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yaoyaochong银虫 (正式写手)
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Pull-Down Assay
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有哪位做过 Pull-Down Assay实验,能不能给提供原理,protocol,和一些注意事项,谢谢了 |
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看一下，或多或少知道一点了： Introduction to Pull-Down Assays Elucidating gene function involves determining the function of each gene encoded protein product. In the cell, proteins participate in extensive networks of protein:protein interactions. These interactions take the form of dynamic protein machines， which assemble and disassemble in concert with an ever-changing influx of intra, inter and extracellular cues. A preliminary step in understanding protein structure and function is to determine which proteins interact with each other, thereby identifying the relevant biological pathways. The pull-down technique has become an invaluable tool for the life scientist interested in studying cellular pathways via protein:protein interactions. The pull-down assay is an in vitro method used to determine physical interaction between two or more proteins. Pull-down assays are useful for both confirming the existence of a protein:protein interaction predicted by other research techniques (e.g., co-immunoprecipitation, yeast two-hybrid and density gradient centrifugation) and as an initial screening assay for identifying previously unknown protein:protein interactions. The minimal requirement for a pull-down assay is the availability of a purified and tagged protein (the bait) which will be used to capture and pull-down a protein-binding partner (the prey). Pull-down assays are a form of affinity purification and are very similar to immunoprecipitation (see previous topic in this section) except that a bait protein is used instead of an antibody. Affinity chromatography (i.e., affinity purification) methodologies greatly enhance the speed and efficiency of protein purification and simultaneously provide the technology platform for performing a pull-down, or co-purification, of potential binding partners. In a pull-down assay, a tagged bait protein is captured on an immobilized affinity ligand specific for the tag, thereby generating a decondary affinity support for purifying other proteins that interact with the bait protein. The secondary affinity support of immobilized bait can be incubated with a variety of other protein sources that contain putative prey proteins. The source of prey protein at this step depends on whether the researcher is confirming previously suspected protein:protein interactions or identifying unknown protein:protein interactions. The Pull-Down Assay as a Confirmatory Tool Confirmation of previously suspected interactions typically utilizes a prey protein source that has been expressed in an artificial protein expression system. This allows the researcher to work with a larger quantity of the protein than is typically available under endogenous expression conditions and eliminates confusing results, which could arise from interaction of the bait with other interacting proteins present in the endogenous system that are not under study. Protein expression system lysates (i.e., E. coli or baculovirus-infected insect cells), in vitro transcription/translation reactions, and previously purified proteins are appropriate prey protein sources for confirmatory studies. The Pull-Down Assay as a Discovery Tool Discovery of unknown interactions contrasts with confirmatory studies because the research interest lies in discovering new proteins in the endogenous environment that interact with a given bait protein. The endogenous environment can entail a plethora of possible protein sources but is generally characterized as a complex protein mixture considered to be the native environment of the bait protein. Any cellular lysate in which the bait is normally expressed, or complex biological fluid (i.e. blood, intestinal secretions, etc.) where the bait would be functional, are appropriate prey protein sources for discovery studies. (back to top) Critical Components of Pull-Down Assays Bait Protein Criteria Bait proteins for pull-down assays can be generated either by linking an affinity tag to proteins purified by traditional purification methods or by expressing recombinant fusion-tagged proteins. Researchers who have access to commercially available purified proteins or frozen aliquots of purified protein from an earlier study can design a pull-down assay without the need for cloning the gene encoding the protein of interest. The purified protein can be tagged with a protein-reactive tag (e.g., Sulfo-NHS-LC-Biotin, Product # 21335) commonly used for such labeling applications. Alternatively, if a cloned gene is available, molecular biology methods can be employed to subclone the gene to an appropriate vector with a fusion tag (e.g., 6xHis or glutathione S-transferase, GST). Recombinant clones can be overexpressed and easily purified, resulting in an abundance of bait protein for use in pull-down assays. Binding Parameters: