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Coupling Ig to CNBr activated Sepharose
Reagents needed:
• CNBr activated sepharose (1 g makes ~3.5 ml) from Pharmacia
• Purified antibody (needs to be very pure and between 1 and 4 mg/ml), dialyzed against coupling buffer
• Coupling buffer, pH 9.0
0.1 M NaHCO3
0.5 M NaCl
• Blocking buffer, pH 8.0 (make fresh just before use)
1 M ethanolamine in coupling buffer (sterile filtered)
or 1 M Tris-Base, pH 9.0 in coupling buffer (sterile filtered)
• 1 mM HCl, sterile-filtered
• Low pH Wash
0.1 M acetic acid
0.5 M NaCl
1) Take an OD280 reading of the antibody solution
2) Swell CNBr Sepharose beads in 1 mM HCl for ~15 min. at room temp.
3) Transfer gel to sintered glass funnel and wash with 200 ml of 1 mM HCl
a) Be prepared to do the next 2 steps immediately -- there cannot be any pausing in the protocol between steps 4 and 5
4) Wash gel with 5 ml coupling buffer
5) Using a clean spatula, immediately transfer gel to tube containing antibody solution after the coupling buffer has passed through the gel
a) the ratio of antibody to gel is dependent upon the antigen size. If the antigen to be purified is large, then you should decrease the amount of antibody used (try ~4 mg antibody/0.286 g gel for 100 kD antigen) and vice versa. For antigens of ~60 kD, use 6 mg antibody/0.286 g gel. Also, you can add some tris (or any free amine group) to the antibody solution when coupling low concentrations of antibody to maintain a more uniform coupling of antibody across the bead surface. 0.286 g of gel will swell to approximately 1 ml volume when hydrated.
6) Rotate tube genetly at room temp. for 2 hours
7) Spin down beads (tabel-top centrifuge, 2000 rpm for 1 min) and take OD280 of supernatants
a) if OD280 is at least 10-fold lower than in Step 1, continue with procedure
b) if OD280 is not at least 10-fold lower than in Step 1, coupling did not proceed as expected
i) this is most commonly caused by not transferring the gel to the antibody solution quickly enough in Step 5
c) when coupling large amounts of antibody/ml of gel, the OD280 reading may be 3-fold lower instead of 10-fold lower
8) Wash coupled beads 3x with coupling buffer
9) Aspirate coupling buffer from beads and incubate with blocking buffer for 2 hr at room temp.
10) Wash beads 4x, alternating between low pH wash buffer and coupling buffer (2x low pH and 2x coupling buffer) and checking OD280 of first and fourth supernatants (OD280 readings should be minimal ~0.01 or lower)
11) Pour gel into column (careful - no bubbles in the column!) and wash with 5 column volumes of either sterile-filtered lysis buffer or sterile-filtered PBS-azide
12) Store column in sterile-filtered PBS-azide
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