CEF, erythrocytes and HEK293 protein extracts, (52 mcg, corresponding to 110 000 cells) along with the protein marker SM0661, Fermentas, were separated by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred to 0.45 mkm Polyvinylidene difluoride membrane for Western blotting (Biotrans, US) using a mini transfer cell (Bio-Rad, Hercules CA). After transfer the membrane was blocked by an 1 h incubation in 5% fat-free dry milk dissolved in PBST (1× PBS, 0.1% Tween-20), washed three times in PBST and incubated with primary antibody anti-CTCF (Cell Signaling Technology, 3418S) or with primary antibody anti-Rad21 (Abcam, ab16-473-100) (3 h, RT). After three washes in PBST the membrane was incubated with affinity purified alkaline phosphatase-conjugated goat anti-rabbit IgG (A3812, Sigma) or with affinity purified alkaline phosphatase-conjugated goat anti-mouse IgG (A3562, Sigma). Three washes in PBST of the membrane was followed by 5 min incubation in the buffer for alkaline phosphatase (100 mM Tris–HCl, pH 9.5, 100 mM NaCl, 10 mM MgCl2) and the bound antibodies were visualized using nitro-blue tetrazolium/5-bromo-4-chloro-3′-indolyphosphate (BCIP/NBT) substrate for alkaline phosphatase (Sigma).
The protein marker was visualized by amido black staining according to a standard protocol.
The protein marker was visualized by amido black staining according to a standard protocol.