Overlay assays

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. 

Screening based on halo formation is not only suited for the identification of single enzyme activities but may also be used to screen for whole metabolic pathways. Aarons et al. recently set up a system to screen for a functional enzyme cascade that is involved in the production of substances that act as antibiotics towards an indicator bacteria strain but not the producer strain. Using the Bacillus overlay-assay [50] they were able to isolate genes capable of complementing P. fluorescens FI 13 gacS and gacA mutants [51]. 

Fig. 4.2. Agar overlay assay for plastninogen activator. B16-F1 and -LS9 cells grown as isolated colonies have been overlaid with agar/milk (see text) in the presence (lower wells) or absence (upper wells) of plasminogen, to detect celt surface associated uPA activity. B16-LS9 cells clearly show a higher activity as compared to B16-F1, in which the activity is undetectable under this assay conditions (Rusciano et al., 1998a).

First, an overlay assay was used to demonstrate binding of photoaffinity labeled FPR to actin from neutrophil cytosol [49]. Second, actin increased the sedimentation rate of Triton X-lOO-solubilized FPR in a sucrose density gradient [49]. Third, anti-actin antibodies were able to immunosediment FPR suggesting... 

Transposon-based approaches have also been applied to myxobacteria (Figure 3). Mutants from transposon libraries can be analyzed chemically by comparison of their secondary metabolite profiles with the wild-type strain, or screened for altered bioactivity, for example, by overlay assays. This strategy was successfully used to identify the biosynthetic gene clusters responsible for the known metabolites tubulysin, disorazol, aurachin, and DKxanthene in the genomes of their respective producer strains, and has the potential to detect novel, as yet unidentified compounds and their corresponding biosynthetic pathways and regulatory elements (see Section 2.07.5). 

LC—MS) analysis of culture broth extracts, and reporter gene activation have all been successfully used as readouts in high-throughput assays designed to find small-molecule-producing clones (see Section 2.13.4). The most frequently used functional assays have been color production and antibacterial activity. While any assay strain can he selected for an overlay assay, Bacillus subtilis is commonly used due to its sensitivity to most known classes of antibiotics. Using simple functional assays, clones that produce new natural products have been recovered from both Escherichia coli- and Streptomyces lividans-h seA eDNA libraries (see Section 2.13.4). 

A simple overlay assay against Staphylococcus aureus (Manohar/5 may be carried out as follows ... 

Figure 2. Agar-overlay assay used to screen for hydantoinase-producing microorganisms.

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). 

---