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). 

Store the radiolabeled protein fractions at 4°C until ready to perform the blot overlay assay for best results, it is recommended that the protein be used as soon after labeling as possible. However, depending on the stability of the radiolabeled probe, it may be used for several weeks after labeling. Resolve the radiolabeled probe on a polyacrylamide gel immediately prior to use to ensure that it is still intact and has not undergone proteolysis. 

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. 

To perform the blot overlay assay, place overlay buffer into a small container with a tight-fitting lid. To the overlay buffer, add radiolabeled probe to a final concentration of 250,000 cpm/ml mix the solution well, and add the nitrocellulose strip. Because the radiolabeled protein is often available in limited quantity, it may be necessary to perform the blot overlay assay in a small volume. For a 5 X 10-cm nitrocellulose strip, 10 ml of solution is usually sufficient for the assay, as long as the container does not exceed these dimensions significantly. 

An attractive feature of the blot overlay assay is that it is a useful method to investigate the specificity of protein-protein interactions. For example, the selective... 

If the recognized ligand can be purified to homogeneity, the blot overlay assay... 

A number of control experiments can be performed to strengthen the interpretation of the results obtained from blot overlay assays (A) A heat-denatured probe (i.e., 95°C for 10 min) should be significantly reduced in its ability to interact with the immobilized ligand (see Crawford et al., 1992). (B) If possible, excess unlabeled protein should be used in the overlay assay to competitively inhibit protein-protein interactions (see Gilmore et al., 1992). (C) If both proteins can be purified to homogeneity, the blot overlay may be performed using each protein as the radiolabeled probe (see Crawford et al., 1992). 

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)... 

Blot Overlay Assay for the Identification of GTP-Binding Proteins... 

Moeremans, M., Daneels, G., Van Dijck, A., Langanger, G., and De Mey, J. (1984) Sensitive visualization of antigen-antibody reactions in dot and blot immuno overlay assays with the immunogold and immu-nogold/silver staining./. Immunol. Meth. 74, 353-360. 

Protein samples to be assayed in the blot overlay procedure should be resolved on SDS-polyacrylamide gels (Laemmli, 1970 see article by Julio E. Celis and EySfinmar Olsen). Prepare duplicate samples one of these will be used for the overlay assay the other will be stained with Coomassie blue to enable evaluation of sample complexity and binding specificity. 

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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