpha/beta will activate STAT-1, STAT-2, STAT-3 ,and STAT-5, whereas IFN-gamma will predominantly activate STAT-1. In this chapter, we describe a procedure to identify IFN-inducible deoxyribonucleic acid (DNA) binding factors independently of any knowledge of their target DNA sequences. This procedure permits the identification of IFN-inducible STAT complexes as well as any other IFN-inducible DNA binding factors. This biochemical technique uses genomic DNA Affinity Chromatography to isolate DNA binding factors from IFN-inducible cytoplasmic or nuclear extracts.
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Application of Genomic DNA Affinity Chromatography Identifies Multiple Interferon-α-regulated Stat2 Complexes
Journal of Biological Chemistry, 1996Co-Authors: Julien J. Ghislain, Eleanor N. FishAbstract:Abstract Interferon-α (IFN-α)-induced signal transduction is mediated by the phosphorylation-activation of the signal transducer and activator of transcription (STAT) proteins Stat1, Stat2, and Stat3. Previous studies have shown that these activated STATs dimerize to form four distinct STAT complexes which translocate to the nucleus and activates transcription by binding to specific promoter elements. The interferon-stimulated gene factor-3 (ISGF3) consists of Stat2 and Stat1 heterodimers in association with a DNA-binding protein, p48, that binds to the interferon stimulated response element. Homo- and heterodimers of Stat1 and Stat3 bind to the palindromic interferon response element (pIRE). In this report we demonstrate the utility of a biochemical procedure that we have developed, based on genomic DNA Affinity Chromatography, for the identification of IFN-α-induced STAT complexes. Using this approach, we identified ISGF3-independent Stat2-containing STAT complexes. Results from the analysis of Stat2 complexes in the electrophoretic mobility shift assay were consistent with genomic DNA Affinity Chromatography results and identified a Stat2:1 complex that binds with low Affinity to the pIRE of the interferon regulatory factor-1 gene. Immunoprecipitation studies of Stat2 revealed an IFN-α dependent co-precipitation of both Stat1 and Stat3. Taken together, our results suggest that IFN-α activates, in addition to ISGF3, other Stat2-containing STAT complexes, one of which binds to an element related to the interferon regulatory factor-1 pIRE.
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Application of Genomic DNA Affinity Chromatography Identifies Multiple Interferon-α-regulated Stat2 Complexes
Journal of Biological Chemistry, 1996Co-Authors: Julien J. Ghislain, Eleanor N. FishAbstract:Abstract Interferon-α (IFN-α)-induced signal transduction is mediated by the phosphorylation-activation of the signal transducer and activator of transcription (STAT) proteins Stat1, Stat2, and Stat3. Previous studies have shown that these activated STATs dimerize to form four distinct STAT complexes which translocate to the nucleus and activates transcription by binding to specific promoter elements. The interferon-stimulated gene factor-3 (ISGF3) consists of Stat2 and Stat1 heterodimers in association with a DNA-binding protein, p48, that binds to the interferon stimulated response element. Homo- and heterodimers of Stat1 and Stat3 bind to the palindromic interferon response element (pIRE). In this report we demonstrate the utility of a biochemical procedure that we have developed, based on genomic DNA Affinity Chromatography, for the identification of IFN-α-induced STAT complexes. Using this approach, we identified ISGF3-independent Stat2-containing STAT complexes. Results from the analysis of Stat2 complexes in the electrophoretic mobility shift assay were consistent with genomic DNA Affinity Chromatography results and identified a Stat2:1 complex that binds with low Affinity to the pIRE of the interferon regulatory factor-1 gene. Immunoprecipitation studies of Stat2 revealed an IFN-α dependent co-precipitation of both Stat1 and Stat3. Taken together, our results suggest that IFN-α activates, in addition to ISGF3, other Stat2-containing STAT complexes, one of which binds to an element related to the interferon regulatory factor-1 pIRE.