Stable vs. Transient Interactions Discovery and confirmation of protein:protein interactions using the pull-down technique depend heavily on the nature of the interaction under study. Interactions can be stable or transient and this characteristic determines the conditions for optimizing binding between bait and prey proteins. Stable interactions make up most cellular structural features but can also occur in enzymatic complexes that form identifiable structures. Transient interactions are usually associated with transport or enzymatic mechanisms. The ribosome illustrates both examples because the structure consists of many stable protein:protein interactions, but the enzymatic mechanism that translates mRNA to nascent protein requires transient interactions. Stable protein:protein interactions are easiest to isolate by physical methods like pull-down assays because the protein complex does not disassemble over time. Since these interactions often contribute to cellular structure, the dissociation constant between proteins is usually low, correlating to a strong interaction. Strong, stable protein complexes can be washed extensively with high ionic strength buffers to eliminate any false positive results due to nonspecific interactions. If the complex interaction has a higher dissociation constant and is a weaker interaction, the interaction strength and thus protein complex recovery can be improved by optimizing the assay conditions related to pH, salt species and salt concentration. Problems of nonspecific interactions can be minimized with careful design of appropriate control experiments (see below). Transient interactions are defined by their temporal interaction with other proteins and are the most challenging protein:protein interactions to isolate. These interactions are more difficult to identify using physical methods like pull-down assays because the complex may dissociate during the assay. Since transient interactions occur during transport or as part of enzymatic processes, they often require cofactors and energy via nucleotide triphosphates hydrolysis. Incorporating cofactors and nonhydrolyzable NTP analogs during assay optimization can serve to 憈rap?interacting proteins in different stages of a functional complex that is dependent on the cofactor or NTP. Elution of the Bait-Prey Complex Identification of bait-prey interactions requires that the complex is removed from the affinity support and analyzed by standard protein detection methods. The entire complex can be eluted from the affinity support by utilizing SDS-PAGE loading buffer or a competitive analyte specific for the tag on the bait protein. SDS-PAGE loading buffer is a harsh treatment that will denature all protein in the sample, and it restricts analysis to SDS-PAGE. This method may also strip excess protein off the affinity support that is nonspecifically bound to the matrix, and this material will interfere with analysis. Competitive analyte elution is much more specific for the bait-prey interaction because it does not strip proteins that are nonspecifically bound to the affinity support. This method is non-denaturing; thus, it can elute a biologically-functional protein complex, which could be useful for subsequent studies. An alternative elution protocol allows selective elution of prey proteins while the bait remains immobilized. This is accomplished using a step-wise gradient of increasing salt concentration or a step-wise gradient of decreasing pH. A gradient elution is not necessary once the critical salt concentration or pH has been optimized for efficient elution. These elution methods are also non-denaturing and can be informative in determining relative interaction strength. Gel Detection of Bait-Prey Complex Protein complexes contained in eluted samples can be visualized by SDS-PAGE and associated detection methods including gel staining with Pierce GelCode Stains (e.g., Product # 24590, 24592), Western blotting detection with SuperSignal Chemiluminescent Substrates (e.g., Product # 34080) and 35S radioisotopic detection. Final determination of interacting proteins often entails protein band isolation from a polyacrylamide gel, tryptic digestion of the isolated protein and mass spectrometric identification of digested peptides. Importance of Control Experiments for the Pull-Down Assay In all pull-down assays, carefully designed control experiments are absolutely necessary for generating biologically significant results. A negative control consisting of a non-treated affinity support (minus bait protein sample, plus prey protein sample) helps to identify and eliminate false positives caused by nonspecific binding of proteins to the affinity support. The immobilized bait control (plus bait protein sample, minus prey protein sample) helps identify and eliminate false positives caused by nonspecific binding of proteins to the tag of the bait protein. The immobilized bait control also serves as a positive control to verify that the affinity support is functional for capturing the tagged bait protein. |
2楼2007-12-17 23:41:03