Preparing Fab and F(ab)’2
SDS PAGE gel of different antibody fragments following papain digestion and purification on Protein A agarose.
- F(ab) and Fc: Incubation with papain results in cleavage of immunoglobulin at the hinge region. Further purification on Protein A-agarose separates the antigen binding Fab from the Fc region. Fragments can be further purified and analyzed using size exclusion chromatography.
- F(ab’)2 : Cleavage with pepsin produces one F(ab’)2 fragment and smaller Fc portions. These fragments are useful for conjugation with detectable labels as the sulfhydryl group is still intact.
- Eliminates binding to Fc receptors
- Improved staining in immunohistochemistry(IHC)
- Easier penetration of tissues
- More useful in some applications when 150 kD antibody proves too large