There are several bits of additional information that are needed to be able to answer this question. Most importantly, what immune cells are you using? If the cells are either B lymphocytes or monocytes/macrophages, activation will increase surface expression of Fc receptors, and your anti-His antibody could be bound by the Fc receptors, giving a positive signal on activated but not treated with the His-tagged protein. You can block that by pretreating the cells with either purified IgG from the same species as the cells you are testing or by using Fc-blocking antibodies. I know that these are available for both human and mouse cells.

Hi Navit!

In order to reduce the background I would recommend to use an Fc-blocker and to titre your antibody, in order to reduce the non specific binding

Thank you. The Fc receptor blocking could be very relevant here

Hi navit,

There are some proteins in cells like YY1 that they have Histidine repeat and maybe its the main reason for background. You can read following article:

Article An endogenous ‘non-specific’ protein detected by a His-tag a...