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Results for “Electrophoretic Mobility Shift Assay”

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At least 19 recordsLinked to original sources

PubMed2000

A fluorescence based non-radioactive electrophoretic mobility shift assay.

Electrophoretic mobility shift assay (EMSA) or gel shift assay is one of the most powerful methods for studying protein-DNA interactions. Typically, 32P-labeled DNA probes containing the sequence bound by the protein of interest are used in EMSA (rEMSA). Although rEMSA is sensitive and practicable, it relies on the handling of hazardous radioisotopes, and does not easily allow quantification. We developed a non-radioactive procedure using fluorescence (Cyano dye Cy5) labeled oligodeoxynucleotide duplexes as specific probes (fEMSA) and an automatic DNA sequencer for analysis. Testing different DNA-binding proteins (restriction endonuclease EcoRII, transcription factor NFkappaB and it's subunit p50) the results in fEMSA and rEMSA are similar in regard to quality, reproducibility, and sensitivity. fEMSA allows a semiquantitative screening of large amounts of samples for specific DNA binding activities and is, therefore, a high throughput technology for semiquantitative analysis of DNA-protein interaction.

Carbocyanines↗

PubMed1998

Examination of DNA-binding activity of neuronal transcription factors by electrophoretical mobility shift assay.

Electrophoretical mobility shift assay (EMSA) is a simple, rapid, and highly sensitive technique for detection of single- or double-stranded DNA-binding proteins such as transcription factors in crude nuclear extracts (F.M. Ausubel, R. Brent, R.E. Kingston, D. D. Moore, J.G. Seidman, J.A. Smith, K. Struhl (Eds.), Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley-Interscience, 1989, pp. 12.0.1-12.2.10 [1]; J. Carey, Gel Retardation. Methods Enzymol., 208 (1991) 103-117 [2]). By using this technique, it is possible to quantify the abundance, relative affinity and binding specificity of DNA-binding proteins. Since proteins which bind specifically to radiolabeled DNA probes retard the mobility of the probe during electrophoresis (it also called gel retardation assay), discrete bands correspond to the individual DNA-protein complexes. Furthermore, EMSA allows one to determine which member(s) of a certain protein family are included in the DNA-protein complex by means of specific antibodies raised against the DNA-binding protein (supershift assay).

Animals↗

PubMed2001

2,4,4'-trichlorobiphenyl increases STAT5 transcriptional activity.

The promoting effects of polychlorinated biphenyls (PCBs) have been studied extensively in a variety of two-stage carcinogenesis models. However, the molecular mechanisms responsible for the promotion effects of PCBs have not been elucidated. We measured the effect of PCBs on DNA-binding proteins involved in cell proliferation and transformation. Male Sprague-Dawley rats were injected intraperitoneally with mono-, di-, tri-, tetra-, or hexachlorobiphenyls (300 micromol/kg/d) each day for 4 d and killed 4 h after the last injection. To detect alterations in nuclear proteins that could explain the tumor-promoter activity of PCBs, liver nuclear extracts were analyzed by electrophoretic mobility shift assays. Electrophoretic mobility shift assay analysis of signal transducers and activators of transcription (STAT)-binding activity to a consensus gamma-interferon-activated sequence (GAS) element was compared in liver nuclear extracts from treated rats. STAT-binding activity was eightfold to tenfold higher in nuclear extracts from animals treated with 2,4,4'-trichloro- (PCB 28) and 2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153). Analysis of the protein complex binding to the GAS element, with antibodies specific for STAT3, STAT5, and STAT6, indicated that the protein complex was made up of STAT5 and STAT6 proteins. HepG2 cells transiently transfected with a luciferase reporter gene construct containing many STAT5 binding sites were treated with PCB 28 and PCB 153. PCB 28 stimulated a greater than 25-fold increase in luciferase activity at the highest concentration tested, 1.0 microg/mL. However, enhanced luciferase activity did not occur with PCB 153 treatment. 4-Chlorobiphenyl (PCB 3), PCB 28, and PCB 153 treatment of Sprague-Dawley rats resulted in a large increase in protein binding to a consensus activated protein-1 (AP-1) element. However, 3,4-dichlorobiphenyl (PCB 12) and 3,3',4,4'-tetrachlorobiphenyl (PCB 77) treatments did not increase AP-1 transcription activity. Further analysis of the proteins binding to the AP-1 consensus sequence with antibodies specific for c-fos, junD, and junB indicated that the protein composition consists of junD proteins. These data showed functional differences between noncoplanar and coplanar PCBs with respect to STAT activation and AP-1-DNA binding.

