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lihaoexe銀蟲 (小有名氣)
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[交流]
請教EMSA的有關(guān)問題 已有2人參與
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由于我們實驗室之前沒有做過EMSA，有很多問題不懂，請教大家 1.做丙烯酰胺電泳可以用做SDS-PAGE的設(shè)備嗎？ 2.探針用什么標(biāo)記比較好啊，比較簡單一點的 3.大家都用哪個公司的試劑盒啊 如果能提供詳細(xì)的protocol那就更好了  |
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qingfengwu
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protocol
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lihaoexe(金幣+3,VIP+0): 11-27 16:09
amisking(金幣+2,VIP+0): 11-27 17:38
lihaoexe(金幣+3,VIP+0): 11-27 16:09
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Introduction to the EMSA (Gel Shift) Technique The interaction of proteins with DNA is central to the control of many cellular processes including DNA replication, recombination and repair, transcription, and viral assembly. One technique that is central to studying gene regulation and determining protein  NA interactions is the electrophoretic mobility shift assay (EMSA).The EMSA technique is based on the observation that protein NA complexes migrate more slowly than free DNA molecules when subjected to non-denaturing polyacrylamide or agarose gel electrophoresis.1,2 Because the rate of DNA migration is shifted or retarded upon protein binding, the assay is also referred to as a gel shift or gel retardation assay. Until conception of the EMSA by Fried and Crothers3 and Garner and Revzin4, proteinNA interactions were studied primarily by nitrocellulose filter-binding assays.5An advantage of studying DNA:protein interactions by an electrophoretic assay is the ability to resolve complexes of different stoichiometry or conformation. Another major advantage for many applications is that the source of the DNA-binding protein may be a crude nuclear or whole cell extract rather than a purified preparation. Gel shift assays can be used qualitatively to identify sequence-specific DNA-binding proteins (such as transcription factors) in crude lysates and, in conjunction with mutagenesis, to identify the important binding sequences within a given gene’s upstream regulatory region. EMSAs can also be utilized quantitatively to measure thermodynamic and kinetic parameters.3,4,6,7 The ability to resolve protein NA complexes depends largely upon the stability of the complex during the brief time (approximately one minute) it is migrating into the gel. Sequence-specific interactions are transient and are stabilized by the relatively low ionic strength of the electrophoresis buffer used. Upon entry into the gel, protein complexes are quickly resolved from free DNA, in effect freezing the equilibrium between bound and free DNA. In the gel, the complex may be stabilized by “caging” effects of the gel matrix, meaning that if the complex dissociates, its localized concentration remains high, promoting prompt reassociation.3,6 Therefore, even labile complexes can often be resolved by this method.Critical EMSA Reaction Parameters Target DNA Typically, linear DNA fragments containing the binding sequence(s) of interest are used in EMSAs. If the target DNA is short (20-50 bp) and well defined, complementary oligonucleotides bearing the specific sequence can be synthesized, purified by gel or HPLC, and annealed to form a duplex. Often, a protein NA interaction involves the formation of a multiprotein complex requiring multiple protein binding sequences. In this situation, longer DNA fragments are used to accommodate assembly of multiprotein complexes. If the sequence is larger (100-500 bp), the DNA source is usually a restriction fragment or PCR product obtained from a plasmid containing the cloned target sequence. ProteinNA complexes formed on linear DNA fragments result in the characteristic retarded mobility in the gel. However, if circular DNA is used (e.g., minicircles of 200-400 bp), the proteinNA complex may actually migrate faster than the free DNA. Gel shift assays are also good for resolving altered or bent DNA conformations that result from the binding of certain protein factors. Gel shift assays need not be limited to DNA:protein interactions. RNA:protein interactions9,10 as well as peptide:protein interactions11 have also been studied using the same electrophoretic principle.