I'm trying to use IAC to purify a protein using an old prepacked NHS column with a potent antibody, that was coupled 2-3 years ago, and getting bad yields (Wide peak over 4 mL at 2 mAU max, protein not visible on a SDS PAGE gel, but shows up on a western blot when probed with the same antibody). I'm wondering if the antibody or column degraded etc. over time, as it was stored in PBS+azide at 4C for two whole years. (We elute with pH2.5 buffer if that makes any difference).
I have the chromatograms from when the column was fresh, and they look relatively good, with a decent area-under-the curve. But this time, it's dropped. Image is attached. I know columns last forever, but I'm just wondering how long a coupled column is good for.
Any good advice is welcomed. Thank you in advance.
That does seem like a long time to use an immunoaffinity column. The antibody may have become degraded, or denatured, or leached off the resin. If possible, prepare a fresh column.
The best option is to prepare a fresh affinity column. The IAC after elution of bound macromolecules must be washed with PBS and thence stored either with sodium azide or thiomerosal as a preservative.
As stated above the column may be degraded or loss of Ab. It may also be a little clogged up with ppt /non specific bound protein and needs a good clean.
As well as using glycine pH 2.7 wash, you can also incorporate a detergent wash such as Tween 20. I have even read that you could use 6M GnHCL to remove ppt protein, maybe worth a go.
If you make a new column obviously keep the old one as a comparator.
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