Animals↗

PubMed1995

The transcription factor, Nm23H2, binds to and activates the translocated c-myc allele in Burkitt's lymphoma.

We have identified an in vivo footprint over the PuF site on the translocated c-myc allele in Burkitt's lymphoma cells. The PuF site on the silent normal c-myc allele was unoccupied. We demonstrated by electrophoretic mobility shift assay, electrophoretic mobility shift assay with antibody, UV cross-linking followed by SDS-gel electrophoresis, and Western analysis that Nm23H2 in B cell nuclear extracts bound to the c-myc PuF site. Transfection experiments with c-myc promoter constructs in both DHL-9 and Raji cells revealed that the PuF site functioned as a positive regulatory element in B cells with a drop in activity with mutation of this site. Access to this site is blocked in the normal silent c-myc allele; these data suggest that the Nm23H2 protein is involved in deregulation of the translocated c-myc allele in Burkitt's lymphoma cells.

Alleles↗

PubMed2004

The transcription factor CCAAT-binding factor CBF/NF-Y and two repressors regulate the core promoter of the human pro-alpha3(V) collagen gene (COL5A3).

To elucidate the mechanisms underlining alpha3(V) collagen chain expression, we performed an initial analysis of the structure and function of the core promoter of the human COL5A3 gene. The core promoter, which lacks a typical TATA motif and has a high GC content, was defined within the -129 bp immediately upstream from the major transcription start site by transient transfection experiments. In this region, we identified four DNA-protein complexes, named A, B, C, and D, by a combination of DNase I footprinting and electrophoretic mobility shift assays. Electrophoretic mobility shift assays using mutant oligonucleotide revealed that the complexes A, B, C, and D bind to -122 to -117, the -101 to -96, the -83 to -78, and the -68 to -57 bp, respectively. The competition assays using consensus oligonucleotides and supershift assays with specific antibodies showed that complex A consists of CBF/NF-Y. In a chromatin immunoprecipitation assay, CBF/NF-Y protein directly bound to this region, in vivo. Functional analysis showed that CBF/NF-Y activated the gene, whereas the proteins of complexes B and C repressed its activity. Furthermore, overexpression of a mutant form of the CBF-B/NF-YA subunit, which forms CBF/NF-Y with CBF-A/NF-YB and CBF-C/NF-YC subunits, inhibited promoter activity.

Amino Acid Motifs↗

PubMed1997

Bacterial protease Lon is a site-specific DNA-binding protein.

The product of the Escherichia coli lon gene is the ATP-dependent Lon protease. Lon contributes to the regulation of several important cellular functions, including radiation resistance, cell division, filamentation, capsular polysaccharide production, lysogeny of certain bacteriophages, and proteolytic degradation of certain regulatory and abnormal proteins. Lon homologues are also found in several widely divergent bacteria, as well as in the mitochondria of yeast and humans. E. coli Lon has long been known to bind to DNA, but this interaction has not been further characterized and has generally been assumed to be nonspecific. We now demonstrate that E. coli Lon can bind to a TG-rich DNA promoter element in a sequence-specific manner. This finding is based on the results of experiments employing SouthWestern blotting, protein purification, "shift-shift" electrophoretic mobility shift assays, electrophoretic mobility shift assays using in vitro transcribed and translated Lon, and DNase footprinting. Site-specific DNA binding is likely to be an additional important biochemical characteristic of the multifaceted Lon protease.

ATP-Dependent Proteases↗

PubMed2006

Electrophoretic mobility shift assays to study protein binding to damaged DNA.

The electrophoretic mobility shift assay (EMSA) can be used to identify proteins that bind specifically to damaged DNA. EMSAs detect the presence of key DNA repair proteins, such as ultraviolet (UV)-damaged DNA binding protein, which is involved in nucleotide excision repair, and Ku and DNA-PKcs, which are involved in double-strand break repair. This chapter describes EMSA protocols for detecting proteins that bind to UV-damaged DNA, cisplatin-damaged DNA, and DNA ends. The chapter also describes variations of the EMSA that can be used to obtain additional information about these important proteins. The variations include the reverse EMSA, which can detect binding of 35S-labeled protein to damaged DNA, and the antibody supershift assay, which can define the composition of protein-DNA complexes.

