iv. Visualization
The last step in the western blot technique is to develop the membrane, i.e. visualize the proteins. If there was any of the protein of interest (GT50) in the samples you analyzed, then there should be primary and secondary antibody attached to this protein. The secondary antibody used in this experiment has been manufactured with an enzyme linked to it that will react with a certain chemical (see below) to produce a bluish precipitate.
The steps are:
- Prepare the DAB solution (the chemical that reacts with the enzyme on the secondary antibody) by dissolving a tablet of DAB in a Tris buffer solution.
- Mix the DAB solution with a small amount of hydrogen peroxide and pour it on the membrane.
- Watch as the membrane develops
- Pour buffer solution (phosphate buffered saline) to slow (and eventually stop) development when it appears adequately developed.
- Transfer the membrane to another culture dish and rinse it with more buffer.
After completion of these steps the membrane may be inspected to determine if the protein of interest is present by looking for dark bands at the appropriate position relative to the molecular weight markers and positive controls (pure GT50).
Figure: An illustration of a developed membrane, showing protein of interest occuring in the tested sample. From left to right, the visualized lanes contain: 1. molecular weight standards markers, 2. sample1, 3. sample2, 4.sample3, 5.pure protein sample, 6.sample1, 7.sample2, 8.sample3, 9.pure protein sample, 10.molecular weight standards markers
Figure: Photograph of developed membrane from an actual laboratory experiment. The membrane development revealed the presence of the protein of interest as can be seen in the row of darker bands. The strip on the right was cut off prior to the addition of the secondary antibody to determine the success of the development procedure.
What’s going on:
The DAB reacts with the enzyme on the secondary antibody to produce a blue colored precipitate. This should indicate where your protein of interest is located, because the secondary antibody binds to the primary antibody, and the primary antibody is bound to the protein you are looking for. This does not always occur as planned, however, and non-specific binding may occur. Non-specific binding is where an antibody attaches to protein other than the intended one.
Notes: DAB is hazardous (i.e. believed carcinogenic), and should be handled with caution!
© Copyright 2001, Georgia Institute of Technology
School of Biomedical Engineering