Detection blot overlay assay

Blot Overlay Assay A Method to Detect Protein-Protein Interactions... 

The blot overlay assay is a powerful method used to study protein-protein interactions and provides an especially useful means by which to identify potential protein ligands. In this approach, a radiolabeled protein probe is used to overlay protein samples immobilized on nitrocellulose. Because this assay allows detection of protein-protein interactions within the context of a complex mixture of proteins, this method is also useful to investigate the specificity of an interaction between two ligands. The blot overlay assay may also be used to define specific domains of a ligand involved in protein-protein interactions for example, proteolytic fragments of proteins or nested deletion fragments may be probed to determine which portion of a molecule contains the interactive site (Crawford ei al., 1992 Gilmore et al., 1992). In addition, the blot overlay procedure may be modified to probe cDNA libraries to identify bacterially expressed proteins that interact with labeled probes (Cicchetti et al., 1992 Blanar and Rutter, 1992). 

The blot overlay assay was first described as a method to detect interacting protein ligands resolved on polyacrylamide gels (Glenney and Weber, 1980 Otto, 1983). In the procedure described here, potential protein ligands are transferred from polyacrylamide gels to nitrocellulose before incubating with labeled probes. 

The blot overlay assay is not a native assay. Thus, it is recommended that interactions detected by this approach be confirmed by employing solution binding assays under native conditions. 

Low-abundance ligands may not be detected in the blot overlay assay. To increase the possibility of detecting low-abundance ligands by this method, enriching for specific proteins by selective biochemical fractionation of protein samples (e.g., ammonium sulfate fractionation, anion exchange chromatography)... 

FIGURE 1 A typical blot overlay experiment to detect protein binding partners. Many proteins are present in the Coomassie blue-stained gel (A) however, I-zyxin recognizes predominantly a 23-kDa protein (the cysteine-rich protein) from this complex protein sample (B). The purity of I-zyxin used in this assay is shown in C (750,000 cpm, exposed for 10 min). 

Fig. 2. Protein-lipid overlay assay to demonstrate the effect of ASAPl tyrosine phosphorylation on phospholipid binding. Protein-hpid overlay assays are performed with immobilized phosphohpids (100 pmol/spot) incubated with 0.5 tg/ml nonphosphorylated (left panel) or phosphorylated ASAPl (middle panel). Binding of Flag-ASAPl to immobilized phospholipids is detected by Western blotting with an anti-Flag antibody. Positions of the individual hpids on the array are shown on the right paneL...

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