Westernbright ecl system

For western blotting of FKBP12, monoclonal RPE1 dCas9‐Kin14VIb and its parental polyclonal population were transfected with 20 nM of siRNA Luciferase (CGUACGCGGAAUACUUCGAdTdT) siRNA FKBP12 (AAACUGGAAUGACAGGAAdTdT) using HyPerFect (Qiagen). 72 h following transfection, cells were lysed in Laemmli buffer for 10 min at 100°C. Protein concentration was determined using a Lowry assay. Proteins were separated on a 15% SDS‐polyacrylamide (PAGE) by electrophoresis and transferred to a nitrocellulose membrane. The membrane was blocked with 5% milk in TBS‐0.01% Tween‐20 for 1 h, before being incubated with rabbit anti‐FKBP12 antibodies (Abcam ab2918) at 4°C overnight. Horseradish peroxidase (HRP)‐conjugated goat anti‐rabbit secondary antibodies (Bioke, cat. no 7074) were then added for 2 h at room temperature. Chemiluminescence was detected using the WesternBright ECL system (Advansta K‐12045‐D20) and visualized using an Amersham Imager 600. The membrane was subsequently stripped by a 1‐h incubation with PBS + 0.02% sodium azide and reprobed with mouse anti‐Histone 3 antibodies (Abcam, ab1791) as a loading control. For the loading control, HRP‐conjugated goat anti‐mouse secondary antibodies (Biorad, 170‐6516) were used.