Preparation of an affinity resin

Please read the instructions given in the Pharmacia booklet, especially in "difficult" and unusual cases.

Solutions, buffer, materials:

  • CNBr-activated Sepharose 4 B (Pharmacia)
  • 1 mM HCl
  • coupling buffer (100 mM NaHCO3/NaOH [pH 8.3], 300 mM NaCl
  • blocking buffer (0.1 M Tris/HCl [pH 8.0])
  • washing buffer I (0.1 M Na-acetate, 0.5 M NaCl, [pH 4.0])
  • washing buffer II (0.1 M Tris/HCl, 0.5 M NaCl, [pH 8.0])
  • protein solution. The concentration should be as high as possible, ideally > 5 mg/ml. Dialyse to coupling buffer.

Protein preparation

  • Native: dissolve proteins in coupling buffer (5-10 mg protein per ml gel). Make sure that your protein is soluable in the coupling buffer. If not adjust the coupling buffer according to the recommendations in the booklet.
  • denatured proteins may be coupled to sepharose as micelles. Following Ni column purification in 8 M urea, add SDS to 1% and dialyse against coupling buffer (3x 100xvolumes). Make sure that all urea is removed completely
  • SDS-treated: dissolve proteins in 1x SDS-PAGE sample buffer without glycerol; if protein was freeze dried, incubate at 37¡C for 1hr; boil for 5 min; dialyse against 4 changes of coupling buffer over a 2-3 day periode.
!!! Avoid any buffer containing amino groups such as Tris because they will react with the BrCN groups.

Preparation of the CNBr-activated Sepharose 4 B (Pharmacia)

Start with these steps only when all materials are ready. The BrCN-sepharose beads are NOT stable in coupling buffer.
  • Let dry material swell in 10 ml 1 mM HCl for 15 min. 1 g material will give about 3.5 ml gel.
  • Wash on a sintered glass filter (porosity G3) with about 200 ml 1 mM HCl per gram dry gel. Add wash in several aliquots.
Note: HCl preserves activity of the reactive groups which hydrolyze at high pH
 

Coupling

  • Wash gel with coupling buffer. Use 5 ml per gram of dry gel.
  • Immediately after wash with coupling buffer add gel to protein suspension at a gel : buffer ratio of 1:2.
  • Mix gel with protein solution with an end-over-end mixer or rocker for 3 hr at r.t. or at 4ºC over night. Use a 15 ml conical tube. Note: avoid magnetic stirrer.
  • Aspirate coupling buffer; check for binding efficientcy by protein assay such as Bradford or A280.
  • Add blocking buffer to gel and mix for 2 hr at r.t. of at 40C over night (see above).
  • Pour suspended beads into column.
  • Wash with 5 cycles of low and high pH (Washing buffers I and II).
  • Wash with 5 volumes of PBS
Storage- Store column in PBS, 0.02% NaN3 at 40C.