Bind or No Bind
Pull down assays offer a binary "yes" or "no" answer regarding any possible interaction between two proteins, thus establishing this technique as essential for the study of cellular pathways via protein-protein interactions. Pull-down assays are a form of affinity purification very similar to immunoprecipitation, except that a "bait" protein is used instead of an antibody. Affinity chromatography (i.e., affinity purification) methodologies enhance the speed and efficiency of protein purification and simultaneously provide the technology platform to perform a pull-down, or co-purification, of potential binding partners.
General Procedure
In a pull-down assay, a bait protein (ex. pRB) is tagged (ex. with GST) and captured on an immobilized affinity ligand specific for the tag (ex. Glutathione-S-transferase Sepharose beads), thereby generating a "secondary affinity support"’ for purifying other proteins that interact with the bait protein. Often, recombinant "bait" proteins may be expressed with the tag (ex. GST-pRB) from bacterial culture, eliminating the need to bind the bait protein to the affinity ligand. Molecular biology methods can be employed to subclone the gene encoding the "bait" protein into an appropriate vector with a fusion tag .Recombinant clones can be overexpressed and easily purified, resulting in an abundance of bait protein for use in pull-down assays.
The secondary affinity support of immobilized bait is then incubated with a protein source (ex. whole cell lysate) that contains putative "prey" proteins (ex. E2Fs). The source of prey protein at this step depends on whether the researcher 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.
General Procedure
In a pull-down assay, a bait protein (ex. pRB) is tagged (ex. with GST) and captured on an immobilized affinity ligand specific for the tag (ex. Glutathione-S-transferase Sepharose beads), thereby generating a "secondary affinity support"’ for purifying other proteins that interact with the bait protein. Often, recombinant "bait" proteins may be expressed with the tag (ex. GST-pRB) from bacterial culture, eliminating the need to bind the bait protein to the affinity ligand. Molecular biology methods can be employed to subclone the gene encoding the "bait" protein into an appropriate vector with a fusion tag .Recombinant clones can be overexpressed and easily purified, resulting in an abundance of bait protein for use in pull-down assays.
The secondary affinity support of immobilized bait is then incubated with a protein source (ex. whole cell lysate) that contains putative "prey" proteins (ex. E2Fs). The source of prey protein at this step depends on whether the researcher 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.
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Stable protein-protein interactions are easiest to isolate by physical methods like pull-down assays because the protein complex does not disassemble over time. 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 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.
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.
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, 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 is to use a step-wise gradient of increasing salt concentration or decreasing pH, which allows the selective elution of prey proteins while the bait remains immobilized. 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 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
Pull-down assays entail multiple steps often using more than one cell lysate, and therefore each experiment 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 passed through the support to identify any nonspecific binding to the support. Each wash should also be analyzed to observe any eluted protein, and bait- and prey-free controls should also be used 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.
http://www.piercenet.com/browse.cfm?fldID=F3FD3612-415F-42A5-8922-736F9FDD36FB
Stable protein-protein interactions are easiest to isolate by physical methods like pull-down assays because the protein complex does not disassemble over time. 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 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.
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.
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, 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 is to use a step-wise gradient of increasing salt concentration or decreasing pH, which allows the selective elution of prey proteins while the bait remains immobilized. 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 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
Pull-down assays entail multiple steps often using more than one cell lysate, and therefore each experiment 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 passed through the support to identify any nonspecific binding to the support. Each wash should also be analyzed to observe any eluted protein, and bait- and prey-free controls should also be used 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.