Coupling to CNBr-Sepharose

1) Weigh out required amount dry powder (1 g swells to 3.5 ml beads) [say you weight 3 g]

2) Swell the beads for 15' at RT in 1 mM HCl immediately before use

3) Centrifuge 10 min at 4,000 rpm WITHOUT BRAKE

4) Wash the beads twice using 50 ml of HCl 1 mM

5) Raise pH by washing in coupling buffer (NaHC0₃ / Na₂C0₃ ph9.5, 0.5 M)

Coupling buffer: 30 ml 0.5 M Na2C03 +70 ml 0.5 M NaHC0₃ check pH

6) Immediately add the Ab diluted in a volume of coupling buffer equal to that of swollen beads. Use 10 mg of Ab or proteins/g beads.

6a. To prepare FBS-sepharose: use 300 µL of FCS (protein content approx 30-45 g/L) per 1 g of beads [use 1 mL for 3 g of beads]

7) Rotate the mixture for 1.5 h at RT or O/N at 4°C.

8) Spin down beads (10 min at 4000 rpm, without brake) and take off supernatants to check coupling efficiency by

measuring OD280 (use coupling buffer as zero). Optimal bond exceeds 90%.

9) Wash away excess ligand with 10 ml of coupling buffer/3.5 ml beads.

10) Block any remaining active groups with Tris-HCl O.1 M, pH 8.0 (10 ml) for 1 h, at RT. (In alternative, use 0.2 M ethanolamine or tri-ethanolamine, pH 8.0)

11) Wash the beads with 3 cycles of: Acetate buffer, then Tris Buffer (10 ml each) = A, B, A, B, A, B.

Acetate buffer: 0.1 M Naacetate + 0.5 M NaCl/ pH 4.0 with acetic acid

Tris buffer: 0.1 M Tris 0.5 M NaCl pH 8.0 with concentrate HCl

12) Store In PBS + 0,1% NaN3 at 4°C.