Figure 2

(A) Coomassie staining of purified HIS-FAAH recombinant protein from Dictyostelium. Dictyostelium cells AX3FAAH expressing HIS-FAAH were lysed and the recombinant protein was bound to Ni-NTA resin. Resin bound protein was eluted using lysis buffer containing 200 mM Imidazole and the eluate fractions S1, S2, S3, S4, S5 were resolved on 10% SDS-PAGE and Coomassie stained. (B) Western blotting analysis. Fractions analysed in Figure 2A were analysed by Western blotting using anti-HIS antibody. (C) Western blotting analysis. Fractions analysed in Figure 2A/2B were pooled together (P1) and analysed by Western blotting using anti-FAAH polyclonal antibody and the same fraction was used in enzyme kinetic assay. (D) Coomassie staining analysis of purified recombinant MBP-FAAH protein from E.coli. Cells expressing recombinant MBP-FAAH were lysed and the recombinant protein was bound to amylose resin. Resin bound recombinant protein was eluted using lysis buffer containing 15 mM maltose and the eluate fractions S6, S7, S8, S9, S10 were resolved on 10% SDS-PAGE and Coomassie stained. (E) Coomassie staining analysis. Fractions analysed in Figure 2D were pooled together (P2) and analysed by Coomassie staining.