Amine coupling in TRIS

Recently, a college gave me a protein and asked for it to be amine coupled to a CM5 chip. A preconcentration evaluation showed that 10mM Na Acetate, pH 5.5 would work well for a coupling buffer. After a 7 min EDC/NHS INJECT we diluted the protein 10-fold (to ~100ug/ml) for immobilization; obtaining >5000 RU amine coupled protein, post ethanolamine. Only then did I discover the stock protein was in 20mM Tris (supposedly). This would mean we did an amine coupling in 2mM Tris. Everything I've read/heard says to stay away from Tris because of the primary amine interference with amine coupling. Undoubtedly there is some very small (trace?) level of Tris that would be acceptable when doing amine coupling, but 2mM ??? Is this possible ?

ProfPlum

Hi,

You did already the immobilization and you should know the amount of immobilized ligand on the chip. Immobilized TRIS will give a little response but with a larger ligand (say > 20.000 Da) it depends on how much you immobilize response is. If it is > 1000 RU, I should try a positive control to check how active the channel is. Obviously it is better to dialyse the ligand against HBS.

Arnoud

The protein I immobilized was ~55,000; small molecule ~450. Max signal of small molecule binding ~30 RU, which is about what you'd expect, given those MWs and immobilization level.

I mean, had I known that you could get away with amine coupling in 2mM Tris, it would have saved a lot of headaches for those generating the proteins for immobilization. I can't remember how many times either I've done dialysis or asked a college to perform dialysis simply because the protein was in Tris.

ProfPlum

In general I would not recommend to have primary amines in the coupling buffer.
When we purify proteins we always dialyse a batch against HBS or PBS because primary amines are also used in coupling recations like biotinylation or adding other labels.

Arnoud