I've recently purified TEV with a his-tag on a Ni-NTA column using our AKTA Pure FPLC. It usually works great. This time I had two peaks of lower absorbance over many fractions in the elution (linear gradient, 10-250 mM imidazole). After running the fractions on SDS-PAGE, both peaks have the TEV at the same MW. When I use the concentration calculator in the results of the AKTA, it shows that we have 23 mg of protein. However, when I use our UV-vis, I calculate 33 mg of protein. Does anyone know what might be causing this and how to fix it?