Anti 6 his

For western blotting, 6His-LbNMNAT and 6His-Δ_241-249LbNMNAT proteins were separated on a 12% SDS-PAGE, blotted onto PVDF membrane at 200 mA for 2 h in transfer buffer (10 mM Tris-HCl pH 8.0, 0.2 M glycine, 10 % (v/v) methanol). The membrane was blocked at room temperature for 2 h with 5 % non-fat milk in TBS-T buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% (v/v) Tween 20). Three washes were performed with TBS-T each for 10 min. The detection of the 6His tag fusion proteins was performed by incubating the membrane with the primary antibody anti-6His (1:5000) (Abcam) and anti-mouse IgG secondary antibody conjugated to biotin (1:10000) (Sigma). Chromogenic detection of complexes was performed with alkaline phosphatase-conjugated streptavidin (1:3000) (Promega) and the substrates nitro-blue tetrazolium (NBT) (50 mg/ml), and 5-bromo-4-chloro-3′-indolyl phosphate (BCIP) (50 mg/ml) (Promega) in substrate buffer (100 mM Tris-HCl pH 9.0, 150 mM NaCl, 1 mM MgCl₂).