| Antibody Solutions | Help Article |
Purifying Antigens using Polyacrylamide Gels
Introduction
Antigens may be purified for antibody production using SDS-polyacrylamide gels (SDS-PAGE). Since the antigen is denatured by SDS-PAGE, the resulting antibodies are usually particularly good for techniques that detect antigen in a denatured form (i.e., Western Blotting). However, the resulting antibodies are less likely to bind to native antigen.
Isolating the antigen
The protein sample is run on a preparative gel. Load the gel with enough sample to obtain at least 100-200 ug of antigen per gel slice per injection. The maximum allowable size of the gel slice per injection is 1.5 inches long by 3-4 mm wide by 1.5 mm thick.
Locating the antigen
After electrophoresis, the protein band of interest must be located in the gel, and the gel slice excised for injection. Two methods are suggested. The Side-Strip Method is recommended only for abundant proteins that are well separated. Side-strips are cut from the edge of the gel and stained using typical procedures. The stained side strip is placed back alongside the unstained gel and the protein band excised from the similar location in the unstained gel. A second method, Reversible Staining Method, is generally recommended, particularly when the protein of interest is less abundant and adjacent to other proteins. In this method the entire gel is stained, the relevant band is excised, and then the gel slice is destained (e.g., Pierce, E-zinc reversible stain kit).
Storage and transport of the gel
The excised gel slice should be wrapped in plastic wrap (i.e., saran wrap) along with a few drops of water. Place the gel slice in a secondary container (50 ml tube, plastic bag) along with a moistened paper tissue or towel. The amount of antigen in the gel slice must be given. The maximum allowable size of the gel slice per injection is 1.5 inches long by 3-4 mm wide by 1.5 mm thick. Store the gel at 4 oC.