Pull-down assay

Fusion protein pull-down assays involve the overexpression of bait and/or fusion proteins in bacteria. Often, the expressed fusion proteins are localized in occlusion bodies and not readily soluble under nondenaturing conditions. The expressed proteins can be extracted using urea, sonication, sodium dodecyl sulfate (SDS), or a combination of all the three. The net result is the denaturation of the recombinant protein and it may need to be refolded if the interaction domain is conformationally dependent. A major advantage of the pull-down assay is that high concentrations of proteins can be easily generated thus favoring protein association for a reversible equilibrium between two proteins. 

For immunoprecipitations from native tissues, one requires antibodies directed against both the fish and the bait proteins. Further, these antibodies should not bind to epitopes within the putative protein-protein BDs. It is technically difficult to determine low affinity or transient association among proteins by immunoprecipitation because low-affinity interactions may be lost by washing immune pellets to remove nonspecifically bound proteins. Also, one cannot manipulate protein concentrations to favor protein association as one can in a pull-down assay. Under these circumstances, probably the best method to use would be FRFT. 

In this pull-down assay, the enzymatic reaction is carried out completely in solution. Samples taken from the reaction mixture are then transferred to a SAM-modified MALDI target, on which the remaining substrate and the reaction product are selectively immobilized. Subsequent to the extraction of the analytes, the target is rinsed, treated with matrix, and MALDI-MS analysis is carried out. A major advantage of this assay scheme is that the inherent danger of negative influences on the reaction kinetics, which may be caused by immobilization of the substrate as in standard SAMDI-MS-based assay formats, is circumvented. Additionally, by selective extraction of the analytes of interest and removal of the other... 

Fig. 8.14 Scheme of a pull-down assay. The enzymatic reaction is completely carried out in solution. Upon enzyme addition, substrate is consumed, and product is formed. Sample aliquots are taken at several time points from the reaction mixture and are taken to a SAM, which has been modified with selective end groups. The latter are able to bind both substrate and product. Finally, matrix is added, and the SAM is analyzed by means of MALDI-MS. 

The interaction between RhoA and its key effector Rho-associated kinase (ROK or ROCK) is decreased upon RhoA phosphorylation, as shown by pull-down assays [71], and RhoA phosphorylation is also inversely correlated with signaling to its downstream effector phosphoh-pase D (PLD) [76,81]. However, not all effects of RhoA phosphorylation can be attributed to increased binding to RhoGDI leading to decreased signaling. RhoA expressed in bacteria (i.e., nonprenylated) and phosphorylated in vitro bound less efficiently to its downstream effector ROK in vitro than did nonphosphorylated RhoA [65]. Since this was done without any RhoGDI present, there must be something intrinsic about RhoA phosphorylation that affects RhoA interactions with at least some of its... 

Physiologic levels of total cellular GTP-bound Ras can be detected with pull-down assays (40) (see Table 2). With these assays, cells are lysed with detergent-containing buffers and then incubated with a recombinant fusion protein that contains the isolated RBD of c-Raf-1 fused to glutathione-S-transferase (GST designated GST-Raf-RBD). The presence of Ras in the... 

Before the development of the RNA three-hybrid system, identification of protein-RNA interactions was limited to in vitro methods such as pull-down assays using radiolabeled RNA. The introduction of the RNA three-hybrid system has allowed not only the detection of well-studied protein-RNA... 

An increasing number of available protein-binding assays, functional cell-based assays, and methods of chemical proteomics (affinity chromatography, three-hybrid assays, pull-down assays) will allow for a better assignment of the specificity and selectivity of a hit compound. It would be desirable that the data collected during these screening... 

GST-Pull Down Assay for Chemoattraclant-Induced Ras Activation... 

Brymora, A., et at. (2001). Enhanced Protein Recovery and Reproducibility from Pull-down Assays and Immu-noprecipitations Using Spin Columns, . 4na/. Biochem. 295 119-122. 

Figure 1.20 The major elements of the pull-down assay. (From Terstappen, G. C. et al., 2007, Target Deconvolution Strategies in Drug Discovery, Nat. Rev. Drug Disc., 6 891-903.)...

Figure 11.3 Identification of protein targets using photo-cross-linking beads, (a) Upon UV Irradiation, aryl-diazirine groups covalently Introduced onto a solid matrix (e.g., agarose beads) are transformed into highly reactive carbenes, which in turn bind to or insert irreversibly into a proximal small molecule in a functionality independent manner, (b) Pull-down assays are performed by mixing...

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