(back to top) Labeling and Detection If large quantities of DNA are used in EMSA reactions, the DNA bands can be visualized by ethidium bromide staining. However, it is usually preferable to use low concentrations of DNA, requiring the DNA to be labeled before performing the experiment. Traditionally, DNA is radiolabeled with 32P by incorporating an [-32P]dNTP during a 3´ fill-in reaction using Klenow fragment or by 5´ end labeling using [-32P]ATP and T4 polynucleotide kinase. Alternatively, DNA can be labeled with a biotinylated or hapten-labeled dNTP, then probed and detected using an appropriately sensitive fluorescent or chemiluminescent substrate. Pierce offers a chemiluminescent EMSA system (LightShift Chemiluminescent EMSA Kit, Product # 20148) and a kit to facilitate labeling DNA with biotin (Biotin 3´ End DNA Labeling Kit, Product # 89818). The LightShift EMSA Kit utilizes Pierce’s patented SuperSignal Chemiluminescent Detection Technology to offer detection levels rivaling that of isotopic-based systems. (back to top) Nonspecific Competitor Nonspecific competitor DNA such as poly(dI•dC) or poly(dA•dT) is included in the binding reaction to minimize the binding of nonspecific proteins to the labeled target DNA. These repetitive polymers provide an excess of nonspecific sites to adsorb proteins in crude lysates that will bind to any general DNA sequence. The order of addition of reagents to the binding reaction is important in that, to maximize its effectiveness, the competitor DNA must be added to the reaction along with the extract prior to the labeled DNA target. Besides poly(dI•dC) or other nonspecific competitor DNA, a specific unlabeled competitor sequence can be added to the binding reaction. A 200-fold molar excess of unlabeled target is usually sufficient to out-compete any specific interactions. Thus, any detectable specific shift should be eliminated by the presence of excess unlabeled specific competitor (Figure 6). The addition of a mutant or unrelated sequence containing a low-affinity binding site, like poly(dI•dC), will not compete with the labeled target and the shifted band will be preserved. (back to top) Lane 1 2 3 4 EBNA Extract - + + + Unlabeled EBNA DNA - - + - Unlabeled Oct-1 DNA - - - + Figure 6. EMSA results using the EBNA control system. Biotin-labeled 60 bp duplex bearing the EBNA-1 binding sequence was incubated with an extract in which the EBNA-1 protein was overexpressed. The binding buffer was supplemented with 50 ng/µl poly (dl•dC), 10% glycerol and 0.05%NP-40. Exposure time was 30 seconds with X-ray film. Binding Reaction Components Factors that affect the strength and specificity of the protein NA interactions under study include the ionic strength and pH of the binding buffer, the presence of nonionic detergents, glycerol or carrier proteins (e.g., BSA), the presence/absence of divalent cations (e.g., Mg2+ or Zn2+), the concentration and type of competitor DNA present, and the temperature and time of the binding reaction. If a particular ion, pH or other molecule is critical to complex formation in the binding reaction, it is often included in the electrophoresis buffer to stabilize the interaction prior to its entrance into the gel matrix.(back to top) Gel Electrophoresis Non-denaturing TBE-polyacrylamide gels or TAE-agarose gels are used to resolve protein NA complexes from free DNA. The gel percentage required depends on the size of the target DNA and the size, number and charge of the protein(s) that bind to it. It is important that the proteinNA complex enters the gel and does not remain in the bottom of the well. Polyacrylamide gels in the range of 4-8% are typically used, although it is not uncommon for higher percentage gels to be used with certain systems. Agarose gels (0.7-1.2%) can be used to resolve very large complexes, as is the case with E. coli RNA polymerase (~460 kDa).Gels are pre-run at a constant voltage until the current no longer varies with time. The primary reasons for pre-running gels is to remove all traces of ammonium persulfate (used to polymerize polyacrylamide gels), to distribute/equilibrate any