Cell Extracts↗

PubMed2003

Transcription factor binding study by capillary zone electrophoretic mobility shift assay.

Regulation of gene expression through interaction of proteins with specific DNA sequences is a central issue in functional genomics. Capillary electrophoretic mobility shift assay is an efficient novel method for the investigation of sequence specific protein-DNA interactions, allowing rapid and sensitive quantification of the complex formation. In this paper, we present a pilot study on capillary zone electrophoretic mobility shift assay (CZEMSA) to investigate the interaction between the transcription factors of HeLa nuclear extract and Sp1-specific fluorescein-labeled oligonucleotide, using the unlabeled probe as competitor. The mobility shift assay was accomplished by CZE in coated capillaries without polymeric buffer additives. Specificity of the DNA protein complex formation was verified by competition experiments, as well as by supershift assay with an anti-Sp1 antibody. The applied electric field strength did not affect the stability of DNA-protein complex during the electrophoretic analysis, allowing rapid identification and quantification of the protein DNA interaction. A practical application to study the interaction between Oryza sativa MADS-box transcription factor 4 (OsMADS4) and its consensus sequence is also reported.

Base Sequence↗

PubMed1995

Alternative to polyacrylamide gels improves the electrophoretic mobility shift assay.

In this paper we outline a simplified protocol for the electrophoretic mobility shift assay utilizing TreviGel 500, a nontoxic alternative to polyacrylamide. The TreviGel 500 matrix combines the strength and resolution of polyacrylamide with the simplicity and flexibility of agarose in the casting of gels. Therefore, this method provides a simple, rapid and nontoxic alternative to current protocols for the investigation of protein: DNA interactions.

Base Sequence↗

PubMed2005

Chemiluminescence-based electrophoretic mobility shift assay of RNA-protein interactions: application to binding of viral capsid proteins to RNA.

A chemiluminescence electrophoretic mobility shift assay was introduced for the study of RNA-protein interactions that include association of genomic RNA and viral capsid proteins. Binding of the capsid protein of Venezuelan equine encephalitis virus (VEEV) to several types of RNA was used as a model system to test the application of the method. The effects of RNA secondary structures and the significance of electrostatic interaction on binding were identified. This method may have wide application to the study of RNA-protein interactions.

Capsid Proteins↗

PubMed2002

Design of a fluorescent electrophoretic mobility shift assay improved for the quantitative and multiple analysis of protein-DNA complexes.

We describe a protocol for the fluorescent electrophoretic mobility shift assay improved for the quantitative analysis of protein-DNA complexes. Fluorescent-labeled oligonucleotide probes incubated with nuclear proteins were followed by electrophoresis. The signals for protein-DNA complexes were measured and normalized with fluorescent-labeled marker using fragment analysis software. This assay proved reliable measurement and multiple detection of DNA binding proteins.

DNA↗

PubMed2003

Suppression of inflammatory cytokine production by carbon monoxide involves the JNK pathway and AP-1.

The stress-inducible protein heme oxygenase-1 provides protection against oxidative stress and modulates pro-inflammatory cytokines. As the sepsis syndrome results from the release of pro-inflammatory mediators, we postulated that heme oxygenase-1 and its enzymatic product CO would protect against lethality in a murine model of sepsis. Mice treated with a lethal dose of lipopolysaccharide (LPS) and subsequently exposed to inhaled CO had significantly better survival and lower serum interleukin (IL)-6 and IL-1beta levels than their untreated counterparts. In vitro, mouse macrophages exposed to LPS and CO had significantly attenuated IL-6 production; this effect was concentration-dependent and occurred at a transcriptional level. The same effect was seen with increased endogenous CO production through overexpression of heme oxygenase-1. Mutation within the AP-1-binding site in the IL-6 promoter diminished the effect of CO on promoter activity, and treatment of macrophages with CO decreased AP-1 binding in an electrophoretic mobility shift assay. Electrophoretic mobility supershift assay indicated that the JunB, JunD, and c-Fos components of AP-1 were particularly affected. Upstream of AP-1, CO decreased JNK phosphorylation in murine macrophages and lung endothelial cells. Mice deficient in the JNK pathway had decreased serum levels of IL-6 and IL-1beta in response to LPS compared with control mice, and no effect of CO on these cytokine levels was seen in Jnk1 or Jnk2 genedeleted mice. In summary, these results suggest that CO provides protection in a murine model of sepsis through modulation of inflammatory cytokine production. For the first time, the effect of CO is shown to be mediated via the JNK signaling pathway and the transcription factor AP-1.

