Dialyze IgG vs 1liter 20mM NaPO4 ; 10mM EDTA pH7.0 at 4C. Concentrate to 4mg/ml (according to 1.2OD280nm per ml to 1mg protein per ml). Use Centriplus cut off 100000. Keep sample in aliquots at -80C.
 
 

Digestion buffer preparation:

Prepare digestion buffer just prior to use because cysteine is very easily oxidized. Buffer: 20mM NaPO4 ; 10mM EDTA ; 20mM Cysteine pH7.0
 
 

Equilibration and Preparation of the Immobilized papain:

Mix the Papain-agarose suspension by inversion.

Add 0.5 ml gel slurry (0.25ml settled gel) to a suitable reaction vessel.

Spin 3min 3500rpm. Discharge supernatant.

Add 1ml digestion buffer. Mix. Spin. Discharge supernatant.

Repeat last washing twice.

Resuspend the Immobilizd papain in 0.4ml digestion buffer.
 

Generation of Fab and Fc fragments:

Add 0.4ml (1.6mg) IgG to the equilibrated papain-agarose. Incubate overnight RT (maintain constant mixing of gel during incubation).

Spin 3min 3500rpm. Keep supernatant.

Add to the resin 1ml 20mM Hepes pH7.0

Mix, spin and keep both supernatants together.
 
 

Separation of Fab and Fc fragments:

Separate both fragments with HiTrap SP column (cation exchange), with NaCl step gradient in buffer 20mM Hepes pH7.0 + 0.02% Na Azide.
 
 

PAGE-SDS analysis + or - Beta Mercapto Ethanol