Summary: Auble Lab
Coupling Py antibody to agarose beads
Use Pharmacia GammaBind Plus Sepharose (cat # 17-0886-02). Binding
capacity is 18 mg/ml but I have never tried to bind antibody to this extent.
The binding capacity of Pharmacia Protein G Sepharose is > 20 mg/ml but
GammaBind is supposed to bind tighter to antibodies and the linkage is protease
Wash 5 ml (50% slurry) with 3 X 25 ml PBS in 50 ml conical tube. Spin to pellet
beads in clinical centrifuge 3 K for 2 minutes.
Add 2.5 ml 5 mg/ml purified Py antibody and allow to bind for 2 hours by
rotating end over end at room temperature.
Pellet beads in clinical centrifuge; save flow-through.
Wash beads 2 X 35 ml with 0.2 M Na Borate pH 9; save each wash.
Resuspend beads in 25 ml 0.2 M Na Borate pH 9 and remove 100 Ál suspension
Add dimethyl pimelimidate (Sigma D-8388) to 20 mM (129.5 mg/25 ml).
Mix 30 minutes at room temperature; remove 100 Ál suspension for gel.
Wash beads with 25 ml 0.2 M ethanolamine pH 8.
Incubate beads for 2 hours in 25 ml 0.2 M ethanolamine pH 8.