Animals↗

PubMed2000

Electrophoretic mobility shift assay for the detection of specific DNA-protein complex in nuclear extracts from the cultured cells and frozen autopsy human brain tissue.

The electrophoretic mobility shift assay (EMSA) is generally used to study the interaction of transcription factors to specific DNA sequences. The preparation of high quality nuclear extracts is an important step before performing the assay. Here we describe a rapid method for the isolation of good-quality DNA-binding proteins from cultured cell lines and autopsy tissue samples from the human brain. The 'rapid method' (RM) utilizes the low salt/detergent lysis steps followed by high salt extraction of nuclei. To test and compare the activity of nuclear extracts prepared by the standard and 'rapid' methods for its ability to form the specific DNA-protein complex, EMSA was carried out with three different oligonucleotide probes: AP1, NF-kappaB and URE. A comparative study indicates that the capacity to form the specific DNA-protein complex with these oligonucleotide probes by standard and RM nuclear extracts was very similar. Each nuclear extract formed the corresponding DNA-protein complex, the specificity of which was checked by the competition experiment. In some cases unspecific bands were observed and which were present in nuclear extracts from both preparations. Thus the simplicity of the 'rapid method' permits the preparation of nuclear extracts from several cell lines and tissue samples at the same time at much shorter time than the 'standard' method without compromising the DNA-binding activity. The RM can be applied to determine the cell type or tissue specificity of transcription factors in an efficient, economical and consistent manner.

5' Untranslated Regions↗

PubMed1997

Quantitating oligonucleotide affinities for duplex DNA: footprinting vs electrophoretic mobility shift assays.

Determining the affinities of oligonucleotides for duplex DNA is an important analytical problem that arises during the design of potential gene repressors based on triple helix recognition. Quantitative DNa-seI footprinting assays (QDFA) offer a rigorous technique for this purpose. Electrophoretic mobility shift assays (EMSA) have proven to be simpler and more rapid. Although EMSA can separate triplex and duplex complexes, there is concern that this technique does not afford as rigorous an equilibrium measurement as is provided by QDFA. We show that QDFA and EMSA techniques provide Kd estimates that agree within one order of magnitude under common experimental conditions. Agreement is best in buffers with low concentrations of monovalent cations. Surprisingly, EMSA appears to slightly overestimate triplex stabilities relative to QDFA in the presence of physiological concentrations of monovalent cations (100 mM). Under these conditions, agreement between the techniques can be improved by quenching EMSA samples with excess unlabeled competitor duplex just prior to gel loading. The data suggest that EMSA can provide results in reasonable agreement with QDFA and offer some insight into sources of deviation between the two methods.

DNA↗

PubMed2006

Electrophoretic mobility shift assay on poly(ethylene glycol)-modified glass microchips for the study of estrogen responsive element binding.

The binding of estrogen receptor (ER) to estrogen response element (ERE) is essential for genomic pathways of estrogens and gel-based electrophoretic mobility shift assay (EMSA) is commonly used for analyzing ERE binding. Gel-based EMSA, however, requires the use of hazard radio isotopes and they are slow, labor-intensive and difficult to quantify. Here, we present quantitative affinity assays based on microchip electrophoresis using PEG-modified glass microchannels, which bear neutral surfaces against the adsorption of acidic DNA molecules and basic ER proteins. We first demonstrated the feasibility of the method by measuring binding constants of recombinant ERalpha and ERbeta with a consensus ERE sequence (cERE, 5'-GGTCAGAGTGACC-3') as well as with an ERE-like sequence (ERE 1576, 5'-GACCGGTCAGCGGACTCAC-3'). Changes in mobility as a function of protein-DNA molar ratios were plotted and the dissociation constants were determined based on non-linear curve fitting. The minimum amount of ER proteins required for one assay was around 0.2 ng and the run time for one chip analysis was less than 2 min. We further measured the estrogenic compound-mediated dissociation constants with recombinant ER proteins as well as with the extracted ERbeta from treated and untreated A549 bronchioloalveolar carcinoma cells. Dissociation constants determined by this method agree with the fact that agonist compounds such as 17beta-estradiol (1.70 nM), diethylstilbestrol (0.14 nM), and genistein (0.80 nM) assist ERE binding by decreasing the constants; while antagonist compounds such as testosterone (140.4 nM) and 4-hydroxytamoxifen (10.5 nM) suppress the binding by increasing the dissociation constant.

