Amersham ecl prime western blot detection reagent

Western blots were performed to determine the cellular protein abundances in the wild-type and genetically modified strains of M. maripaludis. The polyclonal rabbit antisera against the purified proteins were generated by MBL International Corporation. Mid-exponential cells of M. maripaludis were harvested and resuspended in Lysis Buffer [50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 10 (w/v) glycerol, 0.05% (v/v) NP-40 detergent] and lysed by sonication. The cell lysate was centrifuged at 14,000 g for 15 min at 4°C, and proteins in the supernatant were separated on 15% SDS-PAGE and transferred to a nitrocellulose membrane. The antisera to the proteins were diluted 1:5000. A horseradish peroxidase (HRP)-linked secondary conjugate at a 1:5000 dilution was used for the immunoreaction. Immune-active bands were visualized by an Amersham ECL Prime western blot detection reagent (GE Healthcare). For quantifying the cellular content of a protein, cell-free extract of M. maripaludis at indicated amounts was electrophoresed on SDS-PAGE synchronously with loading the corresponding purified recombinant proteins from E. coli as references. Density of each protein band was quantified from photographs using ImageJ software, and the cellular abundance of a protein was calculated by comparison to the respective recombinant protein.

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.

S. oligofermentans cells were first sonicated in RIPA lysis buffer (Beyotime Biotechnology) containing 1 mM PMSF. Cell lysates were then collected by centrifugation. Same amounts of proteins were separated by SDS-PAGE and hybridized with horseradish peroxidase (HRP)-conjugated anti-His tag monoclonal antibody at a 1:2,000 dilution (Abmart, Shanghai, China). Anti-puromycin monoclonal antibody at a 1:10,000 dilution (Millipore Company, Darmstadt, Germany) and HRP-conjugated anti-mice secondary antibody (Abmart) at a 1:2,000 dilution were used to detect puromycin-integrated proteins. Western blotting signals were detected using the Amersham ECL prime Western blot detection reagent (GE Healthcare). Intensities of target bands were quantified using Image J.