Western Blot Analysis of Archaeal Proteins

Western blot assays to detect the expression of SpCas9, aCPSF1b, and aCPSF2 were performed similarly to that described previously (33 (link)). After resuspension in lysis buffer (20 mM Tris base, 150 mM NaCl, 10% [vol/vol] glycerol, 0.1% [vol/vol] Triton X-100), cells of M. maripaludis were lysed by sonication. The cell debris was precipitated by centrifugation at 10,000 × g for 10 min at 4°C, and the supernatant was quantified for the protein and then used for the Western blot assay. Proteins in the supernatant were separated by SDS-PAGE and then transferred to nitrocellulose membranes. Western blotting was performed using the commercial polyclonal rabbit antiserum raised against Cas9 and the customized polyclonal rabbit antiserum raised against the purified recombinant aCPSF1b and aCPSF2. The antibodies were used at a 1:5,000 dilution. Immune-active bands were visualized by an Amersham ECL Prime Western blot detection reagent (GE Healthcare) on a Tanon-5200 multichemiluminescence/fluorescence imaging system. The quantification of Western blot band intensity was performed using ImageJ.