His Tag Cleavage (protein precipitates) - (Jul/04/2012 )

After eluting my 19.4kDa protein (theoretical pI = 5.75 uncleaved) off the Ni NTA His column, I attempted to cleave the tag. My Elution buffer contains 20mM Na2HPO4, 500mM NaCl, 500mM imidazole pH 7.4. It seemed to be stable in the elution buffer, as it was left overnight at 4C without reducing agents (it contains 4 cysteines) and remained soluble. I dialyzed into 2L of thrombin cleavage buffer (20mM Tris, 150mM NaCl, 2.5mM CaCl2, pH 8.4) and left it at 4C overnight. The morning after, the protein precipitated (confirmed by SDS PAGE). I did not include a reducing agent during dialysis as this can disrupt thrombin activity, but I may have to reconsider this. The cleavage was not complete.

I am considering increasing the salt concentration, adding DTT, and including glycerol -- or some combination. Any other suggestions to prevent precipitation?

Thanks.

If your protein is stable in the phosphate buffer I would perform the digestion without dialyzing, no matter if the cleavage buffer is better for the protease. I case of doubt add more protease or incubate longer and add fresh protease the next day. If thrombin is inhibited by DTT it would be better not to add it to the buffer, or maybe in a low concentration. I always use Glycerol in my protein buffers...it's better for the protein when you store it at -20/-80 °C.

I'll give it a shot. Thanks for the suggestion.

I usually make up my buffer so it has 10% glycerol. I am also trying to avoid DTT as the column goes brown when I use it.

So what I would do is to try 10% glycerol, and see how it goes.