Steps of western blotting experiments

Basically, western blotting experiments consist of five steps (cf Figures 1 and 2 and Table 1):

1. Sample preparation and electrophoretic separation in a slab gel.

2. Transfer to a membrane with high protein binding capacity.

3. Blocking (quenching) of the residual binding capacity of the membrane.

4. Incubation with antibody.

5. Detection of bound antibody with secondary reagents.

The procedure combines elements of Immunoelectrophoresis, enzyme-linked immunoassay, and - with respect to the localized dye formation - immuno-histochemistry. The deposition and immobilization

Transverse electrophoresis

Diffusion

Tank

Tank fib

Semidry

Figure 1 Common methods of transferring proteins from a slab gel to an immobilizing membrane. Two methods rely on transverse electrophoresis either by a buffer tank device (buffer tank, plastic pads and supports not shown) or by a semidry blotting apparatus where transfer is mediated by filter paper soaked in buffer. The third method, transfer by diffusion, is particularly useful for thin plastic supported gels. The arrows indicate the direction of the movement of the proteins from the gel towards the immobilizing membrane.

of the proteins on a membrane greatly facilitates access of large molecules (antibodies) to the antigen as well as all washing and any further incubation steps. Western blotting is particularly valuable for characterizing protein antigens which are difficult to analyze by immune precipitation, e.g. antigens not soluble in buffers compatible with immunological reactions or antigens that cannot be radioactively labeled (e.g. complex allergens, protozoa, food components, etc.).

Electrophoretic separation

The numerous variations possible at each step render western blotting a versatile analytical tool. For separation, electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate (SDS) is usually preferred because this detergent solubilizes virtually all proteins and because the system yields information on the molecular weight of the proteins. Western blot ting is also easily adapted to agarose gels (e.g. isoelectric focusing) or to electrophoresis in buffer systems containing urea or physiological buffers.

Transfer

Several methods have been devised to transfer the resolved proteins to membranes. Replicas may be produced by letting the proteins diffuse to a membrane placed on one - or both sides - of the gel. However, diffusion from thick polyacrylamide gels is slow and inefficient. To speed up elution transverse electrophoresis is usually employed (Figure 1). An electric field is applied perpendicularly to the gel surface. The immobilizing membrane and the gel are held in place by plastic sponges and plastic grids to form a rigid sandwich assembly which is placed into a chamber containing buffer and plate electrodes. Alternatively, a horizontal setup is used where gel and membrane are sandwiched between several

Blotted protein

Blocking

Incubation with antitody

Detection of bound antibocfy

Figure 2 Scheme of a typical immunological detection procedure for protein antigens transferred by western blotting. Microscopic views of the membrane are shown on the right.
Table 1 Commonly used components in western blotting

Transfer methods

Transverse electrophoresis

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