special stabilizing factors or ions that were added to the electrophoresis buffer, and to ensure a constant gel temperature. After loading samples onto the gel, it is important to minimize the electrophoretic dead time required for the free DNA to enter the gel matrix, especially when analyzing labile complexes. (back to top) Applications Supershift Reactions While there are characteristic shifts caused by the binding of specific protein(s) to the target DNA, the change in relative mobility does not identify the bound protein. Identification of the DNA-bound protein is frequently accomplished by including an antibody, specific for the DNA-binding protein in question, to the binding reaction. If the protein of interest binds to the target DNA, the antibody will bind to that protein NA complex, further decreasing its mobility relative to unbound DNA in what is called a “supershift” (Figure 7). In some cases, the antibody may disrupt the proteinNA interaction resulting in loss of the characteristic shift but no supershift. However, supported by the proper controls, even this “negative” result can support the identification of the protein of interest.Supershift reactions need not be limited to antibodies, but could include other secondary or indirectly bound proteins as well. An alternative identification process would be to perform a combination “Shift-Western blot.” This involves transferring the resolved protein NA complexes to stacked nitrocellulose and anion-exchange membranes. Protein captured on the nitrocellulose membrane can be probed with a specific antibody (Western blot) while autoradiography or chemiluminescent techniques can detect the DNA captured on the anion-exchange membrane.12Extract - + + + Antibody - - + - Competitor - - - + Figure 7. Supershift assay using the LightShift EMSA kit. Rabbit anti-Oct-1 antibody (1 µg) was added to a binding reaction containing a 22 bp Oct-1 specific biotin-labeled duplex and a HeLa nuclear extract. The supershift observed upon addition of specific antibody identifies the presence of Oct-1 in the original protein NA complex (Oct-1 shift).ELISA-Based Transcription Factor Assays The EMSA or gel shift assay is a versatile tool that can be used to explore and characterize protein:nucleic acid interactions. As stated above, considerable information can be obtained from the intensity, location and identity of the shifted bands. The activation level of well-characterized transcription factors, such as NF-B p50 and p65 and c-Fos and c-Jun, can be monitored either by EMSA or in an ELISA format (Pierce EZ-Detect Transcription Factor Plate Assays, Product # 89858, 89859, 89860, 89861). Active transcription factors are captured as they bind to their DNA consensus sequence immobilized on streptavidin-coated 96-well plates. The captured “active” transcription factor is probed with a specific primary antibody followed by use of HRP-conjugated secondary antibody and substrate detection. (Visit the Endogen web site) References: 1. Hendrickson, W. (1985). BioTechniques 3, 346-354. 2. Revzin, A. (1989). BioTechniques 7, 346-354. 3. Fried, M. and Crothers, D.M. (1981). Nucleic Acids Res. 9, 6505-6525. 4. Garner, M.M. and Revzin, A. (1981). Nucleic Acids Res. 9, 3047-3059. 5. Stockley, P.G. (1994). Filter-binding assays. From Methods in Molecular Biology, Vol. 30: DNA-Protein Interactions: Principles and Protocols. G. Geoff Kneale, Ed., Humana Press Inc., Totowa, NJ. 6. Fried, M.G. and Crothers, D.M. (1984) J. Mol. Biol. 172, 263-282. 7. Fried, M.G. (1989). Electrophoresis 10, 366-376. 8. Revzin, A., et al. (1986). Anal. Biochem. 153, 172-177. 9. Allen, S.V. and Miller, E.S. (1999). Anal. Biochem. 269, 32-37. 10. Park, Y.W., et al. (1999). Proc. Natl. Acad. Sci. USA 96, 6694-6699. 11. Grunwald, M.E., et al. (1998). J. Biol. Chem. 273, 9148-9157. 12. Demczuk, S., et al. (1993). Proc. Natl. Acad. Sci. USA 90, 2574-2578. |
3樓2009-11-27 15:52:48
qingfengwu
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2樓2009-11-27 15:51:30
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4樓2009-11-27 15:53:32
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5樓2009-11-27 15:53:54
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