Immunoblotting validation of research antibodies generated against HS1-associated protein X-1 in the human neutrophil model cell line PLB-985.

HS1-associated protein X-1 (Hax1) is a 35 kDa protein that is ubiquitously expressed. Hax1 is an anti-apoptotic protein with additional roles in cell motility, and autosomal recessive loss of Hax1 results in Kostmann syndrome, a form of severe congenital neutropenia. Because of the important role of Hax1 in neutrophils we demonstrate here validation of two commercially available research antibodies directed against human Hax1 in the human myeloid leukemia cell line PLB-985 cells. We show that both the mouse anti-Hax1 monoclonal IgG directed against amino acids 10-148 of Hax1 and a rabbit anti-Hax1 polyclonal IgG antibody directed against full-length Hax1 reliably and consistently detect Hax1 during immunoblotting of three different PLB-985 cell densities. Using shRNA mediated Hax1 knockdown, we demonstrate the specificity of both Hax1 antibodies. In addition, our results suggest that the rabbit anti-Hax1 polyclonal antibody provides a stronger intensity in detecting Hax1 protein, with detection in as few as 0.1 x 10 6 cells in 6 total replicates we have performed.