Prasad Trivedi :

Thank you,

Actually, I use the following molarity:

1: Tri@@ton 0.4% EDTA 1mM, Tris 50mM and Fresh DTT 1mM (3 Times)

2: deoxycholate 20mM , EDTA 25mM, Tris 50mM and Fresh DTT 1mM (3 Times)

3: PBS

4: Water

I use the same molarity and steps for some recombinant proteins in our lab.

During the washing steps, unlike other proteins, in this case, impurities are not removed or they are removed very little. I use E. coli DE3 strain to express all my proteins. The confusing thing for me is the host proteins (HCP).

Aren't they the same in all cases? if yes, why i can't remove them in this case like others?

Is it possible that something downstream caused this to happen?

Thanks a lot

I decreased pH and increased DTT concentration.