Rabbit polyclonal antibodies against full-length Nap1 were raised by Covance and affinity purified from total serum on a HiTrap N-hydroxysuccinimide–activated HP column (GE Healthcare) coupled with recombinant Nap1. Antibodies were eluted with Gentle Ag/Ab Elution Buffer (Thermo Fisher Scientific) and dialyzed into 50 mM Hepes.
H1M was immunodepleted from egg extracts as previously described (Maresca et al., 2005 (link)). In brief, 55 µl CSF extract was subjected to two successive 45-min incubations with 40 µg anti-H1M antibody coupled to 200 µl protein A Dynabeads (Invitrogen). To deplete Nap1, 11 µg of affinity-purified Nap1 antibody coupled to 40 µl protein A Dynabeads (Invitrogen) was used to deplete 11 µl of egg extract over two rounds of 45-min incubations. For control (mock depleted) reactions, an equal amount of total rabbit IgG antibody was coupled to beads. Recombinant Nap1-L1B or mutants (Nap1N6, Nap1C9, and Nap1N6C9) were added back to depleted extracts at a final concentration of 1 µM to match endogenous Nap1 levels.
H1M was immunodepleted from egg extracts as previously described (Maresca et al., 2005 (link)). In brief, 55 µl CSF extract was subjected to two successive 45-min incubations with 40 µg anti-H1M antibody coupled to 200 µl protein A Dynabeads (Invitrogen). To deplete Nap1, 11 µg of affinity-purified Nap1 antibody coupled to 40 µl protein A Dynabeads (Invitrogen) was used to deplete 11 µl of egg extract over two rounds of 45-min incubations. For control (mock depleted) reactions, an equal amount of total rabbit IgG antibody was coupled to beads. Recombinant Nap1-L1B or mutants (Nap1N6, Nap1C9, and Nap1N6C9) were added back to depleted extracts at a final concentration of 1 µM to match endogenous Nap1 levels.
