All steps up to the dialysis at rt.
- Pour column in TBS (=0.15M NaCl, 20mM TrisCl pH 7.4). We use a 5 ml column for 25 mls serum. Wash extensively in TBS after prewashing as indicated in the protocol for coupling peptide to the resin.
- Thaw serum- dilute 1:1 with TBS and filter through a 0.2 um filter
- Load the serum over the column, taking at least 20 minute total.
- Run the breakthrough over the column five times. Alternatively you can use a parastaltic pump and recirculate the serum ON or just batch bind the serum ON.
- Wash with 5 col vols TBS.
- Wash with 10 col vols 0.5 M NaCl, 20mM TrisCl pH 7.4, 0.2% Triton- X-100.
- Wash 5 col vols TBS
- Elute with 0.15 M NaCl, 0.2 M Glycine-HCl pH 2.0. Collect 1 ml fraction, with each tube containing 0.1ml of 2 M TrisCl pH 8.5
- Wash with TBS until pH is reequillibrated.
- Elute with 6 M GuanidineHCl in TBS, collecting 1ml fractions.
- Wash with TBS + 0.1% NaN3, and store at 4 deg C.
- To determine where to pool fractions, spot 1 ul of each fraction onto nitrocellulose paper and stain with ponceau S. Pool all fractions that show pink color.
- Dialyze ON into TBS or your favorite buffer.
- If necessary the antibody can be concentrated by sweating the dialysis bags, or by spin-concentrating.
- Bring the azide concentration up to 0.1% and store at 4 deg C for up to three months. For longer storage freeze in aliquots and store at -80 degC or add glycerol to 50% and store at -20 deg C.
Note: Do not pool the low pH and GuHCl eluates as they may have significantly different properties. We have found that the GuHCl may have higher affinities, but also may contain a higher fraction of partially denatured antibody that could contribute to staining background. The proportion in each pool varies with the peptide immunogen.
The quality of the anti-peptide serum seems to increase with multiple boosts - the first bleed may be feeble.
Back to top
Back to protocols