I am about to use our Anion Exchange Column to further purify my protein.
We purified our protein using the His column, so it has 150mM NaCl, 150mM Immidizole and 20mM Tris.
There are a few contaminating bands along with our eluted protein, so we are going to run it over our MonoQ column.
My question is, I know that our protein will bind to the anion exchange column and that using a salt gradient will wash off proteins.
But when we load our sample there is already salt present in our buffer. Will this salt affect our protein binding? Another lab suggested diluting down our sample to below 100mM salt concentration by adding starting buffer.
Also the protocol says to wash 5-10CV with starting buffer (20mM Tris pH 8.0). However there is no salt in this buffer, and since salt is important to help solubilize proteins wouldn't this cause stuff to precipitate out of solution and clog our column?