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Study Notes: Western Blotting

Western blotting is a technique in which proteins are detected immunologically in a gel following electrophoresis. That is, antibodies directed against a particular protein or set of proteins are used to selectively detect those, and only those, proteins.

The two main advantages of electrophoresis are:

  • sensitivity - silver staining can detect 10 ng whilst western blotting can detect 0.1 ng
  • specificity - only the protein(s) of interest will be detected.

Antibodies are bulky protein molecules that do not penetrate well into gels. In western blotting the protein bands in the gel are transferred to a nitrocellulose membrane that is then reacted with the antibody molecules. The sample proteins bind to the nitrocellulose membrane with great avidity.

The use of an electric current enhances this procedure and the term for this is electroblotting. Essentially the gel is covered with a nitrocellulose membrane and held in place between filter paper and foam pads.

This is then placed in a buffer chamber with the nitrocellulose on the side of the positive electrode and the gel on the side of the negative electrode. Remember that the SDS-treated proteins carry a negative charge and will move to the positive electrode under an electric current, encounter the membrane and bind to it. Putting the gel and the membrane in the other way spells disaster as the proteins come out of the gel and into the buffer!Skip flash movie


Assembling the western blotting apparatus.

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This technique relies on the antibodies binding specifically to the proteins of interest. One potential problem is that antibodies are also proteins and would bind to the membrane non-specifically. To stop this the membrane is first treated with a blocking agent. Note that between each step in this process the membrane is rinsed with fresh buffer to remove all unbound traces of the previous reagent.

One popular blocking agent is called ‘Blotto’ and contains:

  • 5% w/v non-fat powdered milk
  • 0.05% v/v Tween 20, in
  • 0.05M Tris-HCl, pH 7.4, 0.2M NaCl.

The membrane is then soaked in the specific antibody solution and the antibodies will attach ONLY to specific site(s) on specific protein(s). If no specific protein is present in the sample then no antibody will attach. This provides the specificity of the technique. This antibody is called the primary antibody and the unattached antibody is washed off.

The membrane is then soaked in another antibody called the secondary antibody. This is an antibody directed against the first antibody. An ‘anti-antibody or antibody to an antibody’ if you like. Again, if no primary antibody has attached then no secondary antibody will attach. Unbound antibody is then washed off.

This secondary antibody has a radioactive label (125I - called direct detection) or an antibody-enzyme (see below - called indirect detection) chemically attached to it. Such an enzyme is called a conjugate. Direct detection uses X-ray film as for autoradiography whilst in indirect detection the enzyme will react with a colourless substrate in a fresh buffer solution that is used to soak the membrane and causes a coloured band to appear in the region of bound secondary antibody. This then highlights the area on the gel to which the protein of interest is attached.Skip flash movie

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Commonly used enzymes and substrates are:

Conjugated Enzyme Substrate
Horseradish Peroxidase (HRP)

ABTS
4-chloro-1-napthol
TMB

Alkaline Phosphatase

PNPP
BCIP
NBT

Troubleshooting
Western blotting is a method with many steps and adherence to the method is vitally important to achieve a good result.

Some of the most common problems are:

  • putting membrane on wrong side of apparatus - no transfer
  • moving membrane after contact with the gel - smearing of protein bands on membrane
  • not running transfer for long enough - poor transfer
  • bubbles between the gel and the membrane - ‘holes’ in transfer
  • insufficient blocking (Blotto) - high non-specific back ground staining
  • poorly specific primary antibody - high non-specific background staining
  • poorly reactive secondary antibody - poor detection
  • insufficient washing between steps - high non-specific background staining
  • leaving out either antibody or the substrate - no detection.

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