Julien J. Ghislain - One of the best experts on this subject based on the ideXlab platform.
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Application of Genomic DNA Affinity Chromatography Identifies Multiple Interferon-α-regulated Stat2 Complexes
Journal of Biological Chemistry, 1996Co-Authors: Julien J. Ghislain, Eleanor N. FishAbstract:Abstract Interferon-α (IFN-α)-induced signal transduction is mediated by the phosphorylation-activation of the signal transducer and activator of transcription (STAT) proteins Stat1, Stat2, and Stat3. Previous studies have shown that these activated STATs dimerize to form four distinct STAT complexes which translocate to the nucleus and activates transcription by binding to specific promoter elements. The interferon-stimulated gene factor-3 (ISGF3) consists of Stat2 and Stat1 heterodimers in association with a DNA-binding protein, p48, that binds to the interferon stimulated response element. Homo- and heterodimers of Stat1 and Stat3 bind to the palindromic interferon response element (pIRE). In this report we demonstrate the utility of a biochemical procedure that we have developed, based on genomic DNA Affinity Chromatography, for the identification of IFN-α-induced STAT complexes. Using this approach, we identified ISGF3-independent Stat2-containing STAT complexes. Results from the analysis of Stat2 complexes in the electrophoretic mobility shift assay were consistent with genomic DNA Affinity Chromatography results and identified a Stat2:1 complex that binds with low Affinity to the pIRE of the interferon regulatory factor-1 gene. Immunoprecipitation studies of Stat2 revealed an IFN-α dependent co-precipitation of both Stat1 and Stat3. Taken together, our results suggest that IFN-α activates, in addition to ISGF3, other Stat2-containing STAT complexes, one of which binds to an element related to the interferon regulatory factor-1 pIRE.
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Application of Genomic DNA Affinity Chromatography Identifies Multiple Interferon-α-regulated Stat2 Complexes
Journal of Biological Chemistry, 1996Co-Authors: Julien J. Ghislain, Eleanor N. FishAbstract:Abstract Interferon-α (IFN-α)-induced signal transduction is mediated by the phosphorylation-activation of the signal transducer and activator of transcription (STAT) proteins Stat1, Stat2, and Stat3. Previous studies have shown that these activated STATs dimerize to form four distinct STAT complexes which translocate to the nucleus and activates transcription by binding to specific promoter elements. The interferon-stimulated gene factor-3 (ISGF3) consists of Stat2 and Stat1 heterodimers in association with a DNA-binding protein, p48, that binds to the interferon stimulated response element. Homo- and heterodimers of Stat1 and Stat3 bind to the palindromic interferon response element (pIRE). In this report we demonstrate the utility of a biochemical procedure that we have developed, based on genomic DNA Affinity Chromatography, for the identification of IFN-α-induced STAT complexes. Using this approach, we identified ISGF3-independent Stat2-containing STAT complexes. Results from the analysis of Stat2 complexes in the electrophoretic mobility shift assay were consistent with genomic DNA Affinity Chromatography results and identified a Stat2:1 complex that binds with low Affinity to the pIRE of the interferon regulatory factor-1 gene. Immunoprecipitation studies of Stat2 revealed an IFN-α dependent co-precipitation of both Stat1 and Stat3. Taken together, our results suggest that IFN-α activates, in addition to ISGF3, other Stat2-containing STAT complexes, one of which binds to an element related to the interferon regulatory factor-1 pIRE.
