SulfoLink affinity purification

To affinity purify antibodies raised against cysteine-containing peptides

Column preparation

  1. Clean column several times with destilled water, rinse it with 70% ethanol
  2. >Mix 2 ml of Sulfolink coupling gel (Thermo Scientific, Rockford, USA) with 10 ml of Coupling Buffer (50 mM Tris pH 8.5, 5 mM EDTA) in a small plastic column, invert a few times to equilibrate gel (2 ml of coupling gel beads correspond to about 5 ml of beads in solution from the stock bottle)
  3. Open the bottom of the column to remove Coupling Buffer
  4. Dissolve fresh 2 mg of Cys-containing peptide in 2 ml of Coupling Buffer
  5. Add 2 ml peptide solution and rotate the column at room temperature for 15 min
  6. Incubate at room temperature for 30 min without mixing
  7. Open the column and remove buffer
    Optional: (if peptide contains aromatic amino acid): check peptide concentration (A280) and compare to original peptide solution to determine coupling efficiency
  8. Wash the beads 3 times with 10 ml Coupling Buffer
  9. Add 2 ml of 50 mM cysteine in Coupling Buffer (made fresh, 12.1 mg Cys into 2 ml) and rotate for 15 min at room temperature to quench unreacted sites
  10. Incubate at room temperature for 30 min without mixing
  11. Wash 3 times with 10 ml 1 M NaCl (about 5 min each)
  12. Wash 3 times with 25 ml PBS

Antibody binding

  1. Defrost the antibody serum, and filter through a 0.2 μMfilter (this step will increase flow through speed)
  2. Add the serum to the peptide-coupled beads in the column, close with the lid
  3. Rotate overnight in the cold room
  4. Put the column back to the holder and wait 15 min until the resin sediments
  5. Open the column and allow all the serum to flow through into a Falcon Tube (at room temperature)
  6. Run the flow-through once more through the column
  7. Wash the column 5 times with 25 ml PBS
  8. Wash the column with 15 ml 0.5 M NaCl in PBS to remove weakly bound antibodies
  9. Wash the column twice with 10 ml PBS

Manual elution

  1. Prepare the following solutions, adjust the pH precisely:
    • 100 mM glycine pH 2.7
    • 100 mM glycine pH 2.3
    • 100 mM glycine pH 2.0 0,5 M NaCl
  2. Elute 8x1 ml fractions with 100 mM glycine pH 2.7, into Eppendorf tubes containing 40 μl 1 M Tris, pH 9.5
  3. Elute 8x1 ml fractions with 100 mM glycine pH 2.3, into Eppendorf tubes containing 75 μl 1 M Tris, pH 9.5
  4. Elute 8x1 ml fractions with 100 mM glycine pH 2.0 1 M NaCl, into Eppendorf tubes containing 95  μl 1 M Tris, pH 9.5
  5. Measure protein concentration in each fraction using a Nanodrop machine (Thermo Scientific)
  6. Pool fractions eluted with the same bufferFor the pH 2.7-eluted fractions pool only the fractions starting with the peak fraction, i.e. discard the left most side of the peak, but include the right side minor fractions, which presumably have a higher affinity. For the pH 2.3 and pH 2.0 eluted fractions pool everything with protein (it is expected that the progressively lower pH will elute progressively higher affinity antibodies).
  7. If needed, concentrate separately the glycine pH 2.3, and 2.0 fractions using  Sartorius spin columns with 10,000 cut off, spin at 4,000 g for 10-40 min at 4 °C
  8. Concentrate 10-20 fold, add 5 mL 50 % glycerol-PBS and spin down again to 100-200 mL
  9. Measure protein concentration in the concentrated samples using the Nanodrop

Antibody storage

  1. Dilute antibodies to 50% glycerol (final concentration) and store aliquots at -20°C for mid-term storage (1-2 years) or -80°C for long-term storage
  2. You can test your antibody using our whole-mount immunostaining protocol


The only tricky thing about the prep is getting the Tris and glycine solutions right. You elute the antibody using low pH glycine, but this can damage the antibody if you are not careful. Eluted antibody is collected into tubes containing 1M Tris at pH 9.5, which immediately neutralizes the antibody solution. Well before you come to elute the antibody, prepare the glycine and Tris solutions as accurately as possible and run tests of how much Tris you need to neutralize the glycine - it should be about 50 μl for 950 μl of glycine (i.e. a 2 ml total fraction), but this will vary each time. pH indicator strips are good way of sorting this out.