Ab204448

Vectors expressing the editors (4 μg) and gRNA-puro (2 μg) were co-transfected into HEK293T cells in 6 cm dish (JETBIOFIL). Puromycin (InvivoGen) was added 24 h later to a final concentration of 2 μg/ml. Cells were harvested 72 h after transfection, and total protein extracted by RIPA lysis (EpiZyme). The protein samples were separated by SDS-PAGE, transferred to PVDF membrane (Merck Millipore). After blocking with 5% (w/v) non-fat milk dissolved in TBST (25 Mm Tris, PH 8.0, 150 Mm NaCl, and 0.1% Tween 20) for 1 h, the membranes were incubated overnight with anti-CRISPR-Cas9 antibody (Abcam # ab204448) or anti-GAPDH antibody (Absin #abs132004) at 4 °C. After extensive washing, the membranes were incubated with HRP-conjugated secondary antibodies at room temperature for 1 h. Proteins were visualized using Enhanced Chemiluminescence (ELC) reagent (Merck Millipore) and detected with an Amersham Imager 600.

For Western blotting, after plasmid transfection, HEK293T cells were harvested and lysed in RIPA buffer. The blots were subjected to incubations with anti-Cas9 (Abcam ab204448, 1:2000), anti-β-actin (Absin abs132001, 1:10000), or anti-Flag (Abcam ab205606, EPR20018-251, 1:5000) antibodies in TBST with 5% skim milk. Images were captured with Amersham Imager 600. The original scanned blots for the presented Western results are included in the Source Data file.