Mouse anti his

Kunkel mutagenesis was utilized to make truncated CD99 ECD fragments (23-122, 33-122, and 43-122 ) and a combination of Kunkel mutagenesis and restriction and insertion cloning were utilized to produce the other CD99 fragments (53-122, 63-122, 73-122, 83-122, 93-122, 103-122, and 113-122) ^{77 (link)}. The proteins were expressed and purified as described above, except they were only purified using a Ni-Sepharose resin (Cytivia). SDS-PAGE of 50 ng of each purified protein was performed followed by transfer to Immobilon-P^SQ PVDF membrane (ca# ISEQ00010, Millipore). For Western blot detection, the membranes were blocked with 5% skim milk overnight at 4°C, then washed three times with 1X TBST (50 mM Tris pH 7.5, 150 mM NaCl, and 0.05% Tween-20). The membranes were then incubated with 14 nM of clones 10, 22, and 30, 1 nM of HO36-1.1, or 1:1000 of mouse anti-His (ca# 37-2900, Invitrogen) for 1.5 hours at room temperature, then washed as described above. The membranes were then incubated with goat anti-mouse IgG Fc conjugated with horseradish peroxidase (HRP) (ca# 31437, Invitrogen) or goat anti-mouse IgM-HRP (ca# 626820, Invitrogen), for 1 hour at room temperature, then washed as described above. Pierce ECL 2 Western Blotting Substrate (ca# 80198, Thermo) was added according to manufacturer protocol. Signal detection was analyzed on ChemiDoc Touch imaging system (Biorad).