Western blot and gel overlays

I illustrate Western blots and gel overlayson this page. See Old and Primrose for details.

Western blotting: 

1. An antibody prepared a known protein is used as a probe. The antibody will be typically prepared in rabbits against purified protein (Details unimportant).
2. The protein sample is separated on the basis of molecule size by (typically SDS-PAGE) gel electrophoresis. 
   • i.e. proteins (polypeptides) of the same size will run to the same position on the gel. Proteins are separated into their separate polypeptide subunits.
3. The proteins are then transferred and fixed to a membrane (blotted), preserving the separation pattern.
4. The specific (primary) antibody is added in solution to the membrane and incubated in chemical conditions which allow it bind the corresponding antigen. i.e. the antibody seeks the corresponding antigen (strictly speaking epitope) among a complex mixture and bind it.
5. The membrane is then treated with a detection system usually involving a secondary antibody. The secondary antibody is raised against the primary antibody, e.g. rabbit antibody is used to make an antibody in a goat - hence "goat anti-rabbit". This secondary antibody is chemically tagged, and the tag can involve an enzyme which is then found on the basis of the labelling system used. There are 4-5 separate principals used: gold particles, biotin, fluorescent tags, alkali phosphatase, peroxidase. Some of these detection methods are used for western blotting, others in immunohistological techniques.

Uses:

• Western blotting:
  • Determine in which tissues, or under which physiological conditions a gene is expressed to produce a protein.
  • Determine whether a transgenic organism expresses the inserted gene to produce a protein. (Does it tell you if the protein works?)
• Gel overlay (below)
  • The study of protein-protein interactions - e.g. as a confirmation of a yeast-two hybrid screen.

An example

Characterisation of 14-3-3 from barley, induced in leaves by the powdery mildew fungus. An antibody raised against a protein isolated from cow brain (bovine) finds antigen in pea and barley leaves and barley roots. Note that there are bands of different sizes: these represent different isoforms of the protein. 

from: Brandt, J., Thordal-Christensen, H., Vad, K., Gregersen, P.L., and Collinge, D.B. (1992). A pathogen-induced gene of barley encodes a protein showing high similarity to a protein kinase regulator. The Plant Journal. 2: 815-820. Medline abstract (copyright: Blackwell)

Gel overlay assays

These are a refinement of the western blot technique for studying protein-protein interactions. A specific protein is used as a probe. This is chemically treated to make a  tag (e.g. DIG - digoxigen). The tag is found with an antibody and the antibody detected as for a western blot. 

An example

The 14-3-3 proteins have been shown to accumulate in barley-powdery mildew interactions. The are known to bind other proteins in other plants (and animals). Do they bind something in barley? Panel A is stained for protein. Panels C and E show western blots from (a) total leaf (b) leaf epidermis (where the fungus lives) showing the presence of a protein (H⁺ATPase) in inoculated not uninoculated epidermis. Panels B and D are identical membranes to C and E which have been probed with DIG-14-3-3 protein as described in the figure above. Thus it seems very likely that 14-3-3 is binding the H⁺ATPase in the inoculated epidermis, confirming yeast two-hybrid results.

(unpublished results)

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Created  by David B. Collinge January 2000, revised 25-02-11