Quiescent human colon carcinoma HCT116 cultures were treated with 10% FBS for three time points (0, 15, 30 minutes) or (0, 30, 60 minutes) were used in Matrix-ChIP and real-time PCR assays at EGR1 gene (Exon1) and 15kb upstream site.
Anti-SMYD3 antibody - ChIP Grade (ab85277) at 1 µg/ml (in 5% skim milk / PBS buffer) + human fetal stomach lysate at 10 µgSecondaryHRP conjugated anti-Rabbit IgG at 1/50000 dilution
ICC/IF image of ab85277 stained HeLa cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab85277, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.