Advantages of Fab and F(ab')2 Fragmentation:
- Reduced nonspecific binding resulting from Fc interactions
- Better controlled binding to Protein A in experiments including immunoprecipitation and Western blotting
- More efficient penetration of tissue sections for improved staining for immunohistochemical applications
- Higher sensitivity in antigen detection in solid phase applications
- Simpler system for studying the structural basis for immune recognition using X-ray cyrstallography or NMR,
- Lower immunogenicity than intact antibody for in vivoexperiments
- Fab preparation, utilize immobilized papain to enzymatically cleave IgG molecule just above the Fc hinge region to release two Fab fragments and one Fc fragment;
- F(ab)’2 preparation: utilize immobilized Pepsin to enzymatically cleave IgG molecule to release one F(ab)’2 fragment and one Fc fragment
- Fab and F(ab)'2 Preparation for Mouse IgG1
- Utilize immobilized Pepsin to enzymatically cleave IgG molecule at different cysteine concentration to generate Fab or F(ab)’2 frangments.
All enzymatic reactions are followed by protein A column purification to remove the Fc fragments.