In principal the same as a western blot except that in stead of using a specific antobody to probe the membrane, you use a protein. In the example uillustrated below, the protein of interest is a 14-3-3 protein which has been labelled with DIG (digoxigenin - a steroid isolated from Digitalis pupurea - (Foxglove -fingerbøl). Instead of a secondary antibody, an antibody raised against DIG is used. This can be detected by an appropriate method, e.g. ECL.
A real-life example taken from Finnie et al 2001. Epidermal specific14-3-3-binding activity induced by the powdery mildew fungus. Nitrocellulose blots with total protein extracts from non-inoculated (-) or Bgh-inoculated (+) material from whole leaf (A-C) or epidermis (D,E). A: Ponceau S staining to show all proteins. C,E: western blot with antibody recognising plasma membrane H+-ATPase. B,D: Overlay assay with 14-3-3-DIG. Sizes of molecular weight standards are indicated. In this paper, it can be seen that a protein with the same size as the proton pump binds 14-3-3 protrein in the epidermis of barley leaves infected with Blumeria graminis.
Finnie, C., Andersen, C.H., Borch, J., Gjetting, S., Christensen, A.B., De Boer, A.H., Thordal-Christensen, H. and Collinge, D.B. 14-3-3 Proteins are involved in an epidermis-specific response to the barley powdery mildew fungus. Plant Molecular Biology in press. abstract
David B. Collinge, created 6/11-01 revised 04-12-01