Base Sequence↗

PubMed2005

Oligonucleotide trapping method for transcription factor purification systematic optimization using electrophoretic mobility shift assay.

Oligonucleotide trapping, where a transcription factor-DNA response element complex is formed in solution and then recovered (trapped) on a column, was optimized for the purification of CAAT/enhancer binding protein (C/EBP) from rat liver nuclear extract. Electrophoretic mobility shift assays (EMSAs) with ACEP24(GT)5 oligonucleotide, containing the CAAT element, was used to estimate thebinding affinity and concentration of C/EBP in the nuclear extract and then low concentrations of protein and oligonucleotide, which favor specific binding, were used for all further experiments. Also using EMSA, the highest concentrations of competitors, which inhibit non-specific binding but do not inhibit oligonucleotide binding by C/EBP, were determined to be 932 nM T18 (single-stranded DNA), 50 ng/ml heparin (non-DNA competitor), and 50 microg/ml poly(dI:dC) (duplex DNA). Inclusion of 0.1% Tween-20 improved DNA binding. For complex formation, 110 microg nuclear extract was diluted to 0.2 nM C/EBP (apparent Kd of C/EBP) and 1.34 nM ACEP24(GT)5 was added, along with Tween-20 and the competitors. After incubation, the complex was trapped by annealing the (GT)5 tail of the C/EBP-[ACEP24(GT)5] complex to an (AC)5-Sepharose column under flow at 4 degrees C. The column was washed with 0.4 M NaCl and the protein eluted with 1.2 M NaCl. The purification typically resulted in two proteins of apparent molecular mass 32000 and 38000. The smaller one, the major product, was identified to be C/EBP-alpha. The yield was 2.1 microg (66 pmol) of purified C/EBP-alpha p32. This systematic approach to oligonucleotide trapping is generally applicable for the purification of other transcription factors.

Base Sequence↗

PubMed2006

The identification of nucleic acid-interacting proteins using a simple proteomics-based approach that directly incorporates the electrophoretic mobility shift assay.

Proteins that interact with nucleic acids are central to numerous cellular processes, and their continuing characterization represents one of the foremost challenges in the postgenomic era. Here we describe a simple proteomics-based approach for the identification by mass spectrometry of proteins in crude extracts that interact with nucleic acids. It incorporates the electrophoretic mobility shift assay and is based on the finding that when a protein forms a complex with nucleic acid its electrophoretic mobility is affected as well as that of the nucleic acid. Our method should greatly reduce and in some cases may even eliminate the need for extensive protein purification and as such should contribute significantly to the functional annotation of the proteome. Furthermore it requires no prior knowledge of the molecular mass, quaternary structure, or pI of the interacting protein. Proof of principle is demonstrated using a recently discovered transcription factor; however, the approach should also have application in the identification of proteins that interact with RNA.

Amino Acid Sequence↗

PubMed2003

A sensitive two-color electrophoretic mobility shift assay for detecting both nucleic acids and protein in gels.

DNA-binding proteins are key to the regulation and control of gene expression, replication and recombination. The electrophoretic mobility shift assay (or gel shift assay) is considered an essential tool in modern molecular biology for the study of protein-nucleic acid interactions. As typically implemented, however, the technique suffers from a number of shortcomings, including the handling of hazardous (32)P-labeled DNA probes, and difficulty in quantifying the amount of DNA and especially the amount of protein in the gel. A new detection method for mobility-shift assays is described that represents a significant improvement over existing techniques. The assay is fast, simple, does not require the use of radioisotopes and allows independent quantitative determination of: (i) free nucleic acid, (ii) bound nucleic acid, (iii) bound protein, and (iv) free protein. Nucleic acids are detected with SYBR Green EMSA dye, while proteins are subsequently detected with SYPRO Ruby EMSA dye. All fluorescence staining steps are performed after the entire gel-shift experiment is completed, so there is no need to prelabel either the DNA or the protein and no possibility of the fluorescent reagents interfering with the protein-nucleic acid interactions. The ability to independently quantify each molecular species allows more rigorous data analysis methods to be applied, especially with respect to the mass of protein bound per nucleic acid.

DNA↗