Biological Microscopes

An epitope is a structure that an antibody recognizes as an antigen. Recognition of the antigen occurs when an appropriate structure is formed in an area of a protein in which amino acids are arranged linearly. A protein usually has several kinds of epitopes. The antibody offers an important technique for identifying the distribution and quantity of cell components (proteins, etc.). The antibody is usually obtained by refining the protein of interest and injecting it in a rabbit, etc. However, this takes long time and there are many cases in which antibody creation fails (absence of epitope). But the DNA recombination technology has made it possible to create the fused protein, in which the protein of interest is bonded with a small fragment of protein working as the epitope (peptide). The proteins tagged with epitope can be detected later by using the antibody for the epitope.

Immune assay is an important technique for identifying the distribution and quantity of cell components (proteins, etc.). The relevant antibody is usually obtained by purifying the protein of interest and injecting it in rabbits or other animals. However, this approach is time-consuming and there are many cases in which antibody production fails due to the absence of epitopes. DNA recombination has now made it possible to create a fused protein, in which the protein of interest is bound with a small fragment of protein (peptide) working as the epitope. The proteins thus tagged with the epitope can later be detected by the specific antibody for the epitope.

Fig.1

Fig.2

The use of epitope tagging is identical to that of Green Fluorescent Proteins (GFPs). The gene of the target protein is inserted into the epitope tag vector and the target protein with its tag is expressed in cells by transfection of the vector. The protein is distributed inside the cell according to the original properties of the protein. When using GFPs, the target protein can be observed easily because the GFP tag emits fluorescence; however, when using the epitope tag, the target proteins can be detected only by immunological procedures because the epitope tag is not functional in cells.

Advantages of epitope tagging

• Very short time requirement compared to ordinary antibody preparation, which requires more than one month to immunize the test animal
• Using a single epitope tag, a variety of proteins in specimens can be detected by the same tag-specific antibody
• Lower cost than antibody preparation
• The epitope tag is composed of 3 to 14 amino acids and does not spoil protein functions.
• Can be used to distinguish between two proteins with similar antigenicity
• Can be used to detect proteins that are hard to purify 

The following table shows the amino acid arrangements that are often used as epitope tags:

Detection

Both the direct and indirect methods can be used like the fluorescent antibody technique. With the direct method, the antibody for the tag is labeled with a fluorescent dye, while with the indirect method, the antibody against the tag is applied as the primary antibody and detection is performed on the secondary antibody that is labeled with a fluorescent dye. For more information, please refer to the Experiment Manual in the Roche web site.

Link

Experimental Manual

One-letter codes for amino acids