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BTEC 4P11 (1)
Lecture

A+Primer+on+EMSA+and+ChIP.pdf

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Department
Biotechnology
Course
BTEC 4P11
Professor
Charles Despres
Semester
Winter

Description
Després’ Lab - Primer Series. A Primer on EMSA and Chromatin Immunoprecipitation. Introduction to the EMSA (Gel Shift) Technique The interaction of proteins with DNA is centr al to the control of many cellular processes including DNA replication, recombination and repair, transcription, and viral assembly. One technique that is centra l to studying gene regulation and determining protein:DNA interactions is the electrophoretic mobility shift assay (EMSA). The EMSA technique is based on the observation that protein:DNA complexes migrate more slowly than free DNA mol ecules when subjected to non-denaturing polyacrylamide or agarose gel electrophoresis . Because the rate of DNA migration is shifted or retarded upon protein binding, the assay is also refe rred to as a gel shift or gel retardation assay. An advantage of studying DNA:protein interactions by an electrophoretic assay is the ability to resolve complexes of differ ent stoichiometry or conformation. Another major advantage for many applications is th at the source of th e DNA-binding protein may be a crude nuclear or whole cell extract ra ther than a purified preparation. Gel shift assays can be used qualitatively to id entify sequence-specific DNA-binding proteins (such as transcription factors) in crude lysates and, in conjunction with mutagenesis, to identify the important binding sequences w ithin a given gene’s upstream regulatory region. EMSAs can also be utilized quantitatively to measure thermodynamic and kinetic parameters. The ability to resolve protein:DNA comp lexes depends largely upon the stability of the complex during the brief time (approxima tely 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 buffe r 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, th e complex may be stab ilized by “caging” effects of the gel matrix, meaning that if the complex dissociates, its localized concentration remains high, promoting prompt re-association. Therefore, even labile complexes can often be resolved by this method. Critical EMSA Reaction Parameters Target DNA. Typically, linear DNA fragments cont aining 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 th e specific sequence can be synthesized, purified by gel or HPLC, and annealed to form a duplex. Often, a protein:DNA interaction involves the formation of a multip rotein complex requiring multiple protein binding sequences. In this situation, longe r 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. Protein:DNA 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 protein:DNA complex may actually migrate faster than the free DNA. Gel shift a ssays are also good for resolving altered or bent DNA conformations that result from the bi nding of certain protein factors. Gel shift 1 Després’ Lab - Primer Series. A Primer on EMSA and Chromatin Immunoprecipitation. assays need not be limited to DNA:protei n interactions. Protein:DNA interactions as well as protein:peptide interactions have also been studied using the same electrophoretic principle. 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 32beled before performing the experiment. Traditionally, DNA is radiolabeled with P by incorporating an [α- P]dNTP during a 3´ fill-in reaction using Klenow fragment or by 5´ end labeling using [γ-32P]ATP and T4 polynucleotide kinase. Alte rnatively, DNA can be labeled with a biotinylated or hapten-labeled dNTP, then probed and detected using an appropriately sensitive fluorescent or chemiluminescent s ubstrate. DNA can also be labeled directly with a fluorophore. Nonspecific Competitor . Nonspecific competitor DNA such as poly(dI•dC) or poly(dA•dT) is included in the binding reac tion to minimize the bi nding of nonspecific proteins to the labeled target DNA. These repetitive polyme rs 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 1). The addition of a mutant or unrelated sequence containing a low-affin ity binding site, like poly(dI•dC), will not compete with the labeled target and the shifted band will be preserved. Binding Reaction Components . Factors that affect the st rength and specificity of the protein:DNA interactions under study include the ionic strength and pH of the binding buffer, the presence of nonionic detergents, glycerol or carrier pr oteins (e.g., BSA), the presence/absence of divalent cations (e.g., Mg 2+or Zn 2+ ), the concentration and type of competitor DNA present, and the temperature and time of the binding reaction. If a particular ion, pH or
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