Coupling antibodies and proteins to CNBr activated Sepharose 4B

[Luke]

Coupling antibodies to CNBr-activated Sepharose 4B

Buffers/Reagents
•   Cleaning Buffer
o   1mM HCl

•   Coupling Buffer
o   0.1M NaHCO3 pH 8.3
o   0.5M NaCl

•   Blocking Buffer
o   0.1M Tris-HCl pH 8.0

•   Acetate Wash Buffer
o   0.1M Sodium Acetate pH 4.0
o   0.5M NaCl

•   Tris Wash Buffer
o   0.1M Tris-HCl pH 8
o   0.5M NaCl

1.   Weigh out appropriate amount of beads.  1g freeze dried beads gives 2.5mL final volume medium.  5-10 mg antibody can be coupled per mL medium.
2.   Dissolve beads in cleaning buffer, it swells immediately
3.   Wash beads on scintered glass funnel using 200mL of cleaning buffer per g freeze dried beads for 15 minutes.  Wash slowly, more can be used.
4.   Collect beads in tube.
5.   Wash beads in coupling buffer.
6.   Centrifuge 1000 RPMs 5 minutes
7.   Aspirate buffer
8.   Dissolve Ligand into coupling buffer, use 5mL coupling buffer per g freeze dried beads.
9.   Alternatively antibodies can be dialyzed to coupling buffer to assure maximal coupling.
10.   Incubate beads and antibody overnight at 4 C with constant rotation.
11.   Centrifuge 1000 RPM 5 minutes
12.   Aspirate supernatant
13.   Block remaining active groups on beads using blocking buffer.  5-10 bed volumes is sufficient at RT while rotating or shaking.
14.   Centrifuge 1000 RPM 5 minutes, aspirate supernatant
15.   Wash 1st with Acetate wash buffer, centrifuge, aspirate
16.   Wash 2nd with Tris wash buffer, centrifuge, aspirate
17.   Repeat 15 and 16 for a total of at least 5 washes.
18.   Add PBS to create 50% slurry (ie 1mL PBS for 1mL beads)
19.   Azide should be added to final 0.03% as a preservative, store at 4 C