Western blotting


Western blotting (also immunoblotting) is a technique used in molecular biology to detect a specific protein in tissue or homogenate mixture, using gel electrophoresis to separate the proteins by polypeptide length (where they have been denatured) or by 3D structure (where they are in native and undenatured form) and then reporter enzyme-linked anitbodies to probe for the protein of interest.

THE PROCEDURE:

Extracting a protein mixture:

First of all, the tissue extract containing the protein(s) of interest is broken down mechanically either in a blender, a homogeniser or by sonication. Various detergents and salts may also be used to encourage lysis where cell membranes are involved. This is done at low temperatures to prevent denaturation and with the presence of protease and phosphatase inhibitors to prevent such enzymes damaging the proteins of interest when the cells are de-compartmentalised.

Electrophoresis (SDS-PAGE):

Electrophoretic separation of the proteins may then occur in a polyacrylamide gel, usually using the SDS-page technique which involves first reducing proteins to remove disulphide interactions (i.e. converting S-S bridges to -SH SH- sulphydryl groups).

The proteins move towards the positively charged end of the gel, differentially according to molecular weight (KDa). The resolution of lower molecular weight proteins is improved by increasing the concentration of acrylamide in the gel. Samples are loaded into wells in the gel. One lane is usually reserved for a marker or ladder, a commercially available mixture of proteins having defined molecular weights, typically stained so as to form visible, coloured bands. When voltage is applied along the gel, proteins migrate into it at different speeds. These different rates of advancement (different electrophoretic mobilities) separate into bands within each lane.

Transfer from polyacrylamide gel to a nylon membrane:

In order to make the proteins accessible to antibody detection, they are moved from within the gel onto a membrane made of nitrocellulose or polyvinylidene difluoride (PVDF). The membrane is placed on top of the gel, and a stack of filter papers placed on top of that. The entire stack is placed in a buffer solution which moves up the paper by capillary action, bringing the proteins with it.

Another method for transferring the proteins is called electroblotting and uses an electric current to pull proteins from the gel into the PVDF or nitrocellulose membrane. The proteins move from within the gel onto the membrane while maintaining the organization they had within the gel. As a result of this "blotting" process, the proteins are exposed on a thin surface layer for detection (see below).

Both varieties of membrane are chosen for their non-specific protein binding properties (i.e. binds all proteins equally well). Protein binding is based upon hydrophobic interactions, as well as charged interactions between the membrane and protein. Nitrocellulose membranes are cheaper than PVDF, but are far more fragile and do not stand up well to repeated probings.

Block antibodies from binding to the membrane:

Since the membrane has been chosen for its ability to bind protein and as both antibodies and the target are proteins, steps must be taken to prevent interactions between the membrane and the antibody used for detection of the target protein. Blocking of non-specific binding is achieved by placing the membrane in a dilute solution of protein - typically 3-5% Bovine serum albumin (BSA) or non-fat dry milk (both are inexpensive) in Tris-Buffered Saline (TBS), with a minute percentage of detergent such as Tween 20 or Triton X-100. The protein in the dilute solution attaches to the membrane in all places where the target proteins have not attached. Thus, when the antibody is added, there is no room on the membrane for it to attach other than on the binding sites of the specific target protein. This reduces "noise" in the final product of the Western blot, leading to clearer results, and eliminates false positives.

Binding of complementary antibodies (and reporter enzymes) to confirm the presence of a particular protein:

Protein-specific antibodies are harvested and used to bind to the proteins attached to the membrane. These are 'primary antibodies' and, after being left attached to the membrane for a few hours and then the excess rinsed off, 'secondary antibodies', with reporter enzymes attached, are allowed to bound to species-specific sites on the primary antibodies (i.e. detection is a two-step process). The reporter enzyme confirms antibody-protein binding as, when exposed to an appropriate substrate, it drives a colourimetric reaction and produces a colour.