Pull-Down Assays
全国大学生四六级官网
Elucidating gene function involves determining the function of the encoded protein product.
In the cell, proteins participate in extensive networks of protein-protein interactions that take
the form of dynamic “protein machines”, which asmble and disasmble 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.
Page Contents:
Introduction to Pull-Down Assays
Critical Components of Pull-Down Assays
Pull-Down Methodologies
References
Order
Protein Interaction Technical
Handbook Introduction to Pull-Down Assays
The pull-down assay is an in vitro method ud to determine a physical interaction between two or more proteins. Pull-down assays are uful for both confirming the existence of a protein-protein interaction predicted by other rearch techniques (e.g., co-immunoprecipitation) and as an initial screening assay for identifying previously unknown protein-protein interactions.
Pull-down assays are a form of affinity purification and are very similar to immunoprecipitation, except that a "bait" protein is ud instead of an antibody. Affinity chromatography (i.e., affinity purification) methodologies greatly enhance the speed and efficiency of protein purification and simul
taneously provide the technology platform to perform a pull-down, or co-purification, of potential binding partners. In a pull-down assay, a bait protein is tagged and captured on an immobilized affinity ligand specific for the tag, thereby generating a "condary affinity support"’ for purifying other proteins that interact with the bait protein. The condary affinity support of immobilized bait is then incubated with a protein source that contains putative "prey" proteins, such as a cell lysate. The source of prey protein at this step depends on whether the rearcher is confirming a previously suspected protein-protein interaction or identifying an unknown interaction. The method of protein elution depends on the affinity ligand and ranges from using competitive analytes to low pH or reducing buffers.
Besides investigating the interaction of two or more proteins, pull-down assays are a powerful tool to detect the activation status of specific proteins. For example, proteins that are activated in respon to tyrosine phosphorylation can be pulled down using an immobilized SH2 domain that targets the phosphorylated tyrosine on a given protein. Additionally, GTPas, which act as molecular switches that regulate cell signaling by cycling between a GTP-bound (active) and GDP-bound (inactive) state, can be pulled down using an immobilized GTPa-binding domain of downstream proteins that are recruited to GTP-bound, activated GTPas. In both types of pull-down assays, becau the
specificity of the interaction is dependent on the quence of the binding domain, the approaches are highly specific in detecting the activation of distinct proteins.
Overview of Protein-Protein Interaction Analysis Immunoprecipitation
Co-Immunoprecipitation
Tag-bad pull-down kits Active GTPa pull-down and detection kits
D3612-415F-42A5-8922-736F9FDD36FB（第 1／3 页）2012-2-17 11:12:21
General schematic of a pull-down assay. A pull-down assay is a small-scale affinity purification technique similar to immunoprecipitation, except that the antibody is replaced by some other affinity system. In this ca, the affinity system consists of a glutathione S-transfera (GST)-, polyHis- or streptavidin-tagged protein or binding domain that is captured by glutathione-, metal chelate (cobalt or nickel)- or biotin-coated agaro beads, respectively. The immobilized fusion-tagged protein acts as the "bait" to capture a putative binding partner (i.e., the "prey"). In a typical pull-down assay, the immobilized bait protein is incubated with a cell lysate, and after the prescribed washing steps, the c
ompexes are lectively eluted using competitive analytes or low pH or reducing buffers for in-gel or Western blot analysis.
The Pull-Down Assay as a Confirmatory Tool
The confirmation of previously suspected interactions typically utilizes a prey protein source
that has been expresd in an artificial protein expression system. This allows the
rearcher to work with a larger quantity of the protein than is typically available under
endogenous expression conditions and eliminates confusing results that could ari from
interaction of the bait with other interacting proteins prent in the endogenous system that
are not under study. Protein expression system lysates (i.e., E. coli or baculovirus-infected
inct 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
The discovery of unknown interactions contrasts with confirmatory studies becau the
rearch 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
behind>成人高考现场确认
bait is normally expresd, or complex biological fluid (i.e. blood, intestinal cretions, etc.)
where the bait would be functional, is an appropriate prey protein source for discovery
studies.
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. Rearchers who have access to commercially available purified protein 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 commonly ud 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. Recombinant clones can be
overexpresd and easily purified, resulting in an abundance of bait protein for u in pull-
down assays.
