Summary: Batch Cross-linking of Antibody to Protein G Sepharose
· Warm bottle of DMP to room temperature before opening.
· Determine protein concentration of antibody solution (i.e., ascites, supernatant, etc).
· This method is easily scaled down 10-20 fold and done in microfuge tubes.
· Use different buffers with protein G-sepharose or protein A-sepharose (see step 2).
1. Plan for 1 ml of packed protein G-Sepharose beads per antibody. Place slurry of beads in a 15 ml
red cap tube, spin for 5 minutes in centrifuge at 2000 rpm at 4°C. Aspirate supernatant to obtain 1
ml of packed beads. Do not let the beads become dry; leave a small volume of buffer over beads.
2. Begin with a volume containing 2-5 mg of antibody. Ascites is ~20-25 mg/ml total protein, of
which ~10-20% is IgG (2-5 mg/ml IgG), so try 1 ml ascites fluid. Dilute to 9 ml with Borate
buffer. Spin for 10 minutes in centrifuge at 2000 rpm at 4°C. Place on ice.
· If using protein A-Sepharose beads, dilute antibody in 9 ml Borate/NaCl buffer. In all steps
below use Borate/NaCl buffer in place of Borate buffer.
· Save 100 µl of supernatant on ice for analysis by SDS-PAGE.
3. Transfer antibody solution (supernatant) to 15 ml red cap tube containing 1 ml packed beads.
4. Mix on rotator at room temperature for 30 minutes to 1 hour to bind antibody to Sepharose.
5. Spin beads 5 minutes in centrifuge at 2000 rpm at 4°C. Save supernatant at 4°C to analyze later.
6. Resuspend beads in 10 ml of Borate buffer. Rotate for 2 minutes at room temperature to wash.