V. Bouriotis - One of the best experts on this subject based on the ideXlab platform.
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Sequence-specific DNA Affinity Chromatography: Application of a group-specific adsorbent for the isolation of restriction endonucleases
Journal of chromatography, 1993Co-Authors: Charalambos Pozidis, G. Vlatakis, V. BouriotisAbstract:Abstract The use of sequence-specific DNA Affinity adsorbents for the isolation of restriction endonucleases Eco RI and Sph I to near homogeneity has been reported. However, the high cost of these adsorbents is a limiting factor for their wider application. This paper reports the application of sequence-specific DNA Affinity ligands containing recognition sequences for 34 restriction endonucleasesas group-specific ligands in the isolation of restriction endonucleases. Crude samples of six restriction endonucleases, namely Bsh FI, Bam HI, Sma I, Sac II, Pvu II and Sal I, were shown to bind to these adsorbents and could be eluted at different KCl concentrations. High purification factors and recoveries were obtained. Restriction endonuclease Bsh FI, an isoschizomer of Hae III, from the microorganism Bacillus sphaericus was purified to near homogeneity employing a two-step procedure which involves DNA-cellulose Chromatography and oligonucleotide- ligand Affinity Chromatography. The enzyme exists as a monomer with an apparent relative molecular mass of 34 000 as determined by both sodium dodecyl sulphate-polyacrylamide gel electrophoresis and size-exclusion Chromatography.
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Sequence-specific DNA Affinity Chromatography: Application to the purification of EcoRI and SphI
Analytical biochemistry, 1991Co-Authors: G. Vlatakis, V. BouriotisAbstract:Several rapid and effective methods have been described to obtain restriction endonucleases suitable for commercial exploitation. However, lengthy and laborious protocols have been necessary to obtain homogeneous enzymes. We now report the use of sequence-specific DNA Affinity Chromatography to purify restriction endonucleases to near homogeneity. Restriction endonucleases EcoRI and SphI from the microorganisms Escherichia coli RY 13 and Streptomyces phaeochromogenes, respectively, were purified to near homogeneity employing a two-step procedure which involves DNA-cellulose Chromatography and oligonucleotide-ligand Affinity Chromatography.
Brian Stevenson - One of the best experts on this subject based on the ideXlab platform.
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identification of novel DNA binding proteins using DNA Affinity Chromatography pull down
Current protocols in microbiology, 2012Co-Authors: Brandon L Jutras, Ashutosh Verma, Brian StevensonAbstract:This units presents methods through which one may isolate and identify novel bacterial DNA-binding proteins. Briefly, the DNA sequence of interest is affixed to beads, and then incubated with bacterial cytoplasmic extract. Washes with buffers containing nonspecific DNA and low-salt concentrations will remove non-adhering and low-specificity DNA-binding proteins, while subsequent washes with higher salt concentrations will elute more specific DNA-binding proteins. Eluted proteins may then be identified by standard proteomic techniques.
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Identification of novel DNA-binding proteins using DNA-Affinity Chromatography/pull down.
Current protocols in microbiology, 2012Co-Authors: Brandon L Jutras, Ashutosh Verma, Brian StevensonAbstract:This units presents methods through which one may isolate and identify novel bacterial DNA-binding proteins. Briefly, the DNA sequence of interest is affixed to beads, and then incubated with bacterial cytoplasmic extract. Washes with buffers containing nonspecific DNA and low-salt concentrations will remove non-adhering and low-specificity DNA-binding proteins, while subsequent washes with higher salt concentrations will elute more specific DNA-binding proteins. Eluted proteins may then be identified by standard proteomic techniques.
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Current Protocols in Microbiology - Identification of novel DNA-binding proteins using DNA-Affinity Chromatography/pull down.
Current Protocols in Microbiology, 2012Co-Authors: Brandon L Jutras, Ashutosh Verma, Brian StevensonAbstract:This units presents methods through which one may isolate and identify novel bacterial DNA-binding proteins. Briefly, the DNA sequence of interest is affixed to beads, and then incubated with bacterial cytoplasmic extract. Washes with buffers containing nonspecific DNA and low-salt concentrations will remove non-adhering and low-specificity DNA-binding proteins, while subsequent washes with higher salt concentrations will elute more specific DNA-binding proteins. Eluted proteins may then be identified by standard proteomic techniques.