Common fusion tags and their affinity binding ligands
Fusion tag Affinity ligand
Glutathione S-transfera (GST)Glutathione
Poly-histidine (polyHis or 6xHis)Nickel or cobalt chelate complexes
Biotin Streptavidin
Binding Parameters: Stable vs. Transient Interactions
The 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 the bait and prey proteins. Transient interactions are usually associated with transport or enzymatic mechanisms. The ribosome illustrates both examples becau the structure consists of many stable protein-protein interactions, but the enzymatic mechanism that translates mRNA to nascent protein requires transient interactions.抛出窗外
Stable protein-protein interactions are easiest to isolate by physical methods like pull-down assays becau the protein complex does not disasmble over time. Strong, stable protein complexes can be washed extensively with high ionic strength buffers to eliminate any fal positive results due to n
onspecific interactions. If the complex interaction has a higher dissociation constant and a weaker interaction, the interaction strength and thus the 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 the careful design of appropriate control experiments.
Transient interactions are defined by their temporal interaction with other proteins and are the most challenging protein-protein interactions to isolate. The interactions are more difficult to identify using physical methods like pull-down assays becau the complex may dissociate during the assay. Since transient interactions occur during transport or as part of enzymatic process, they often require cofactors and energy via nucleotide triphosphates hydrolysis. Incorporating cofactors and nonhydrolyzable nucleoside triphosphate (NTP) analogs during assay optimization can rve to ‘trap’ interacting proteins in different stages of a functional complex that is dependent on the cofactor or NTP.
Weak or transient protein-protein interactions can be strengthened by covalently crosslinking the interacting proteins prior to pull-down. While this strategy is more advanced than performing the pull-down assay without crosslinking, freezing protein interactions by crosslinking may make or break the success of a pull-down assay.
Protein labeling reagents
Additional methods to study protein-protein interactions
Elution of the Bait-Prey Complex
The 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 using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (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 restricts the sample to SDS-PAGE analysis only. This method may also strip excess protein off the affinity support that is nonspecifically bound to the matrix, and this material can interfere with analysis. Competitive analyte elution is much more specific for the bait-prey interaction, becau 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 uful for subquent studies.
ladykiller
An alternative elution protocol is to u a step-wi gradient of increasing salt concentration or decreasing pH, which allows the lective elution of prey proteins while the bait remains immobilized.
conduction A gradient elution is not necessary once the critical salt concentration or pH has been optimized for efficient elution. The elution methods are also non-denaturing and
injectionOverview of Crosslinker-bad Interaction Analysis
Overview of Western Blotting Mass Spectrometry Workflow
can be informative in determining the 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, Western blotting detection and S-35 radioisotopic detection. The 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.
Pull-Down Assay Controls罗马假日英文版
Pull-down assays entail multiple steps often using more than one cell lysate, and therefore each exp
eriment must be properly controlled to demonstrate that the final results are not artifactual. Each experiment should analyze the lysate(s) both before and after being pasd through the support to identify any nonspecific binding to the support. Each wash should also be analyzed to obrve any eluted protein, and bait- and prey-free controls should also be ud to confirm that there are no bait-prey interactions in the bait lysate and that the prey protein does not bind to the immobilized support.
GelCode Blue Staining Reagent GelCode Blue Safe Staining Reagent
SuperSignal West Substrates
Pull-Down Methodologies
Homemade pull-down approaches for confirming or identifying protein-protein interactions are ubiquitous in contemporary scientific literature. The homemade pull-down assay reprents a collection of reagents from multiple commercial vendors that cannot be validated together as a functional asmbly except by extensive assay development by the rearcher, and troubleshooting this combination of reagents can be tedious and time-
加勒比海盗4主题曲
consuming. Commercial pull-down kits contain complete, validated ts of reagents specifically developed for performing pull-down assays. The buffers provided in each kit allow complete flexibility to determine the optimal conditions for isolating interacting proteins. The working solutions for washing and binding are physiologic in pH and ionic strength, providing a starting point from which specific buffer conditions for each unique interacting pair can be optimized. Many commercial kits also incorporate spin columns for efficient handling of small volumes of affinity support, complete retention of the affinity support during the pull-down assay and thorough washing of the protein complexes for minimal nonspecific protein pull-down, all of which are common sources of variability and high background using traditional pull-down assay formats.
Tag-bad pull-down kits Active GTPa pull-down and detection kits
References
1. Einarson, M.B. and Orlinick, J.R. (2002). Identification of Protein-Protein Interactions
with Glutathione S-Transfera Fusion Proteins. In Protein-Protein Interactions: A
Molecular Cloning Manual, Cold Spring Harbor Laboratory Press, pp. 37-57.
2. Einarson, M.B. (2001). Detection of Protein-Protein Interactions Using the GST
Fusion Protein Pulldown Technique. In Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press, pp.18.55-18.59.
3. Vikis, H.G. and Guan, K.-L. (2004). Glutathione-S-Transfera-Fusion Bad Assays
for Studying Protein-Protein Interactions. In Protein-Protein Interactions, Methods
and Applications, Methods in Molecular Biology, 261, Fu, H. Ed. Humana Press,
Totowa, N.J., pp. 175-186.2016年4月2日
