Commercial mouse and human nerve growth factors contain nerve growth factor prohormone isoforms

Highly sensitive chemiluminescence immunoblot analysis was utilized to examine the purity of mouse 2.5S-, beta- and 7S nerve growth factors as well as that of recombinant human beta-nerve growth factor obtained from commercial vendors. Three polyclonal antisera and two monoclonal antibodies to 13 kDa nerve growth factor (2.5S NGF and beta-NGF) were employed for assessing the purity of each preparation. In addition, polyclonal antisera against two prepro-NGF specific domains were used for immunoblotting analysis to ascertain the identity of high molecular weight nerve growth factor immunoreactive proteins as prohormones. Both the mouse and human NGF preparations contained 53 and 60 kDa immunoreactive proteins. Of these, the mouse 60 kDa and the human 53 kDa proteins strongly immunoreacted with both prepro-nerve growth factor specific domain antibodies suggesting that they are two NGF prohormone isoforms. In addition, both the mouse and human nerve growth factor preparations contained proteins that were immunoreactive to polyclonal antisera and monoclonal antibodies to mouse 2.5S and/or beta-NGF. High molecular weight aggregates of prohormones were also observed in mouse and human nerve growth factor samples. In summary, none of the ten NGF samples examined were pure as stated. Our study cautions investigators in the field to be aware of the presence of nerve growth factor prohormones and other proteins in various mouse and human nerve growth factors sold commercially.